Development and evolution of melanophore patterns in fishes of the genus Danio (Teleostei: Cyprinidae)

Pigmentation patterns in vertebrates have become an important model for those interested in mechanisms of pattern determination. I present detailed information on the development of melanophore patterns in the zebrafish, Danio rerio, five close relatives of that species, and an outgroup. The comparison of the ontogeny of melanophore patterns in this group is an important first step towards understanding the developmental basis of the interspecific variation. Pigment patterns in this group range from no distinct patterning at all to stripes of differing numbers and widths to reticulated stripes. Species examined form identical larval patterns and follow a common sequence of events from which different elements are eliminated or altered to produce the variety of patterns seen in the group. As flexion is completed, melanophores move from larval positions onto the flanks of the fish. In D. rerio, D. rerio ‘leo,’ D. kerri, and D. malabaricus, xanthophores become established on the body of the fish as the melanophores move; erythrophores become established on the flanks of D. albolineatus and D. sp. cf. aequipinnatus. An increase in melanophore number, begun at this time, continues at a higher rate in D. rerio, D. kerri, D. sp. cf. aequipinnatus and Tanichthys albonubes than in the other three species. This results in a greater number of melanophores on adults in those species with a higher rate of melanophore increase. No distinct pattern forms, except on the caudal peduncle, in D. albolineatus. In all other Danio species, melanophore stripes form first below then above the horizontal myoseptum. Additional stripes are added first below then above these initial two stripes. D. kerri develops fewer, wider melanophore stripes than D. rerio. After initial stripe formation, D. malabaricus and D. sp. cf. aequipinnatus both developed vertical pattern elements and reticulations in the melanophore pattern. Differences in patterns between species are similar in several cases to described mutants of the zebrafish, suggesting that some aspects of interspecific pigmentation pattern variation may be under relatively simple genetic control. J. Morphol. 241:83–105, 1999. © 1999 Wiley‐Liss, Inc.     

Aggregation Behavior in Wildtype and Transgenic Zebrafish

Recent advances in the development and availability of genetically modified animals enable researchers to examine the effects of phenotypic characters on social behavior. In fish, shoaling behavior is known to be influenced by characteristics such as body coloration, striping pattern, body shape, and size. GloFish^TM are genetically engineered zebrafish (Danio rerio) that express red fluorescent protein (RFP), resulting in on overall red coloration under the dark longitudinal stripes. The GloFish pattern is distinct from the light body coloration underlying the dark longitudinal stripes seen in wildtype zebrafish. We presented wildtype and transgenic RFP zebrafish with same‐sex shoals of both strains of fish in dichotomous choice tests. No preference for either of the shoals was shown, however, both strains showed significant preferences for swimming near shoals vs. swimming near an empty tank compartment. When presented with opposite‐sex individuals of both strains, no preference was shown by either sex of either strain. Thus, the red body coloration of transgenic zebrafish does not appear to affect choice of social partner, in either a shoaling or a potentially reproductive context.     

Ultrastructure and distribution of chromatophores in the skin of the leopard gecko (Eublepharis macularius)

The aim of this study was to describe the ultrastructure and arrangement of pigment cells in the leopard gecko (Eublepharis macularius) skin to explain how wild‐type coloration is formed. The study also attempted to explain, on a morphological level, how skin colour changes occur. Samples of leopard gecko skin were collected from wild‐type coloration adult specimens. The morphology of pigmented cells was determined using light microscopy on haematoxylin and eosin (H&E) stained sections and in transmission electron microscopy. These studies indicate that skin of E. macularis contains xanthophores and melanophores but lacks iridophores and that this is probably related to nocturnal activity. The number and distribution of xanthophores and melanophores determines the skin colour and pigmentation pattern. The colour changes depend on the arrangement of characteristic protrusions of melanophores and the degree of filling them with melanosomes.     

Expression patterns of the creatine metabolism‐related molecules AGAT,GAMT and CT1 in adult zebrafish Danio rerio

AGAT, GAMT and CT1, three creatine synthesis and transport‐related molecules, have been widely studied in mammals. To explore their homologous genes in adult zebrafish Danio rerio, the gene expression patterns of these three genes in D. rerio were investigated. The results reveal that AGAT, GAMT and CT1 are expressed widely in diverse tissues of D. rerio where the homologous genes in mammals are also expressed.     

