Age-dependent winner-loser effects in a mangrove rivulus fish, Kryptolebias marmoratus.

The outcomes of recent fights can provide individuals information about their relative fighting ability and affect their contest decisions (winner-loser effects). Most studies investigate the presence/absence of the effects in populations/species, but here we examine how they vary between individuals of a species in response to age-dependent growth rate. Many animals' fighting ability is highly dependent on body size, so rapid growth makes information from previous fights unreliable. Furthermore, fast-growing individuals are often at earlier developmental stages and are relatively smaller and weaker than most other individuals but are growing larger and stronger quickly. We therefore predicted winner-loser effects to be less detectable in individuals with high than low growth rates and to decay more quickly. Fast-growing individuals should also display stronger winner than loser effects, because a victory when small indicates a strength which will grow, whereas a loss might soon become irrelevant. We tested these predictions using naïve individuals of a mangrove killifish, Kryptolebias marmoratus, in different growth stages. Measures of contest intensity revealed winner/loser effects only for slow-growth individuals. Both fast- and slow-growth fish with a winning experience won more of the subsequent non-escalated contests than those with a losing experience; in fast-growth individuals this effect disappeared in 3 days, but in slow-growth fish it did not. Fast-growth individuals also displayed winner effects but not loser effects. The fish therefore responded to their contest experiences in a way which reflected value of the information from these experiences to them, consistent with our predictions.

Age-dependent genetic variation in aggression.

Understanding the extent to which behavioural variance is underlain by genotypic, environmental and genotype-by-environment effects is important for predicting how behavioural traits might respond to selection and evolve. How behaviour varies both within and among individuals can change across ontogeny, leading to differences in the relative contribution of genetic and environmental effects to phenotypic variation across ages. We investigated among-individual and among-genotype variation in aggression across ontogeny by measuring, twice as juveniles and twice as adults, both approaches and attacks against a three-dimensional-printed model opponent in eight individuals from each of eight genotypes (N = 64). Aggression was only significantly repeatable and heritabile in juveniles. Additionally, how aggression changed between juvenile and adult life-history stages varied significantly among individuals and genotypes. These results suggest that juvenile aggression is likely to evolve more rapidly via natural selection than adult aggression and that the trajectory of behavioural change across the lifespan has the potential to evolve. Determining when genetic variation explains (or does not explain) behavioural variation can further our understanding of key life-history stages during which selection might drive the strongest or swiftest evolutionary response.

Positive feedback promotes terrestrial emergence behaviour in an amphibious fish.

Major ecological transitions such as the invasion of land by aquatic vertebrates may be facilitated by positive feedback between habitat choice and phenotypic plasticity. We used the amphibious fish Kryptolebias marmoratus to test the hypothesis that aquatic hypoxia, emergence behaviour and respiratory plasticity create this type of positive feedback loop that causes fish to spend increasing amounts of time on land. Terrestrially acclimated fish were more sensitive to aquatic hypoxia (emergence at higher PO2) and were less hypoxia tolerant (shorter time to loss of equilibrium) relative to water-acclimated fish, which are necessary conditions for positive feedback. Next, we tested the prediction that exposure to aquatic hypoxia causes fish to emerge frequently, reduce gill surface area, and become less hypoxia tolerant. Indeed, fish exposed to severe aquatic hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Fish exposed to aquatic hypoxia that could emerge from water were also more sensitive to subsequent acute aquatic hypoxia and were less hypoxia tolerant than normoxia-exposed controls. These results are opposite those of fish that cannot escape from aquatic hypoxia and presumably arise owing to plastic changes that occur during air exposure. Together, these results indicate that emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. This type of positive feedback may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

Habitat choice promotes and constrains phenotypic plasticity.

Habitat choice can either speed up or slow rates of phenotypic evolution, depending on which trait is measured. We suggest that habitat choice plays an analogous, and generally overlooked, role in shaping patterns of phenotypic plasticity. Using our work with an amphibious fish, we discuss two case studies that demonstrate how habitat choice can both promote and constrain expression of plasticity. First, habitat choice during the dry season accentuates adaptive metabolic plasticity and minimizes maladaptive changes to muscle, ultimately increasing survival time out of water. Second, a trade-off between water- and air-breathing drives matching habitat choice, resulting in positive feedback that reinforces respiratory specialization and environmental preference. Overall, these case studies demonstrate that we must consider the interactions between plasticity and habitat choice to fully understand how animals survive in the face of environmental change. Without considering both processes simultaneously, the performance of animals in challenging conditions can be either under- or over-estimated. Finally, because habitat choice shapes the frequency and predictability of environmental changes that animals experience, feedback between habitat choice and expressions of phenotypic plasticity may be an important factor that influences how plasticity evolves.

