Plasma levels of androgens and cortisol in relation to breeding behavior in parental male bluegill sunfish, Lepomis macrochirus

Like many teleosts, male bluegill sunfish (Lepomis macrochirus) provide sole parental care. To understand some of the proximate costs of parental care, we measured body condition and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and cortisol in nesting bluegill males during pre-spawning, spawning and parental care stages. T and 11KT were at their highest mean levels during the pre-spawning period and decreased to lower levels early during the parental care period before rising again when the eggs hatched. Cortisol levels fluctuated across the breeding stages, but there was a noticeable increase from low levels on the day of spawning during the first 2 days of parental care when egg fanning is most intense. Levels of all hormones varied considerably among males, with androgen levels often correlating positively with a male's body condition. We also demonstrate, using a brood reduction experiment and repeated sampling of known individuals, that the presence of eggs affects hormone levels shortly after eggs hatch. Parental males in better body condition had higher levels of androgens during parental care. Males that were known to renest later in the season also had higher androgen levels and were in better body condition during the first nesting bout than males only known to have nested once. However, circulating levels of cortisol did not differ significantly between these groups. We discuss our findings in the context of proximate and ultimate costs of parental care and propose several reasons why elevated androgen levels may not be as incompatible with the expression of paternal care in male teleosts, as compared with avian and mammalian fathers.     

Temporal Variation in Decisions about Parental Care in Bluegill, Lepomis macrochirus

Parental investment theory states that an individual will trade‐off present and future reproductive potential to maximize lifetime reproductive success. Only when parental care is costly in terms of reduced future reproductive potential should individuals be sensitive to changes in the value of current offspring and adjust their care. Here, we examine temporal variation in parental care decision‐making in bluegill (Lepomis macrochirus), in which care is provided by males called ‘parentals’. Previous research has shown that parentals that nest early in the breeding season are in higher energetic condition than those that nest later, and early nesting males appear not to pay an opportunity cost to their care in terms of reduced future reproductive potential. Early nesting males also may have higher paternity in their broods than later nesting males. To examine the parental care decisions made by early and mid‐season nesting parentals, we experimentally reduced males’ perceived paternity by swapping eggs between nests. We found that experimental males that nested early in the breeding season adjusted their brood defence behaviour similarly to control males, which had sham egg swaps performed. Conversely, experimental males that nested mid‐season significantly decreased their brood defence behaviour after the manipulation as compared with control males. Thus, unlike mid‐season nesting males, early nesting males appear relatively insensitive to changes in brood value (paternity), possibly because early nesting males pay little cost in terms of reduced future reproductive potential to providing full care or because these males have a predisposition to high paternity.     

Effects of Exogenous Testosterone on Parental Care Behaviours in Male Bluegill Sunfish (Lepomis macrochirus)

Androgens are known to mediate aggressive and defensive behaviour in many vertebrate species. However, high concentrations of androgens might also conflict with the expression of nurturing behaviours and therefore a trade‐off can exist between aggressive and nurturing behaviours during parental care. We explored the role of testosterone in paternal care in bluegill sunfish (Lepomis macrochirus), where males provide both sole defence of the young from predators and sole nurturing behaviour such as fanning of the eggs. At the onset of parental care, we manipulated testosterone levels in males using testosterone propionate implants. We then observed the frequency of nurturing and aggressive behaviours displayed by the males over 6 d of parental care. Testosterone‐implanted fish were more aggressive when presented with a brood predator, performing more bites, opercular flares and lateral displays than control males. Testosterone‐implanted males, however, were not less nurturing than control fish, performing similar levels of fanning and nest‐cleaning behaviours. Thus, our results support a positive relationship between testosterone and paternal aggression but no testosterone‐mediated trade‐off between paternal nurturing and aggression.     

Paternity and paternal effort in the pumpkinseed sunfish

Theoretical models suggest that males should adjust their parentaleffort according to paternity when parental effort is costly,paternity varies among clutches, and males have a cue to assesspaternity. To date, nearly all tests of this theory have beenconducted using birds as model organisms. In this study we examinedthese three factors and the relationship between paternity andmale parental care in a fish system. In the pumpkinseed sunfish(Lepomis gibbosus), parental care is provided exclusively bymales (parentals), but some males (sneakers) parasitize othersby sneaking fertilizations. Parental males significantly lostweight during the parental care period. Clutch size and amountof parental effort did not affect a male's probability of obtainingmore eggs. Paternity was variable among broods. The proportionof young sired by a parental male was not associated with frequencyof fanning eggs or defense of hatched young, but was positivelycorrelated with levels of nest defense during the egg stage.Egg survivorship might restrict an adjustment of fanning behavior,and a general decline in parental behavior (with brood age)might explain the lack of adjustment once the eggs hatch. Parentalmales did not adjust their care when we experimentally manipulatedone possible cue of paternity. Together, these results indicatethat male pumpkinseeds do adjust their care in relation to paternity,but the cues used to assess paternity are not clear.     

