Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologically identified neurons

In one species of vocalizing (sonic) fish, the midshipman (Porichthys notatus), there are two classes of sexually mature males--Types I and II--distinguished by a number of traits including body size, gonad size, and reproductive tactic. The larger Type-I males (unlike Type-II males and females) build nests, guard eggs, and generate several types of vocalizations. Sound production by Type-I males is paralleled by a proportionate increase of 600% in their sonic muscle mass. The motor volley from ventral occipital roots innervating the sonic muscles establishes their contraction rate and, in turn, the fundamental frequency of emitted sounds. Electrical stimulation of the midbrain in every male and female elicited a rhythmic sonic discharge as recorded in the occipital roots; however, the fundamental frequency was slightly, but significantly, higher (20%) in Type-I males. Intracellular recording from identified motoneurons and presumed presynaptic "pacemaker" neurons showed their synaptic and action potentials had the same frequency as that of the nerve volley in every male and female. Reconstructions of physiologically identified motoneurons and pacemaker neurons following intracellular horseradish-peroxidase (HRP) filling showed their somata and dendrites to be 100-300% larger in Type-I males. These data unambiguously show that the size of a target muscle is correlated with the size of both the respective motoneurons and their presynaptic afferent neurons. As discussed, this implies that the dramatic increase in neuron size in the sonic motor system of Type-I males is causally dependent upon expansion of the sonic muscle. It is further likely that the more modest sex difference in the rhythmic central discharge is established by the intrinsic membrane properties of sonic neurons. These results also corroborate, at a number of behavioral, morphological, and neurophysiological levels, that the sonic motor system of "sneak spawning" Type-II males is similar to that of females. Thus, unlike the vocalizing Type-I males, sexual differentiation of the reproductive system in Type-II males is not linked to concomitant changes in the neurophysiological and morphological features of the sonic motor circuit.     

1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish, the common carp (Cyprinus carpio)

All 20.000 different fish species vary greatly in their ability to tolerate and survive fluctuating oxygen concentrations in the water. Especially fish of the genus Carassius, e.g. the crucian carp and the goldfish, exhibit a remarkable tolerance to limited/absent oxygen concentrations. The metabolic changes of anoxia-tolerant crucian carp were recently studied and published. Contrary to crucian carp, the hypoxia-tolerant common carp cannot survive a complete lack of oxygen (anoxia). Therefore, we studied the ¹H-NMR-based metabolomics of brain, heart, liver and white muscle extracts of common carp, subjected to anoxia (0 mg O₂ l^?1) and hypoxia (0.9 mg O₂ l^?1) at 5 °C. Specifically, fish were exposed to normoxia (i.e. 9 mg O₂ l^?1; controls 24 h, 1 week and 2 weeks), acute hypoxia (24 h), chronic hypoxia (1 week) and chronic hypoxia (1 week) with normoxic reoxygenation (1 week). Additionally, we also investigated the metabolic responses of fish to anoxia for 2 h. Both anoxia and hypoxia significantly changed the tissue levels of standard energy metabolites as lactate, glycogen, ATP/ADP and phosphocreatine. Remarkably, anoxia induced increased lactate levels in all tissues except for the heart whereas hypoxia resulted in decreased lactate concentrations in all tissues except for brains. Furthermore, hypoxia and anoxia influenced amino acids (alanine, valine/(iso)leucine) and neurotransmitters levels (GABA, glutamate). Lastly, we also detected ‘other’ i.e. previously not reported compounds to play a role in the present context. Scyllo-inositol levels changed significantly in heart, liver and muscle, providing novel insights into the anoxia/hypoxic responses of the common carp.     

Intra‐ and Intersexual swim bladder dimorphisms in the plainfin midshipman fish (Porichthys notatus): Implications of swim bladder proximity to the inner ear for sound pressure detection

The plainfin midshipman fish, Porichthys notatus , is a nocturnal marine teleost that uses social acoustic signals for communication during the breeding season. Nesting type I males produce multiharmonic advertisement calls by contracting their swim bladder sonic muscles to attract females for courtship and spawning while subsequently attracting cuckholding type II males. Here, we report intra‐ and intersexual dimorphisms of the swim bladder in a vocal teleost fish and detail the swim bladder dimorphisms in the three sexual phenotypes (females, type I and II males) of plainfin midshipman fish. Micro‐computerized tomography revealed that females and type II males have prominent, horn‐like rostral swim bladder extensions that project toward the inner ear end organs (saccule, lagena, and utricle). The rostral swim bladder extensions were longer, and the distance between these swim bladder extensions and each inner‐ear end organ type was significantly shorter in both females and type II males compared to that in type I males. Our results revealed that the normalized swim bladder length of females and type II males was longer than that in type I males while there was no difference in normalized swim bladder width among the three sexual phenotypes. We predict that these intrasexual and intersexual differences in swim bladder morphology among midshipman sexual phenotypes will afford greater sound pressure sensitivity and higher frequency detection in females and type II males and facilitate the detection and localization of conspecifics in shallow water environments, like those in which midshipman breed and nest.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Steroid hormones and paternal care in the plainfin midshipman fish (Porichthys notatus)