Does leaving water make fish smarter? Terrestrial exposure and exercise improve spatial learning in an amphibious fish.

Amphibious fishes transition between aquatic and terrestrial habitats, and must therefore learn to navigate two dramatically different environments. We used the amphibious killifish Kryptolebias marmoratus to test the hypothesis that the spatial learning ability of amphibious fishes would be altered by exposure to terrestrial environments because of neural plasticity in the brain region linked to spatial cognition (dorsolateral pallium). We subjected fish to eight weeks of fluctuating air-water conditions or terrestrial exercise before assessing spatial learning using a bifurcating T-maze, and neurogenesis in the dorsolateral pallium by immunostaining for proliferating cell nuclear antigen. In support of our hypothesis, we found that air-water fluctuations and terrestrial exercise improved some markers of spatial learning. Moreover, air-water and exercised fish had 39% and 46% more proliferating cells in their dorsolateral pallium relative to control fish, respectively. Overall, our findings suggest that fish with more terrestrial tendencies may have a cognitive advantage over those that remain in water, which ultimately may influence their fitness in both aquatic and terrestrial settings. More broadly, understanding the factors that promote neural and behavioural plasticity in extant amphibious fishes may provide insights into how ancestral fishes successfully colonized novel terrestrial environments before giving rise to land-dwelling tetrapods.

Novel spikey ionocytes are regulated by cortisol in the skin of an amphibious fish.

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na+-K+-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes.

Extensive hybridization and past introgression between divergent lineages in a quasi-clonal hermaphroditic fish: Ramifications for species concepts and taxonomy.

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self-fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self-fertilization for 1-3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.

Acclimation to prolonged aquatic hypercarbia or air enhances hemoglobin­oxygen affinity in an amphibious fish.

When the amphibious mangrove rivulus (Kryptolebias marmoratus) leaves water for extended periods, hemoglobin-O2 binding affinity increases. We tested the hypothesis that the change in affinity was a consequence of hemoglobin isoform switching driven by exposure to environments associated with increased internal CO2 levels. We exposed K. marmoratus to either water (control, pH 8.1), air, aquatic hypercarbia (5.1 kPa CO2, pH 6.6-6.8), or aquatic acid (isocarbic control, pH 6.6-6.8), for 7 days, and measured hemoglobin-O2 affinity spectrophotometrically. We found that mangrove rivulus compensated for elevated CO2 and aquatic acid exposure by shifting hemoglobin-O2 affinity back to aquatic (control) levels when measured at an ecologically-relevant high CO2 level that would be experienced in vivo. Using proteomics, we found that the hemoglobin subunits present in the blood did not change between treatments, but air and aquatic acid exposure altered the abundance of cathodic hemoglobin subunits. We therefore conclude that hemoglobin isoform switching is not a primary strategy used by mangrove rivulus to adjust P50 under these conditions. Abundances of other RBC proteins also differed between treatment groups relative to control fish (e.g. Rhesus protein type A, band 3 anion exchanger). Overall, our data indicate that both aquatic hypercarbia and aquatic acidosis create similar changes in hemoglobin-O2 affinity as air exposure. However, the protein-level consequences differ between these groups, indicating that the red blood cell response of mangrove rivulus can be modulated depending on the environmental cue received.

Smell of Infection: A Novel, Noninvasive Method for Detection of Fish Excretory-Secretory Proteins.

Chemical signals are produced by aquatic organisms following predatory attacks or perturbations such as parasitic infection. Ectoparasites feeding on fish hosts are likely to cause release of similar alarm cues into the environment due to the stress, wounding, and immune response stimulated upon infection. Alarm cues are often released in the form of proteins, antimicrobial peptides, and immunoglobulins that provide important insights into bodily function and infection status. Here we outline a noninvasive method to identify potential chemical cues associated with infection in fish by extracting, purifying, and characterizing proteins from water samples from cultured fish. Gel free proteomic methods were deemed the most suitable for protein detection in saline water samples. It was confirmed that teleost proteins can be characterized from water and that variation in protein profiles could be detected between infected and uninfected individuals and fish and parasite only water samples. Our novel assay provides a noninvasive method for assessing the health condition of both wild and farmed aquatic organisms. Similar to environmental DNA monitoring methods, these proteomic techniques could provide an important tool in applied ecology and aquatic biology.