Circulating androgens are influenced by parental nest defense in a wild teleost fish

While social interactions influence vertebrate endocrine regulation, the dynamics of regulation in relation to specific behaviors have not been clearly elucidated. In the current study, we investigated whether androgens (testosterone) or glucocorticoids (cortisol) play a functional role in aggressive offspring defense behavior in wild smallmouth bass (Micropterus dolomieu), a teleost fish with sole paternal care. We measured circulating testosterone and cortisol concentrations in plasma samples taken from parental males following a simulated nest intrusion by a common nest predator, the bluegill sunfish (Lepomis macrochirus). To understand whether endocrine regulation changes across the parental care period, we looked both at males guarding fresh eggs and at males guarding hatched embryos. Plasma testosterone levels increased in males subjected to a simulated nest intrusion when compared to sham controls. Testosterone concentrations in males guarding embryos were lower than in males guarding fresh eggs, but circulating testosterone was positively correlated with the level of aggression towards the nest predator at both offspring development stages. However, there was no increase in cortisol levels following a simulated nest intrusion, and no relationship between cortisol and any measured parameter. These results suggest that androgens play an important role in promoting aggressive nest defense behavior in teleost fish.     

Dynamic adjustment of parental care in response to perceived paternity

Theories of parental care evolution predict that genetic relatedness will be an important variable in the amount of care a parent provides. However, current inferences of relatedness-based parental investment from studies in humans and birds remain challenged. No study has yet demonstrated parental care adjustment in a manner uncomplicated by life-history correlates or experimental design. We now present a unique test that controls for individual life histories and demonstrates paternity-related dynamic adjustments in parental care. Brood-rearing male bluegill sunfish (Lepomis macrochirus) that are cuckolded to a varying degree will either increase or decrease their parental investment in response to changing information on paternity during brood development. Specifically, as parental males detect paternity lost to cuckolders and, hence, a reduction in the value of their brood, they adaptively lower their level of parental care. Conversely, if they detect that their paternity is higher than previously assessed, they adaptively raise their level of parental care. This dynamic adjustment during brood rearing indicates the importance of genetic relatedness in parental investment decisions and provides needed empirical support for theoretical predictions.     

Cuckoldry incites cannibalism: male fish turn to cannibalism when perceived certainty of paternity decreases

Perceived certainty of paternity is expected to influence a male's behavior toward his offspring: if he is uncertain of his reproductive success with a current brood due to the presence of cuckolders, it may benefit him to invest instead in future reproduction. A decrease in perceived certainty of paternity incites filial cannibalism (the eating of one's own offspring) in some teleost fishes that provide parental care; however, no work has demonstrated that cannibalism increases proportionately with increased levels of cuckoldry. Here we show for the first time in a fish with no parental care that as the number of cuckolders at a spawning event increases, so does the probability that a male will cannibalize eggs. In field observations of Telmatherina sarasinorum, a small fish endemic to Sulawesi, Indonesia, males increased filial cannibalism behavior threefold in the presence of one cuckolder and nearly sixfold in the presence of two or more cuckolders. This suggests that males may use detection of cuckolders as an indication that the paternity of current offspring has been compromised.     

Genetic evidence for cuckoldry in bluegill Lepomis macrochirus

In colonies of bluegill Lepomis macrochirus , some males provide parental care for broods in nests, whereas other males steal fertilizations and do not provide parental care. Using experimental pond populations of bluegill of known genotype (determined through protein electrophoresis), we demonstrate that cuckolder males successfully fertilize eggs in parental male nests. Using electrophoretic techniques to assess the fertilization success of nesting parental male bluegill in Lake Opinicon, Ontario, we demonstrate that paternity of these males ranges from 41% to 100% among four colonies studied. This difference among colonies is related to the density of cuckolder males.     