Ephemeral Sexual Dichromatism in Zebrafish (Danio rerio)

Secondary sexual displays may be overlooked in many species, especially when they are ephemerally expressed or imperceptible to human senses. Zebrafish (Danio rerio), like many schooling fish, do not appear sexually dichromatic, but previous anecdotal observations indicate that sexual colouration is expressed briefly during courtship (ephemeral nuptial colouration). Our goals were to compare colour estimates of male vs. female zebrafish using digital photography in situ, computer software and human observations. We found that both sexes changed their colour estimates during spawning (dark and light stripes) and that some sex differences (light stripes) were larger or only became apparent during this time. We also found that individual males that appeared more colourful and conspicuous to the human eye engaged in courtship more often than less conspicuous males. We detected differences in the colour estimates between wild‐derived vs. a laboratory strain of zebrafish and reduced individual variation in the laboratory strain. This is the first study to systematically and objectively quantify body colour in zebrafish by utilizing colour estimates, although further studies are needed to determine the underlying mechanisms and signalling functions of this sexual dichromatism.     

Interaction between female mating preferences and predation may explain the maintenance of rare males in the pentamorphic fish Poecilia parae

Variation in mating preferences coupled with selective predation may allow for the maintenance of alternative mating strategies. Males of the South American live‐bearing fish Poecilia parae fall in one of five discrete morphs: red, yellow, blue, stripe‐coloured tail (parae) and female mimic (immaculata). Field surveys indicate that the red and yellow morphs are the rarest and that their rarity is consistent across years. We explored the role of variable female mating preference and selective predation by visual predators in explaining the rarity of red and yellow males, and more generally, the maintenance of this extreme colour polymorphism. We presented wild‐caught P. parae females and Aequidens tetramerus, the most common cichlid predator, with the five male colour morphs in separate trials to determine mating and prey preferences, respectively. We found that a large proportion of females shared a strong preference for the rare carotenoid‐based red and yellow males, but a distinct group also preferred the blue and parae morphs. The cichlid predator strongly preferred red and yellow males as prey. Together, these results suggest that the interaction between premating sexual selection favouring and predation acting against the red and yellow morphs may explain their rarity in the wild. The trade‐off between sexual and natural selection, accompanied by variation in female mating preferences, may therefore facilitate the maintenance of the striking colour polymorphism in P. parae.     

No evidence for size‐assortative mating in the wild despite mutual mate choice in sex‐role‐reversed pipefishes

Size‐assortative mating is a nonrandom association of body size between members of mating pairs and is expected to be common in species with mutual preferences for body size. In this study, we investigated whether there is direct evidence for size‐assortative mating in two species of pipefishes, Syngnathus floridae and S. typhle, that share the characteristics of male pregnancy, sex‐role reversal, and a polygynandrous mating system. We take advantage of microsatellite‐based “genetic‐capture” techniques to match wild‐caught females with female genotypes reconstructed from broods of pregnant males and use these data to explore patterns of size‐assortative mating in these species. We also develop a simulation model to explore how positive, negative, and antagonistic preferences of each sex for body size affect size‐assortative mating. Contrary to expectations, we were unable to find any evidence of size‐assortative mating in either species at different geographic locations or at different sampling times. Furthermore, two traits that potentially confer a fitness advantage in terms of reproductive success, female mating order and number of eggs transferred per female, do not affect pairing patterns in the wild. Results from model simulations demonstrate that strong mating preferences are unlikely to explain the observed patterns of mating in the studied populations. Our study shows that individual mating preferences, as ascertained by laboratory‐based mating trials, can be decoupled from realized patterns of mating in the wild, and therefore, field studies are also necessary to determine actual patterns of mate choice in nature. We conclude that this disconnect between preferences and assortative mating is likely due to ecological constraints and multiple mating that may limit mate choice in natural populations.     

Gravidity‐Associated Shoaling Decisions in Three‐Spined Sticklebacks (Gasterosteus aculeatus)

Group living has evolved in many animal species as an antipredator behavior, an evolutionary effect that might be augmented by grouping with similarly looking individuals. Consequently, groups are often composed according to species, size, or coloration. During egg ripening or embryo growth, the outer appearance of females often changes drastically within days, which makes them more prone to predation. Thus, a female's group preference should change according to her reproductive state, an issue that has seldom been investigated. To test this, we gave gravid and non‐gravid three‐spined sticklebacks (Gasterosteus aculeatus) the choice to shoal either with a gravid or a non‐gravid conspecific. The results showed that shoaling preferences of gravid and non‐gravid individuals differed significantly. Non‐gravid females preferred gravid fish as shoaling partners, which might rely on the fact that gravid sticklebacks show reduced escape performance, which in turn might increase a non‐gravid female's chance of survival. However, in contrast to our predictions, gravid fish did not show any significant preference. A reason for this pattern might be competition between gravid females for mating partners, which might overrule benefits of shoaling with similar looking individuals. Hence, gravidity influences social preferences in a shoaling fish, which might pose a largely overlooked form of the cost of reproduction.     