Prolonged survival out of water is linked to a slow pace of life in a self-fertilizing amphibious fish.

Metabolic rate and life-history traits vary widely both among and within species, reflecting trade-offs in energy allocation, but the proximate and ultimate causes of variation are not well understood. We tested the hypothesis that these trade-offs are mediated by environmental heterogeneity, using isogenic strains of the amphibious fish Kryptolebias marmoratus that vary in the amount of time each can survive out of water. Consistent with pace of life theory, the strain that survived air exposure the longest generally exhibited a 'slow' phenotype, including the lowest metabolic rate, largest scope for metabolic depression, slowest consumption of energy stores and least investment in reproduction under standard conditions. Growth rates were fastest in the otherwise slow strain, however. We then tested for fitness trade-offs between 'fast' and 'slow' strains using microcosms where fish were held either with constant water availability or under fluctuating conditions where water was absent for half of the experiment. Under both conditions the slow strain grew larger and was in better condition, and under fluctuating conditions the slow strain produced more embryos. However, the fast strain had larger adult population sizes under both conditions, indicating that fecundity is not the sole determinant of population size in this species. We conclude that genetically based differences in the pace of life of amphibious fish determine survival duration out of water. Relatively slow fish tended to perform better under conditions of limited water availability, but there was no detectable cost under control conditions. Thus, pace of life differences may reflect a conditionally neutral instead of antagonistic trade-off.

Phenotypic flexibility in respiratory traits is associated with improved aerial respiration in an amphibious fish out of water.

Amphibious fishes have evolved multiple adaptive strategies for respiring out of water, but there has been less focus on reversible plasticity. We tested the hypothesis that when amphibious fishes leave water, enhanced respiratory performance on land is the result of rapid functional phenotypic flexibility of respiratory traits. We acclimated four isogenic strains of Kryptolebias marmoratus to air for 0, 1, 3 or 7 days. We compared respiratory performance out of water with traits linked to the O2 cascade. Aerial O2 consumption rate was measured over a step-wise decrease in O2 levels. There were significant differences between strains, but time out of water had the largest impact on measured parameters. Kryptolebiasmarmoratus had improved respiratory performance [lower aerial critical oxygen tension (Pcrit), higher regulation index (RI)] after only 1 day of air exposure, and these changes were strongly associated with the change in hematocrit and dorsal cutaneous angiogenesis. Additionally, we found that 1 h of air exposure induced the expression of four angiogenesis-associated genes - vegfa, angpt2, pecam-1 and efna1 - in the skin. After 7 days in air, respiratory traits were not significantly linked to the variation in either aerial Pcrit or RI. Overall, our data indicate that there are two phases involved in the enhancement of aerial respiration: an initial rapid response (1 day) and a delayed response (7 days). We found evidence for the hypothesis that respiratory performance on land in amphibious fishes is the result of rapid flexibility in both O2 uptake and O2 carrying capacity.

Real or fake? Natural and artificial social stimuli elicit divergent behavioural and neural responses in mangrove rivulus, Kryptolebias marmoratus.

Understanding how the brain processes social information and generates adaptive behavioural responses is a major goal in neuroscience. We examined behaviour and neural activity patterns in socially relevant brain nuclei of hermaphroditic mangrove rivulus fish (Kryptolebias marmoratus) provided with different types of social stimuli: stationary model opponent, regular mirror, non-reversing mirror and live opponent. We found that: (i) individuals faced with a regular mirror were less willing to interact with, delivered fewer attacks towards and switched their orientation relative to the opponent more frequently than fish exposed to a non-reversing mirror image or live opponent; (ii) fighting with a regular mirror image caused higher expression of immediate-early genes (IEGs: egr-1 and c-Fos) in the teleost homologues of the basolateral amygdala and hippocampus, but lower IEG expression in the preoptic area, than fighting with a non-reversing mirror image or live opponent; (iii) stationary models elicited the least behavioural and IEG responses among the four stimuli; and (iv) the non-reversing mirror image and live opponent drove similar behavioural and neurobiological responses. These results suggest that the various stimuli provide different types of information related to conspecific recognition in the context of aggressive contests, which ultimately drive different neurobiological responses.

Oxygen drives skeletal muscle remodeling in an amphibious fish out of water.

