DOI: 10.5061/dryad.ht76hdrd2
title: Environmental complexity during early life shapes average behavior in adulthood

description: Personality has been identified in a range of animal taxa during the last
 few decades, with important ecological and evolutionary implications.
 Investigating the effects of environmental factors during early life can
 provide important insights into the ontogeny of animal personality. We
 reared newborn mosquitofish, Gambusia affinis, in tanks of different
 structural complexities, and measured their behavioral traits (i.e.,
 shyness, exploration, and sociability) when they reached sexual maturity.
 Univariate linear mixed-effects models were fitted to test the effects of
 environmental complexity and sex on population-average behavior, whereas
 multivariate models were fitted to quantify behavioral repeatability (i.e.
 personality) and among-individual correlations (i.e., behavioral
 syndromes). On average, females were shyer and more social than males, and
 the fish reared in complex environments were shyer, less explorative, and
 more social than those reared in open environments. Among-individual
 differences were consistently large across trials for all behaviors,
 indicating that personality variation was present in mosquitofish of both
 sexes reared in different environments. Repeatability did not differ among
 behaviors, and there were no differences in repeatability in any behavior
 between sexes or among environments. A negative correlation between
 shyness and exploration was found in mosquitofish from all treatments at
 both phenotypic and among-individual levels, with the latter indicating a
 strong shyness-exploration behavioral syndrome. Our study provides robust
 evidence that average levels of personality might vary when mosquitofish
 are raised in different levels of structural complexity during early life.
description: <b>Study species and
 rearing conditions</b> A total of 150 gravid female
 mosquitofish were caught during May 2018 in artificial ponds on the campus
 of Anhui University (117.18°E, 31.77°N) and were housed in three 200-L
 holding tanks filled with oxygenated tap water at approximately 26°C. They
 were fed fine-grained commercial food (TIDDLER, Weifang YEE Pet Products
 Co., Ltd., China; 42% crude protein, 5% crude fat, 5% crude fibers, 11%
 ash) twice per day and exposed to a natural photoperiod (~ 14:10 L:D). We
 checked the housing tank frequently for newborn fry and transferred them
 on the day after birth to 30 net tanks (blocks: 80 × 80 × 80 cm, mesh
 size: 0.177 mm), which were placed in an undisturbed artificial pond (60 ×
 10 m) on the same campus. To avoid potential genetic effects, we randomly
 and evenly assigned the fry born in each day to the 30 tanks. Due to this
 assignment procedure, the number of mothers contributing to the fry and
 the sex ratio of the fry in each tank were unknown but were assumed to be
 approximately equal. In total, 1500 newborns were collected within one
 week and were reared, that is, 50 individuals in each tank with a maximum
 age gap of one week, until they were sexually mature. No predation on the
 fish occurred during the rearing period, and the mortality was assumed to
 not differ among the rearing tanks. The 30 rearing
 tanks were equally divided into three groups with different levels of
 structural complexity, that is, open (A), medium (B) and complex tanks
 (C). Two locally common plants, <i>Nymphaea tetragona</i> and
 <i>Alternanthera philoxeroides</i>, were placed into the
 medium and complex tanks to enrich the environment, with approximately 30%
 of the water area covered in the medium tanks and 70% in the complex
 tanks. Three complexity levels were found in the ponds where the mother
 fish were captured. To maintain the level of complexity, plant coverage
 inside the tanks was checked visually each day, and the extra parts due to
 plant growth were manually removed. To avoid the effect of the tank location,
 tanks housing different groups were placed alternately (i.e., ABCABCABC).
 There was little plant coverage in the rearing pond with a water
 depth of approximately 40 cm, and a large natural population of
 mosquitofish lived in this pond for several years. The water temperature
 ranged from 20°C to 32°C and the pH ranged from 7.4 to 7.6 during the
 rearing period. The fish in the tanks were fed brine shrimp nauplii and
 zooplankton during early ontogeny and then fine-grained commercial food
 two weeks later. Apart from being fed additional food, the ambient
 environment of the reared fish was identical to that of the natural
 population. Such a design allows fish to experience a near-identical
 environment in the field, thus avoiding behavioral abnormalities that
 might arise in laboratory conditions (Carere et al. 2015). Two months
 after the experiment started, we randomly caught four females and four
 males from each rearing tank, for a total of 240 individuals at an average
 age of 50 days, to characterize their behavioral traits in the following
 experiments. The sampled subjects were individually kept in labeled,
 black, opaque cylindrical tanks (height: 9 cm; diameter: 15 cm; hereafter,
 the holding container) with transparent lids filled with oxygenated tap
 water before and between experiments. A black, opaque, cylindrical refuge
 chamber (height: 5 cm; diameter: 7 cm; hereafter, temporary refuge) was
 placed in the center of each holding container, and fish were given 24-34
 h (varying due to test orders on the first day of the experiment) to
 acclimate to the chamber before experiments. All fish were kept in a
 laboratory room with a natural day-night photoperiod, and were not fed 12
 h before the experiments. In China,
 mosquitofish is an invasive species that is prohibited from being released
 into natural water bodies; thus, ethics committee review was not required
 to capture mosquitofish (Ministry of Environmental Protection of the
 People’s Republic of China 2016, Index No.000014672/2016-01463). The
 behavioral experiments were approved by the Institutional Animal Care and
 Use Committee of Anhui University (IACUC, AHU). No harm was done to the
 fish during the experiments.   <b>Personality
 assays</b> We measured shyness, exploration,