The present study investigated the relationship between plasma steroid hormone levels and the expression ofpaternal behavior in the plainfin midshipman fish (Porichthys notatus), where males may simultaneously care for multiple clutches in different stages of development. Blood samples were collected from free-living parental males during that part of the breeding season when males may be found in various stages of parental care. Plasma 11-ketotestosterone levels were significantly higher in males with empty nests and nests containing only eggs than in males with nests containing embryos. All males with nests containing embryos had undetectable testosterone levels, whereas testosterone levels were detectable in many males with empty nests or nests containing only eggs. Estradiol levels were detectable in only a few males from nests with no eggs or nests containing only eggs. Cortisol levels were not correlated with stage of paternal care or with handling time. These results follow the frequently reported vertebrate pattern of declining androgen levels over the course of the breeding season or during the period of parental care. However, many male midshipman guarding nests containing only eggs had androgen levels similar to those of males whose nests contained no offspring. Thus the pattern of androgen levels exhibited by reproductively active parental male midshipman may reflect a compromise between investment in paternal care versus courtship and/or territoriality.     

Male-Biased Sexual Size Dimorphism,Resource Defense Polygyny,and Multiple Paternity in the Emei Moustache Toad (Leptobrachium boringii)

We tested the hypotheses that the Emei moustache toad (Leptobrachium boringii) exhibits resource defense polygyny and that combat led to the evolution of male-biased sexual size dimorphism. Between February and March of 2011 and 2012, 26 female and 55 male L. boringii from Mount Emei UNESCO World Heritage Site, Sichuan, China, were observed throughout the breeding season. Prior to the breeding season, males grow 10–16 keratinized maxillary nuptial spines, which fall off once the season has ended. Throughout this time, males construct and defend aquatic nests where they produce advertisement calls to attract females. In a natural setting, we documented 14 cases involving a total of 22 males where males used their moustaches for aggressive interaction, and nest takeover was observed on seven occasions. Males were also observed to possess injuries resulting from combat. Genetic analysis using microsatellite DNA markers revealed several cases of multiple paternity, both within nest and within clutch. This observation indicated that some alternative male reproductive strategy, such as satellite behaviour, is occurring, which may have led to the multiple paternity. Larger males were observed to mate more frequently, and in multiple nests, suggesting that females are selecting for larger males, or that larger males are more capable of defending high quality territories.     

Globins and hypoxia adaptation in the goldfish, Carassius auratus

Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin alpha and beta and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po(2) ~ 6.7 kPa, 8 h at Po(2) ~ 1.7 kPa, 16 h at Po(2) ~ 6.7 kPa) at 20 degrees C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may contribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin).     

Body Size Correlates with Fertilization Success but not Gonad Size in Grass Goby Territorial Males

In fish species with alternative male mating tactics, sperm competition typically occurs when small males that are unsuccessful in direct contests steal fertilization opportunities from large dominant males. In the grass goby Zosterisessor ophiocephalus, large territorial males defend and court females from nest sites, while small sneaker males obtain matings by sneaking into nests. Parentage assignment of 688 eggs from 8 different nests sampled in the 2003–2004 breeding season revealed a high level of sperm competition. Fertilization success of territorial males was very high but in all nests sneakers also contributed to the progeny. In territorial males, fertilization success correlated positively with male body size. Gonadal investment was explored in a sample of 126 grass gobies collected during the period 1995–1996 in the same area (61 territorial males and 65 sneakers). Correlation between body weight and testis weight was positive and significant for sneaker males, while correlation was virtually equal to zero in territorial males. That body size in territorial males is correlated with fertilization success but not gonad size suggests that males allocate much more energy into growth and relatively little into sperm production once the needed size to become territorial is attained. The increased paternity of larger territorial males might be due to a more effective defense of the nest in comparison with smaller territorial males.     

Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish,Danio rerio

In recent years, natural and anthropogenic factors have increased aquatic hypoxia the world over. In most organisms, the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). HIF-1 also plays a critical role in the normal development of the cardiovascular system of vertebrates. We tested the hypothesis that hypoxia exposures which resulted in HIF-1 induction during embryogenesis would be associated with enhanced hypoxia tolerance in subsequent developmental stages. We exposed zebrafish (Danio rerio) embryos to just 4 h of severe hypoxia or total anoxia at 18, 24 and 36 h post-fertilization (hpf). Of these, exposure to hypoxia at 24 and 36 hpf as well as anoxia at 36 hpf activated the HIF-1 cellular pathway. Zebrafish embryos that acutely upregulated the HIF-1 pathway had an increased hypoxia tolerance as larvae. The critical window for hypoxia sensitivity and HIF-1 signalling was 24 hpf. Adult male fish had a lower critical oxygen tension (P_crit) compared with females. Early induction of HIF-1 correlated directly with an increased proportion of males in the population. We conclude that mounting a HIF-1 response during embryogenesis is associated with long-term impacts on the phenotype of later stages which could influence both individual hypoxia tolerance and population dynamics.     