Behavioral and physiological responses of a wild teleost fish to cortisol and androgen manipulation during parental care

Proximate mediators of reproductive behaviors in vertebrates have a long history of study. In fishes, relatively few studies have focused on hormonal control of parental care, despite a comprehensive background on the general physiology of fishes, and the frequent occurrence of parental care behaviors. Studies on this taxon have repeatedly found that the relationships between androgens and paternal care do not follow the predictions made in the avian and mammalian literature. Glucocorticoids may also have a role in mediating parental behaviors, possibly through their role as regulators of metabolism. As such, we investigated the role of 11-ketotestosterone (11-KT) and cortisol in mediating parental effort of male smallmouth bass (Micropterus dolomieu) by manipulating hormone titers in wild fish. In smallmouth bass, males spawn annually with a single female and defend a single brood for up to 30 days. Treatment of parental fish with cyproterone acetate (CYA; an androgen receptor antagonist) resulted in a decrease in nest defense in response to a simulated brood predator; however, no changes in nest success, nest tending or biochemical indicators of nutritional status were detected. Treatment with exogenous cortisol did not change parental behavior, but did increase the rate of nest failure, possibly owing to the energetic cost of chronically elevated cortisol concentrations. We discuss these findings in the context of resource-driven trade-offs and highlight life history as an important factor controlling parental effort in species with costly parental care behaviors.     

In Vitro Fertilization Reveals Offspring Recognition via Self-Referencing in a Fish with Paternal Care and Cuckoldry

Male bluegill sunfish (Lepomis macrochirus) exhibit alternative life histories: some males (parentals) delay maturation for up to 7 yr, then build nests, court females, and care for the eggs and fry, whereas other males (cuckolders) mature precociously, attempt to steal fertilizations from parentals, and provide no parental care. Parental males could avoid misdirecting their nepotism (i.e. caring for unrelated young) by abandoning entire broods if they were sired mainly by cuckolders or by discriminating between offspring and non‐kin fry within broods for which they care. We tested for kin discrimination by obtaining sperm from parental and cuckolder males and eggs from several females, and using them to conceive fry in vitro. In ‘blind’ laboratory tests, parental males (but not cuckolder males) distinguished between sources of dripping water that had been conditioned by their own offspring vs. unrelated fry. Parental males that were in the best physical condition were especially choosy. Because the only referents available to our experimental subjects were chemical cues emanating from their own body, our results imply that parental males can use self‐referent phenotype matching for kin recognition. This mechanism enables males to make the adaptive, nepotistic adjustments in paternal care that have been documented in previous studies.     

Steroid hormones in bluegill, a species with male alternative reproductive tactics including female mimicry

The proximate mechanisms underlying the evolution and maintenance of within-sex variation in mating behaviour are still poorly understood. Species characterized by alternative reproductive tactics provide ideal opportunities to investigate such mechanisms. Bluegill (Lepomis macrochirus) are noteworthy in this regard because they exhibit two distinct cuckolder (parasitic) morphs (called sneaker and satellite) in addition to the parental males that court females. Here we confirm previous findings that spawning cuckolder and parental males have significantly different levels of testosterone and 11-ketotestosterone. We also report, for the first time, that oestradiol and cortisol levels are higher in cuckolders than in parental males. The two cuckolder morphs did not differ in average levels of any of the four hormones. However, among satellite males which mimic females in appearance and behaviour, there was a strong negative relationship between oestradiol levels and body length, a surrogate for age. This finding suggests that for satellite males, oestradiol dependency of mating behaviour decreases with increasing mating experience. Although such decreased hormone dependence of mating behaviour has been reported in other taxa, our data represent the first suggestion of the relationship in fishes.     

MOLECULAR GENETIC DISSECTION OF SPAWNING,PARENTAGE, AND REPRODUCTIVE TACTICS IN A POPULATION OF REDBREAST SUNFISH,LEPOMIS AURITUS

Despite a great diversity of reproductive behaviors in fishes, few studies have examined the genetic consequences of alternative reproductive tactics. Here we develop and employ microsatellite markers to assess genetic paternity and maternity of progeny cohorts in a population of redbreast sunfish (Lepomis auritus), a species in which males build and tend nests. Nearly 1000 progeny from 25 nests, plus nest-attendant males and nearby adults, were genotyped at microsatellite loci that displayed more than 18 alleles each. The genetic data demonstrate that multiple females (at least two to six) spawned in each nest, their offspring were spatially dispersed across a nest, and more than 90% of the young were sired by the attendant male. However, about 40% of the nests also showed genetic evidence of low-level reproductive parasitism, and two nests were tended by males that had fathered none of the sampled offspring. Genetically deduced reproductive behaviors in this population of redbreast sunfish contrast with those reported previously in bluegill sunfish (L. macrochirus) wherein heteromorphic males specialized for parasitism or for parental care coexist in high frequency. Thus, nest-parasitic reproductive behaviors in fishes appear to be evolutionary labile.     