 and sociability for each subject in an individual arena, which was a white
 opaque plastic tank (37 cm long × 30 cm wide × 20 cm high; Figure 1). A
 camera (Sony HDR-CX510, 55× extended zoom, Sony Corporation, Tokyo, Japan)
 was suspended above each identical arena to record the behavior of the
 fish. The tanks were filled with oxygenated tap water (3-cm deep for
 shyness and exploration, 5-cm for sociability), and the water was
 exchanged between trials to exclude any effect of chemical signals
 released by previous individuals on subsequent animals. Behavior
 recordings were carried out in a laboratory with sufficient light (for
 clear observations), constant temperature (26°C), and no disturbances. To
 avoid observer effects, the experimenters were shielded from the tested
 subjects by a 1.5-m high opaque curtain during each trial. Behavioral
 trials for each subject were repeated three times every other day, with
 the test of shyness and exploration on alternate days to the test of
 sociability. To achieve a large sample size (i.e., three times for the
 three behaviors of 240 individuals) within one week, experiments were
 performed by six experimenters with each in charge of four arenas
 simultaneously. To avoid potential observer effects, the experimenters
 were trained before experiments to follow the standard procedures detailed
 as follows, and the fish observed by a certain experimenter were exchanged
 between days. The trial orders were randomized and fish were fed
 commercial food after each trial. At the end of all three behavioral
 trials, standard body length (accurate to 0.1 mm) was measured for each
 subject before fish were released into the artificial pond where they had
 been reared.   Assay 1: shyness and
 exploration Shyness and exploration for each
 subject were tested in one continuous trial. Before the experiment, one
 black, opaque, cylindrical refuge chamber (the same refuge placed in the
 holding container, hereafter referred to as starting refuge) was fixed at
 the middle position of one end of each tank. There was a sliding trapdoor
 (3 × 3 cm) on the side of the starting refuge facing the arena. A piece of
 fishing line was connected to the trapdoor, and hence, experimenters
 behind the opaque curtain could pull the fishing line to remotely open the
 refuge without disturbances to allow fish to swim out of the chamber and
 toward the arena. The rest of the tank (approximately 27 × 30 cm) was
 furnished with novel objects to enrich the environment; thus, the fish
 could not see through the arena (Figure 1a). To avoid potential effects of
 habituation toward the arena, we used different novel materials, that is,
 gravel, plastic sheets, and fresh lotus leaves in the three repeated
 trials. The materials were scattered in the same configuration for
 different subjects. At the
 beginning of each trial, a randomly selected fish was gently (in water)
 coaxed into the closed starting refuge, and then the camera began
 recording. The subject was allowed to acclimate for 5 min, after which the
 observer remotely opened the trapdoor of the starting refuge and left it
 open until the end of the trial. The time taken by the subject to emerge
 out of the starting refuge onto the arena (latency time) was used to
 measure shyness, with shyer subjects emerging later from the shelter.
 Whereas some researchers have defined shyness-boldness as an individual’s
 reaction to explicit risks (Réale et al. 2007), we followed many previous
 studies (Brown and Braithwaite 2004; Brown, Burgess et al. 2007; Brown,
 Jones et al. 2007; Toms et al. 2010; Cote et al. 2011; Huntingford et al.
 2013; Polverino, Cigliano et al. 2016; Polverino, Ruberto et al. 2016)
 that measured shyness in small fish as an individual’s latency to emerge
 from a familiar refuge outside which there might be predation risk. We
 considered a fish to have emerged from the starting refuge when its entire
 body crossed the trapdoor. All tested fish swam out of the refuge within
 13 min. The camera continued recording the movement of the subject for
 another 10 min after it left the refuge. After the exploration assay, the
 subject was immediately transferred back to its holding container. We
 extracted 600 image stacks from the 10-min movement videos (one frame per
 second) and used Image J (http://rsbweb.nih.gov/ij/) to mark the location
 of the fish head in each frame to delineate its movement pathway. Similar
 to Edenbrow and Croft (2013), we used the total pathway length to quantify
 the exploration score of the focal subject. We acknowledge that the
 measurement of exploration could not be unambiguously differentiated from
 activity because animals should be active while exploring (Carter et al.
 2013). While this measurement might concurrently include activity, we
 referred to this test as exploration because it was performed in an
 unfamiliar environment in which the distance covered has been proposed to
 be a measure of exploration (Sih et al. 2004; Réale et al.
 2007).  
 Assay 2:
 sociability Sociability was tested on the day following
 the assay for shyness and exploration. The arena was not furnished with
 novel structures, and no starting refuge was placed in it. Similar to the
 method described by Sommer-Trembo et al. (2017), a perforated,
 transparent, plastic, cylindrical bottle (diameter: 6 cm; length: 23 cm)
 containing 10 non-experimental conspecific individuals (five females and
 five males) as a stimulus shoal was fixed in the middle of the arena. One
 dark line was marked on each side of the bottle to define a social zone
 (width: 13 cm; Figure 1b). The width of the social zone at each side of
 the stimulus shoal was smallest near the water surface (i.e., 3.5 cm,
 approximately twice the average body length of the test fish) and
 increased with water depth. A randomly selected subject was gently
 introduced into one side of the arena (exchanged between trials) and was
 given 5 min to habituate to the new environment. After 5 min, the camera
 was switched on to record the location of the subject for 10 min. We
 determined the sociability of each subject as the time the focal
 individual spent in the social zone during the 10-min period.
description: There are no missing values. We encourage anyone wanting to use
 this data to contact the authors.