Skeletal muscle remodeling in response to terrestrial acclimation improves the locomotor performance of some amphibious fishes on land, but the cue for this remodeling is unknown. We tested the hypothesis that muscle remodeling in the amphibious Kryptolebias marmoratus on land is driven by higher O2 availability in atmospheric air, and the alternative hypothesis that remodeling is induced by a different environmental or physiological condition that fish experience on land. Fish were acclimated to 28 days of air, or to aquatic hyperoxia, hypercapnia, hypoxia, elevated temperature or fasting conditions. Air, fasting and hyperoxic conditions increased (>25%) the size of oxidative fibers in K. marmoratus while hypoxia had the reverse effect (23% decrease). Surprisingly, hyperoxia acclimation also resulted in a transformation of the musculature to include large bands of oxidative-like muscle. Our results show that K. marmoratus is highly responsive to environmental O2 levels and capitalizes on O2-rich opportunities to enhance O2 utilization by skeletal muscle.

Whole-genome sequencing reveals the extent of heterozygosity in a preferentially self-fertilizing hermaphroditic vertebrate.

The mangrove rivulus, Kryptolebias marmoratus, is one of only two self-fertilizing hermaphroditic fish species and inhabits mangrove forests. While selfing can be advantageous, it reduces heterozygosity and decreases genetic diversity. Studies using microsatellites found that there are variable levels of selfing among populations of K. marmoratus, but overall, there is a low rate of outcrossing and, therefore, low heterozygosity. In this study, we used whole-genome data to assess the levels of heterozygosity in different lineages of the mangrove rivulus and infer the phylogenetic relationships among those lineages. We sequenced whole genomes from 15 lineages that were completely homozygous at microsatellite loci and used single nucleotide polymorphisms (SNPs) to determine heterozygosity levels. More variation was uncovered than in studies using microsatellite data because of the resolution of full genome sequencing data. Moreover, missense polymorphisms were found most often in genes associated with immune function and reproduction. Inferred phylogenetic relationships suggest that lineages largely group by their geographic distribution. The use of whole-genome data provided further insight into genetic diversity in this unique species. Although this study was limited by the number of lineages that were available, these data suggest that there is previously undescribed variation within lineages of K. marmoratus that could have functional consequences and (or) inform us about the limits to selfing (e.g., genetic load, accumulation of deleterious mutations) and selection that might favor the maintenance of heterozygosity. These results highlight the need to sequence additional individuals within and among lineages.

Natural hybridization between divergent lineages in a selfing hermaphroditic fish.

By definition, mating between individuals is infrequent in highly selfing organisms, and so too, therefore, hybridization should be rare between genetically divergent lineages in predominantly self-fertilizing species. Notwithstanding these expectations, here we report a remarkable case of natural hybridization between highly diverged phylogeographic lineages of the mangrove rivulus, a small killifish that reproduces predominantly by self-fertilization and typically is found as highly homozygous lines in most parts of its extensive geographical range. Two distinctive genetic lineages (Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus) previously were not known in sympatry, but were found by us to co-occur on San Salvador, Bahamas. Genetic analyses of a mitochondrial and multiple nuclear markers determined the direction of a cross producing a hybrid fish. Furthermore, we show that this hybrid individual was viable, as it successfully reproduced by self-fertilization for two generations. Additional sampling of this population will be necessary to determine if backcrossing of hybrids to the parental lineages occurs in nature and to analyse whether such backcross progeny are viable. Application of the biological species concept (BSC) is traditionally difficult in clonally reproducing organisms. Our results show that although mangrove rivulus fish are mostly highly selfing in nature (resulting in isogenic, effectively clonal and homozygous progeny), classification within this taxonomic complex need not be incompatible with the BSC.

Contest experience and body size affect different types of contest decisions.

This study examined the relative importance of contest experience and size differences to behavioral decisions over the course of contests. Using a mangrove rivulus fish, Kryptolebias marmoratus, we showed that although contest experience and size differences jointly determined contest outcomes, they affected contestants' interactions at different stages of contests. Contest experience affected behavioral decisions at earlier stages of contests, including the tendency and latency to launch attacks, the tendency to escalate contests into mutual attacks and the outcome of non-escalated contests. Once contests were escalated into mutual attacks, the degree of size difference affected the fish's persistence in escalation and chance of winning, but contest experience did not. These results support the hypothesis that contest experience modifies individuals' estimation of their fighting ability rather than their actual strength. Furthermore, (1) in contests between two naïve contestants, more than 60 % of fish that were 2-3 mm smaller than their opponent escalated the contest to physical fights, even though their larger opponents eventually won 92 % of escalated fights and (2) fish with a losing experience were very likely to retreat in the face of an opponent 2-3 mm smaller than them without escalating. The result that a 2-3 mm size advantage could not offset the influence of a losing experience on the tendency to escalate suggests that, as well as depending on body size, the fish's physical strength is influenced by other factors which require further investigation.