Patterns of multiple paternity and maternity in fishes

The characterization of patterns of multiple mating is a major facet of molecular ecology and is paramount to understanding the evolution of behaviours associated with parental care and mate choice. Over the last 15 years, fishes have been particularly well studied with respect to multiple maternity and paternity thanks to the widespread application of microsatellite markers. The present review focusses on the impressive literature on genetic parentage in fishes. In studies of natural populations, we find that multiple paternity is extremely common across fish species, whereas rates of multiple maternity are much more variable. In species with nest defence, for example, rates of multiple maternity are strongly bimodal, and the occurrence of multiple dams per brood is either rare or the rule. The sex of the care‐giving parent is correlated with the rate of multiple parentage: when males provide uniparental care, rates of multiple paternity are low compared to rates of multiple maternity; when females provide parental care, either alone or assisted by males, rates of multiple paternity are highly variable, whereas rates of multiple maternity are quite low. These patterns may reflect conflicts between the reproductive interests of males and females. We also find that fishes in which females brood the offspring internally display much higher rates of multiple paternity compared to mammals or birds, whereas reptiles are intermediate. Male‐nesting fish species, however, show rates of multiple paternity more similar to those found in other vertebrates. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 735–760.     

Coevolution of female fidelity and male help in populations with alternative reproductive tactics

In socially monogamous species, pair-bonded males often continue to provide care to all offspring in their nests despite some degree of paternity loss due to female extra-pair copulation. Previous theoretical models suggested that females can use their within-pair offspring as ‘hostages'' to blackmail their social mates, so that they continue to provide care to the brood at low levels of cuckoldry. These models, however, rely on the assumption of sufficiently accurate male detection of cuckoldry and the reduction of parental effort in case of suspicion. Therefore, they cannot explain the abundant cases where cuckolded males continue to provide extensive care to the brood. Here we use an analytical population genetics model and an individual-based simulation model to explore the coevolution of female fidelity and male help in populations with two genetically determined alternative reproductive tactics (ARTs): sneakers that achieve paternity solely via extra-pair copulations and bourgeois that form a mating pair and spend some efforts in brood care. We show that when the efficiency of mate guarding is intermediate, the bourgeois males can evolve to ‘specialize'' in providing care by spending more than 90% of time in helping their females while guarding them as much as possible, despite frequent cuckoldry by the sneakers. We also show that when sneakers have tactic-specific adaptations and thus are more competitive than the bourgeois in gaining extra-pair fertilizations, the frequency of sneakers and the degrees of female fidelity and male help can fluctuate in evolutionary cycles. Our theoretical predictions highlight the need for further empirical tests in species with ARTs.     

Paternity and the evolution of male parental care

It is generally believed that level of paternity (the proportion of zygotes in a brood that were fertilized by the male providing parental care) has an important role in the evolution of parental care. We have used population genetics models to investigate this role. The models indicate that only in mating systems where a parental male “sacrifices” promiscous matings can paternity influence the evolution of male parental care. This is because level of paternity can reflect the number of opportunities for these promiscuous fertilizations. For example, high paternity can mean few opportunities and therefore a low cost for paternal care.Certain behaviors may preadapt a species for the evolution of male parental care because they decrease the costs of providing care. For example, in fish species where male care has evolved from spawning territories, the very establishment of territories may have precluded males from gaining promiscuous matings, thereby eliminating the promiscuity costs and facilitating the evolution of care. Without a promiscuity cost, level of paternity will not have influenced the evolution of male care in fishes.Because paternity has limited influence in the evolution of male care, differences in reliability of parentage between males and females are unlikely to explain the prevalence of female care. Our analysis suggests that paternity differences between species cannot serve as a general explanation for the observed patterns of parental care behavior.     

Influence of brood size and offspring size on parental investment in a biparental cichlid fish,Neolamprologus moorii

Both males and females ofNeolamprologus moorii, a substrate-spawning cichlid fish in Lake Tanganyika, tend their eggs and fry. In this study, the influence of brood size and fry size on parental investment of this species was examined under natural conditions. Breeding parents intensively attacked fry-eating fishes that approached their broods. The attack rate by the parents against the approaching fry-eaters was positively correlated with brood size and negatively with fry size, but was much more strongly related to brood size. This suggests that the parental decision for brood defense appeared to be primarily determined by brood size. The reason for fry size being less important in the parental decision may be that even large fry have a low survival ability on their own under the high predation pressure in the study sites.     