Upper thermal tolerance of closely related Danio species

The main finding of this study was that measuring maximum heart rate during incremental warming was an effective tool to estimate upper thermal limits in three small cyprinid Danio species, which differed significantly. Arrhenius breakpoint temperature for maximum heart rate, purportedly an index of optimum temperature, was 21·2 ± 0·4, 20·1 ± 0·4 and 18·9 ± 0·8° C (mean ± s.e .) for zebrafish Danio rerio, pearl danio Danio albolineatus and glowlight danio Danio choprae, respectively. The temperature where cardiac arrhythmias were first induced during warming (T_arr) was 36·6 ± 0·7, 36·9 ± 0·8 and 33·2 ± 0·8° C (mean ± s.e .) and critical thermal maximum (T_Cm) was 39·9 ± 0·1, 38·9 ± 0·1 and 37·2 ± 0·1° C (mean ± s.e .) for D. rerio, D. albolineatus and D. choprae, respectively. The finding that T_arr was consistently 3–4° C lower than T_Cm suggests that collapse of the cardiac life support system may be a critical trigger for upper temperature tolerance. The upper thermal limits established here, which correlate well with a broad natural environmental temperature range for D. rerio and a narrow one for D. choprae, suggest that upper thermal tolerance may be a genetic trait even among closely related species acclimated to common temperatures.     

Sexual imprinting and speciation between two Peromyscus species

Sexual isolation, a reproductive barrier, can prevent interbreeding between diverging populations or species. Sexual isolation can have a clear genetic basis; however, it may also result from learned mate preferences that form via sexual imprinting. Here, we demonstrate that two sympatric species of mice—the white‐footed mouse (Peromyscus leucopus) and its sister species, the cotton mouse (P. gossypinus)—hybridize only rarely in the wild despite co‐occurrence in the same habitat and lack of any measurable intrinsic postzygotic barriers in laboratory crosses. We present evidence that strong conspecific mating preferences in each species result in significant sexual isolation. We find that these preferences are learned in at least one species: P. gossypinus sexually imprints on its parents, but in P. leucopus, additional factors influence mating preferences. Our study demonstrates that sexual imprinting contributes to reproductive isolation that reduces hybridization between otherwise interfertile species, supporting the role for learning in mammalian speciation.     

Sympatric colour polymorphisms associated with nonrandom gene flow in cichlid fish of Lake Victoria

Colour polymorphisms have fascinated evolutionary ecologists for a long time. Yet, knowledge on the mechanisms that allow their persistence is restricted to a handful of well‐studied cases. We studied two species of Lake Victoria cichlid fish, Neochromis omnicaeruleus and Neochromis greenwoodi, exhibiting very similar sex‐linked colour polymorphisms. The ecology and behaviour of one of these species is well studied, with colour‐based mating and aggression preferences. Here, we ask whether the selection potentially resulting from female and male mating preferences and aggression biases reduces gene flow between the colour morphs and permits differentiation in traits other than colour. Over the past 14 years, the frequencies of colour morphs have somewhat oscillated, but there is no evidence for directional change, suggesting the colour polymorphism is persistent on an ecological timescale. We find limited evidence of eco‐morphological differentiation between sympatric ancestral (plain) and derived (blotched) colour morphs. We also find significantly nonrandom genotypic assignment and an excess of linkage disequilibrium in the plain morph, which together with previous information on mating preferences suggests nonrandom mating between colour morphs. This, together with negative frequency‐dependent sexual selection, found in previous studies, may facilitate maintenance of these polymorphisms in sympatry.     

Size‐dependent reproductive success of wild zebrafish Danio rerio in the laboratory