Conservation of Hox gene clusters in the self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae).

In this study, whole Hox gene clusters in the self-fertilizing mangrove killifish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae), a unique hermaphroditic vertebrate in which both sex organs are functional at the same time, were identified from whole genome and transcriptome sequences. The aim was to increase the understanding of the evolutionary status of conservation of this Hox gene cluster across fish species.

Genomic organization and transcriptional modulation in response to endocrine disrupting chemicals of three vitellogenin genes in the self-fertilizing fish Kryptolebias marmoratus.

Vitellogenin (Vtg) is the precursor of egg yolk proteins, and its expression has been used as a reliable biomarker for estrogenic contamination in the aquatic environment. To examine the biomarker potential of the self-fertilizing killifish Kryptolebias marmoratus Vtgs (Km-Vtgs), full genomic DNAs of Km-Vtgs-Aa, Km-Vtgs-Ab, and Km-Vtgs-C were cloned, sequenced, and characterized. Three Vtg genes in K. marmoratus are tandemly placed in a 550 kb section of the same chromosome. In silico analysis of promoter regions revealed that both the Km-Vtgs-Aa and Km-Vtgs-Ab genes had an estrogen response element (ERE), but the Km-Vtgs-C gene did not. However, all three Km-Vtgs genes had several ERE-half sites in their promoter regions. Phylogenetic analysis demonstrated that the three deduced amino acid residues were highly conserved with conventional Vtgs protein, forming distinctive clades within teleost Vtgs. Liver tissue showed the highest expression of Km-Vtg transcripts in all tested tissues (brain/pituitary, eye, gonad, intestine, skin, and muscle) in response to endocrine disrupting chemical (EDC)-exposed conditions. Km-Vtg transcripts were significantly increased in response to 17ß-estradiol (E2), tamoxifen (TMX), 4-n-nonylphenol (NP), bisphenol A (BPA), and octylphenol (OP) over 24hr exposure. The Km-Vtg-A gene was highly expressed compared to the control in response to NP and OP. EDC-induced modulatory patterns of Km-Vtg gene expression were different depending on tissue, gender, and isoforms.

Fighting experience alters brain androgen receptor expression dependent on testosterone status.

Contest decisions are influenced by the outcomes of recent fights (winner-loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner-loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner-loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/ß (ERα/ß), glucocorticoid (GR) and serotonin (5-HT1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner-loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ERα/ß, 5-HT1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly.

Opponent familiarity and contest experience jointly influence contest decisions in Kryptolebias marmoratus.

INTRODUCTION: Individual recognition and winner/loser effects both play important roles in animal contests, but how their influences are integrated to affect an individual's contest decisions in combination remains unclear. Individual recognition provides an animal with relatively precise information about its ability to defeat conspecifics that it has fought previously. Winner/loser effects, conversely, rely on sampling information about how an animal's ability to win compares with those of others in the population. The less precise information causing winner/loser effects should therefore be more useful to an individual facing an unfamiliar opponent. In this study, we used Kryptolebias marmoratus, a hermaphroditic mangrove killifish, to test whether winner/loser effects do depend on opponent familiarity. In addition, as previous studies have shown that subordinates that behave aggressively sometimes suffer post-retreat retaliation from contest winners, we also explored this aspect of contest interaction in K. marmoratus. RESULTS: In the early stages of a contest, subordinates facing an unfamiliar dominant were more likely to signal their aggressiveness with either gill displays or attacks rather than retreating immediately. A winning experience then increased the likelihood that the most aggressive behavioral pattern the subordinates exhibited would be attacks rather than gill displays, irrespective of their opponents' familiarity. Dominants that received a losing experience and faced an unfamiliar opponent were less likely than others to launch attacks directly. And subordinates that challenged dominants with more aggressive tactics but still lost received more post-retreat attacks from their dominant opponents. CONCLUSIONS: Subordinates' contest decisions were influenced by both their contest experience and the familiarity of their opponents, but these influences appeared at different stages of a contest and did not interact significantly with each other. The influence of a losing experience on dominants' contest decisions, however, did depend on their subordinate opponents' familiarity. Subordinates and dominants thus appeared to integrate information from the familiarity of their opponents and the outcome of previous contests differently, which warrants further investigation. The higher costs that dominants imposed on subordinates that behaved more aggressively toward them may have been to deter them from either fighting back or challenging them in the future.