Allocation of Male Parental Care in Relation to Paternity Within and Among Broods of the Common Yellowthroat (Geothlypis trichas)

The relationship between male parental care and paternity has been investigated in a number of avian species, but in many cases the influences of confounding factors, such as variation in male and territory quality, were not addressed. These sources of variation can be controlled for by making within-male comparisons between successive broods or within-brood comparisons between groups of fledglings in a divided brood. We studied the relationship between male parental care and paternity in the common yellowthroat ( Geothlypis trichas ) at three levels: between groups of fledglings in divided broods, between first and second broods of the same pair, and among all broods in the population. In this study we proposed three hypotheses: first, males in double-brooded pairs should provide relatively more parental care to broods in which they have higher paternity; secondly, after fledging and brood division, males should provide more care to related offspring; and finally, among all broods in the population, paternity should be related positively to male parental care. Brood division occurred in many of the broods studied; however, broods were not divided according to fledgling size or paternity. Furthermore, within divided broods, males fed within-pair and extra-pair fledglings at similar rates. For sequential broods of the same pair, male feeding rates were not associated with differences in paternity between broods. Among all broods in the population, males did not provide relatively less care to broods containing unrelated young. The lack of a relationship between male parental care and paternity suggests that either males cannot assess their paternity or the costs of reducing male parental care outweigh the benefits.     

The genetic mating system of spotted sunfish (Lepomis punctatus): mate numbers and the influence of male reproductive parasites

In nest-building fish species, mature males often exhibit one of two alternative reproductive behaviours. Bourgeois males build nests, court females, and guard their eggs. Parasitic cuckolders attempt to steal fertilizations from bourgeois males and do not invest in parental care. Previous evidence from the bluegill sunfish (Lepomis macrochirus) suggests that adult males are morphologically specialized for these two tactics. Here, we used microsatellite markers to determine genetic parentage in a natural population of the spotted sunfish (L. punctatus) that also displayed both bourgeois and parasitic male morphs. As gauged by relative investments in gonadal vs. somatic tissues, between 5 and 15% of the mature adult males were parasites. Multi-locus genotypes were generated for more than 1400 embryos in 30 nests, their nest-guardian males, and for other adults in the population. Progeny in approximately 57% of the nests were sired exclusively by the guardian male, but the remaining nests contained embryos resulting from cuckoldry as well. Overall, the frequency of offspring resulting from stolen fertilizations was only 1.3%, indicating that the great majority of paternity is by bourgeois nesting males. With regard to maternity, 87% of the nests had at least three dams, and computer simulations estimate that about 7.2 dams spawned per nest.     

Correlated evolution in parental care in females but not males in response to selection on paternity assurance behaviour

According to classical parental care theory males are expected to provide less parental care when offspring in a brood are less likely to be their own, but empirical evidence in support of this relationship is equivocal. Recent work predicts that social interactions between the sexes can modify co‐evolution between traits involved in mating and parental care as a result of costs associated with these social interactions (i.e. sexual conflict). In burying beetles (Nicrophorus vespilloides), we use artificial selection on a paternity assurance trait, and crosses within and between selection lines, to show that selection acting on females, not males, can drive the co‐evolution of paternity assurance traits and parental care. Males do not care more in response to selection on mating rate. Instead, patterns of parental care change as an indirect response to costs of mating for females.     

High frequency of multiple paternity in broods of a socially monogamous cichlid fish with biparental nest defence

In several animal taxa, genetic analyses have demonstrated that social monogamy and biparental brood care do not preclude polygamous reproduction. Few studies have been conducted in fish, but in fish species without alternative reproductive phenotypes, social monogamy was largely congruent with genetic parentage. In contrast to these findings, we report an exceptionally high level of multiple paternity in a socially monogamous cichlid fish with biparental nest defence ( Variabilichromis moorii ), inferred from microsatellite and mitochondrial data of 10 broods. Whereas all offspring in a nest shared a common mother, each brood was sired by 2 to > 10 males. None of the inferred sires was assigned a large proportion of the brood. Paternity was estimated as the minimum number of sires required to explain multilocus offspring genotypes, and as the maximum-likelihood number of sires given population allele frequencies. Analysis of simulated brood genotypes suggested that, although these two methods tend to under- and overestimate, respectively, the true number of sires, primary sires with many offspring in a brood would have been detected. Hence, the genetic data indicate that the nest tending males suffer substantial cuckoldry and provide alloparental care for a large number of unrelated fry. We have no data on the social status of the cuckolding males, but due to synchronous spawning of pairs and commitment to brood care of paired males, it is possible that most of the parasitic spawners are solitary males.