Size‐dependent reproductive success of wild zebrafish Danio rerio was studied under controlled conditions in the laboratory to further understand the influence of spawner body size on reproductive output and egg and larval traits. Three different spawner size categories attained by size‐selective harvesting of the F₁‐offspring of wild D. rerio were established and their reproductive performance compared during a 5 day period. As to be expected, large females spawned more frequently and had significantly greater clutch sizes than small females. Contrary to expectations, small females produced larger eggs when measured as egg diameter with similar amounts of yolk compared to eggs spawned by large spawners. Eggs from small fish, however, suffered from higher egg mortality than the eggs of large individuals. Embryos from small‐sized spawners also hatched later than offspring from eggs laid by large females. Larval standard length (L_S)‐at‐hatch did not differ between the size categories, but the offspring of the large fish had significantly larger area‐at‐hatch and greater yolk‐sac volume indicating better condition. Offspring growth rates were generally similar between offspring from all size categories, but they were significantly higher for offspring spawned by small females in terms of L_S between days 60 and 90 post‐fertilization. Despite temporarily higher growth rates among the small fish offspring, the smaller energy reserves at hatching translated into lower condition later in ontogeny. It appeared that the influence of spawner body size on egg and larval traits was relatively pronounced early in development and seemed to remain in terms of condition, but not in growth, after the onset of exogenous feeding. Further studies are needed to explore the mechanisms behind the differences in offspring quality between large‐ and small‐sized spawners by disentangling size‐dependent maternal and paternal effects on reproductive variables in D. rerio.     

Incomplete behavioural isolation and asymmetric female preference in darter sister species (Percidae: Etheostoma)

In this study, female preferences and behavioural isolation were estimated in a pair of allopatric sister species, Etheostoma duryi and Etheostoma flavum. Dichotomous mate preference trials were conducted to determine whether females prefer to associate with conspecific over heterospecific males and free‐spawning assays were conducted to determine whether those preferences translated into behavioural isolation. Dichotomous mate choice trials revealed asymmetric female preference, as female E. flavum preferred conspecific males, whereas female E. duryi showed no preference. Free‐spawning assays indicated that behavioural isolation remains incomplete between E. duryi and E. flavum (I_B = 0·19). In addition to female mating preferences, male behaviour also appeared to influence mating outcomes as male E. flavum consistently courted conspecific females more often in free‐spawning assays whereas male E. duryi did not. The data therefore suggest that despite marked divergence in male nuptial colour, divergence in female preferences between these species may not be sufficient to maintain species boundaries upon secondary contact. These results contrast with similar work in a sympatric darter species pair and may be explained by considering the contributions of reinforcement and differences in colour pattern as well as colour value.     

Hunting behaviour in West African forest leopards

The leopard (Panthera pardus) is a major predator of mammals within the rainforest ecosystem of West Africa. Most of the available information on leopard hunting behaviour comes from studies conducted in open savannah habitats, while little is known about forest leopards. Our radio‐tracking data and scat analysis show that forest leopards differ in various ways from the savannah populations. Forest leopards are diurnal and crepuscular hunters who follow the activity pattern of their prey species. They exhibit seasonal differences in activity patterns, and they develop highly individualized prey preferences. These findings challenge the widespread notion of leopards as opportunistic nocturnal predators.     

Alarm substance from adult zebrafish alters early embryonic development in offspring

Alarm substances elicit behavioural responses in a wide range of animals but effects on early embryonic development are virtually unknown. Here we investigated whether skin injury-induced alarm substances caused physiological responses in embryos produced by two Danio species (Danio rerio and Danio albolineatus). Both species showed more rapid physiological development in the presence of alarm substance, although there were subtle differences between them: D. rerio had advanced muscle contraction and heart function, whereas D. albolineatus had advanced heart function only. Hence, alarm cues from injured or dying fish may be of benefit to their offspring, inducing physiological responses and potentially increasing their inclusive fitness.     

Behavioral Plasticity in Response to Environmental Manipulation among Zebrafish (Danio rerio) Populations

Plastic responses can have adaptive significance for organisms occurring in unpredictable environments, migratory species and organisms occupying novel environments. Zebrafish (Danio rerio) occur in a wide range of habitats and environments that fluctuate frequently across seasons and habitats. We expect wild populations of fish to be behaviorally more flexible than fish reared in conventional laboratory and hatchery environments. We measured three behavioral traits among 2 wild (U and PN) and 1 laboratory bred (SH) zebrafish populations in four environments differing in water flow and vegetation regimes. We found that the degree of plasticity varied with the type of behavior and also among populations. In general, vegetation increased aggression and water flow decreased latency to feed after a disturbance, but the patterns were population dependent. For example, while wild U fish fed more readily after a disturbance in vegetated and/or flowing habitats, fish from the wild PN population and lab-reared SH strain showed little variation in foraging across different environmental conditions. Zebrafish from all the three populations were more aggressive when tested in an arena with vegetation. In contrast, while there was an inter- population difference in shoaling distances, variation in shoaling distance across environmental conditions within populations was not significant. These results suggest that both foraging and aggression in zebrafish are more plastic and influenced by immediate context than is shoaling distance, which may have a stronger genetic basis. Our findings point to different underlying mechanisms influencing the expression of these traits and warrants further investigations.     

