The zebrafish is a premier model organism yet lacks a system for assigning postembryonic fish to developmental stages. To provide such a staging series, we describe postembryonic changes in several traits that are visible under brightfield illumination or through vital staining and epiflourescent illumination. These include the swim bladder, median and pelvic fins, pigment pattern, scale formation, larval fin fold, and skeleton. We further identify milestones for placing postembryonic fish into discrete stages. We relate these milestones to changes in size and age and show that size is a better indicator of developmental progress than is age. We also examine how relationships between size and developmental progress vary with temperature and density, and we document the effects of histological processing on size. To facilitate postembryonic staging, we provide images of reference individuals that have attained specific developmental milestones and are of defined sizes. Finally, we provide guidelines for reporting stages that provide information on both discrete and continuous changes in growth and development. Developmental Dynamics 238:2975–3015, 2009. © 2009 Wiley‐Liss, Inc.

Loose aggregations of fishes, or shoals, are a basal social organization of vertebrates and offer a valuable opportunity to determine how individual perceptions influence group formation. We used zebrafish, Danio rerio, to comprehensively investigate the preference space for shoaling related to adult pigment pattern variation, presented in the form of 17 zebrafish pigment pattern mutants or closely related species. We examined all combinations of these phenotypes in 2,920 initial and replicated preference tests, and used as subjects both domesticated laboratory stocks and wild-caught fish. By using multidimensional scaling and other approaches, we show that laboratory and wild zebrafish exhibit similar preferences, yet, unexpectedly, these preferences differ markedly between sexes, and also from how human observers perceive the same pigment patterns. Whereas zebrafish males respond to two traits (species and stripe patterning) in deciding whether to join a shoal, zebrafish female preferences do not correlate with a priori identifiable traits, and neither perceptual world is correlated with that of human observers. The observed zebrafish sex differences run counter to the most commonly accepted explanations for the individual selective advantages gained by shoaling. More generally, these data describe very different perceptual worlds between sexes and reveal the importance of sex differences in social group formation, as well as the critical importance of defining species specificity in visual signaling.

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.

The zebrafish, Danio rerio, has emerged as a major model organism for biomedical research, yet little is known about its natural history. We review the literature pertaining to the geographic range, biotic and abiotic habitats, and life cycle of the zebrafish. We also report our own field study to document several aspects of zebrafish natural history across sites in northeast India. We found zebrafish particularly abundant in silt-bottomed, well-vegetated pools and rice paddies adjacent to slow moving streams at a range of elevations. We further identified co-occurring fishes likely to be zebrafish competitors and predators. Finally, we present observations that indicate substantial habitat degradation and loss, and suggest guidelines for documenting and preserving natural zebrafish populations.

Here, we used a pigment pattern mutant that differs How social aggregations arise and persist is central dramatically from wild-type to determine if D. rerio exto our understanding of evolution, behavior, and psyhibits variation in their shoaling preference, if that preferchology [1–3]. When social groups arise within a speence is mediated through visual signals, and what roles cies, evolutionary divergence and speciation can reinternal and external factors play in the acquisition of sult [4, 5]. To understand this diversifying role of social such a preference. behavior, we must examine the internal and external As a first step in dissecting the behavioral roles of the influences that lead to nonrandom assortment of phezebrafish pigment pattern, we compared the shoaling notypes [6]. Many fishes form aggregations called preferences of wild-type fish and nacre mutants (Figshoals that reduce predation risk while enhancing forure 1). nacre mutants completely lack melanophore aging and reproductive success [7–9]. Thus, shoaling stripes owing to a recessive point mutation in mitfa, is adaptive, and signals that maintain shoals are likely which encodes a basic helix-loop-helix transcription to evolve under selection. Given the diversity of pigfactor that normally acts autonomously to the neural ment patterns among Danio fishes [10–13], visual sigcrest-melanophore lineage to specify melanophore fate nals might be especially important in mediating social [16, 29]. We generated families segregating the nacre behaviors in this group. Our understanding of pigment phenotype by backcrossing nacre heterozygotes (mainpattern development in the zebrafish D. rerio [14, 15] tained in the wild-type strain AB background) to nacre allows integrative analyses of how molecular variation homozygotes. Offspring from these crosses were pheleads to morphological variation among individuals notypically either wild-type (nacre/ ) or nacre mutant and how morphological variation influences social in(nacre/nacre). This design randomizes across effects of teractions. Here, we use the zebrafish pigment mutant other loci that are not linked to the nacre mutation. nacre/mitfa [16] to test roles for genetic and environWe sorted subject fish prior to hatching into three mental determinants in the development of shoaling treatments: controls (reared with three siblings of the preference. We demonstrate that individuals discrimisame phenotype), isolates (reared alone), and crossnate between shoals having different pigment pattern rears (reared with three siblings of the alternate phenophenotypes and that early experience determines type). Fish were maintained in these conditions throughshoaling preference. These results suggest a role for out the experiment. When fish developed adult pigment social learning in pigment pattern diversification in patterns, we tested shoaling preferences by placing indanios. dividual subject fish (n 219) in a test tank containing