Body length rather than routine metabolic rate and body condition correlates with activity and risk‐taking in juvenile zebrafish Danio rerio

In this study, the following hypotheses were explored using zebrafish Danio rerio: (1) individuals from the same cohort differ consistently in activity and risk‐taking and (2) variation in activity and risk‐taking is linked to individual differences in metabolic rate, body length and body condition. To examine these hypotheses, juvenile D. rerio were tested for routine metabolic rate and subsequently exposed to an open field test. Strong evidence was found for consistent among‐individual differences in activity and risk‐taking, which were overall negatively correlated with body length, i.e. larger D. rerio were found to be less active in a potentially dangerous open field and a similar trend was found with respect to a more direct measure of their risk‐taking tendency. In contrast, routine metabolic rate and body condition were uncorrelated with both activity and risk‐taking of juvenile D. rerio. These findings suggest that body length is associated with risk‐related behaviours in juvenile D. rerio for which larger, rather than smaller, individuals may have a higher risk of predation, while the role for routine metabolic rate is relatively limited or non‐existent, at least under the conditions of the present study.     

Differences in biological and behavioural characteristics of Harmonia axyridis (Coleoptera: Coccinellidae) according to colour patterns of elytra

Samples of overwintering populations of Harmonia axyridis were classified into five colour patterns, succinea 1, succinea 2, conspicua, spectabilis and axyridis. Generally, the succinea 1 colour pattern was dominant at six collecting sites in Daejon and Chungnam provinces of Korea and their occurrence proportion was over 50%. Non‐melanic forms such as succinea 1 and 2 were noted in about 90% of collected individuals. On the other hand, conspicua and spectabilis as melanic forms were dominant in the laboratory. One hypothesis is that the difference between field and laboratory populations is related to non‐random mating and environmental conditions. Practically, in mate‐choice experiments, most colour pattern ladybirds seemed to mate with the melanic form, especially the conspicua form. The body size (length) of H. axyridis in colour patterns was slightly different, but in females, there was no significant difference among colour patterns. However, both lengths of males were significantly different among colour patterns. In reproductive ability (fecundity), there was no difference among colour patterns.     

Genetic difference in female choice between two guppy populations

The pattern of mating preferences of female guppies, Poecilia reticulata, differs genetically between two Trinidadian populations. Males from the Aripo and Paria rivers of Trinidad differ in the extent and intensity of orange pigment in their colour patterns. The relative area of orange in Paria colour patterns was nearly twice that in Aripo colour patterns. To test the hypothesis that a difference in female mating preferences could have contributed to the difference in colour patterns, patterns of female choice between laboratory-reared Paria and Aripo females were compared. The frequency of sexual responses to male courtship displays was used to measure the average preference of females for individual males. Paria females discriminated significantly between Paria males based upon differences in the extent of orange in the colour pattern. Aripo females showed no evidence of discrimination between Paria males, and thus differed significantly from Paria females in choice behaviour. Neither group of females discriminated between Aripo males. These results are evidence that female choice behaviour can vary genetically within a species and suggest that differences in preferences may have contributed to divergence in colour patterns among guppy populations.     

Circadian rhythms in zebrafish (Danio rerio) behaviour and the sources of their variability

Over recent decades, changes in zebrafish (Danio rerio) behaviour have become popular quantitative indicators in biomedical studies. The circadian rhythms of behavioural processes in zebrafish are known to enable effective utilization of energy and resources, therefore attracting interest in zebrafish as a research model. This review covers a variety of circadian behaviours in this species, including diurnal rhythms of spawning, feeding, locomotor activity, shoaling, light/dark preference, and vertical position preference. Changes in circadian activity during zebrafish ontogeny are reviewed, including ageing-related alterations and chemically induced variations in rhythmicity patterns. Both exogenous and endogenous sources of inter-individual variability in zebrafish circadian behaviour are detailed. Additionally, we focus on different environmental factors with the potential to entrain circadian processes in zebrafish. This review describes two principal ways whereby diurnal behavioural rhythms can be entrained: (i) modulation of organismal physiological state, which can have masking or enhancing effects on behavioural endpoints related to endogenous circadian rhythms, and (ii) modulation of period and amplitude of the endogenous circadian rhythm due to competitive relationships between the primary and secondary zeitgebers. In addition, different peripheral oscillators in zebrafish can be entrained by diverse zeitgebers. This complicated orchestra of divergent influences may cause variability in zebrafish circadian behaviours, which should be given attention when planning behavioural studies.