Within‐season variation in sexual selection in a fish with dynamic sex roles

The strength of sexual selection may vary between species, among populations and within populations over time. While there is growing evidence that sexual selection may vary between years, less is known about variation in sexual selection within a season. Here, we investigate within‐season variation in sexual selection in male two‐spotted gobies (Gobiusculus flavescens). This marine fish experiences a seasonal change in the operational sex ratio from male‐ to female‐biased, resulting in a dramatic decrease in male mating competition over the breeding season. We therefore expected stronger sexual selection on males early in the season. We sampled nests and nest‐holding males early and late in the breeding season and used microsatellite markers to determine male mating and reproductive success. We first analysed sexual selection associated with the acquisition of nests by comparing nest‐holding males to population samples. Among nest‐holders, we calculated the potential strength of sexual selection and selection on phenotypic traits. We found remarkable within‐season variation in sexual selection. Selection on male body size related to nest acquisition changed from positive to negative over the season. The opportunity for sexual selection among nest‐holders was significantly greater early in the season rather than late in the season, partly due to more unmated males. Overall, our study documents a within‐season change in sexual selection that corresponds with a predictable change in the operational sex ratio. We suggest that many species may experience within‐season changes in sexual selection and that such dynamics are important for understanding how sexual selection operates in the wild.     

Alternative Male Spawning Tactics and Acoustic Signals in the Plainfin Midshipman Fish Porichthys notatus Girard (Teleostei,Batrachoididae)

The plainfin midshipman Porichthys notatus has two male reproductive morphs, ‘Type I’ and ‘Type II’, which are distinguishable by their physical traits alone. Type I males are eight times larger in body mass than Type II males and have a six-fold larger relative sonic (vocal) muscle mass than Type II males. In contrast, the testicles of Type II males are seven times larger than those of Type I males. This study demonstrates morph-specific patterns of reproduction, including acoustic signals, for Type I and II males. Field censuses of nests showed that only Type 1 males maintained nests. Type II males and females transiently appeared in these nests in association with each other. Infra-red video and hydrophone recordings in aquaria showed that Type I males maintained nests and readily vocalized. Long-duration ‘hums’ and sequences of short-duration ‘grunts’ were produced during advertisement and agonistic contexts, respectively. Humming Type I males attracted females to their nests, pair-spawned, and then guarded egg clutches alone. By contrast, Type II males neither acoustically courted females nor maintained available nest sites, but rather ‘sneak-’ or ‘satellite-spawned’ at the nests of Type I males. Type II males infrequently produced low amplitude, short duration grunts that were similar in spectral, temporal and amplitude characteristics to the grunts of females. Type II males appear to be obligate sexual parasites of the nest-building, mate-calling, and egg-guarding Type I males. The dimorphic body and vocal muscle traits of the two male morphs in the plainfin midshipman are thus paralleled by a divergence in their reproductive tactics and the properties of their acoustic signals.     

Physiological responses to acute experimental hypoxia in the air-breathing Indian catfish, Clarias batrachus (Linnaeus, 1758)

With an aim to study the mechanism of adaptation to acute hypoxic periods by hypoxia-tolerant catfish, Clarias batrachus, the mass-specific metabolic rate (VO₂) along with its hematological parameters, metabolic response and antioxidant enzyme activities were studied. During progressive hypoxia, C. batrachus was found to be an oxyconformer and showed a steady decline in its aquatic oxygen consumption rate. When C. batrachus was exposed for different periods at experimental hypoxia level (0.98?±?0.1 mg/L, DO), hemoglobin and hematocrit concentrations were increased, along with decrease in mean cellular hemoglobin concentration, which reflected a physiological adaptation to enhance oxygen transport capacity. Significant increase in serum glucose and lactate concentration as well as lactate dehydrogenase activity was observed. Antioxidant enzymes were found to operate independently of one another, while total glutathione concentration was unaffected in any of the tissues across treatments. These observations suggested that hypoxia resulted in the development of oxidative stress and C. batrachus was able to respond through increase in the oxygen carrying capacity, metabolic depression and efficient antioxidant defense system to survive periods of acute hypoxia.     

Sub‐lethal effects on fish provide insight into a biologically‐relevant threshold of hypoxia

Hypoxia (low dissolved oxygen) is a mounting concern for aquatic ecosystems as its prevalence increases with rising anthropogenic nutrient inputs. Hypoxia is most commonly defined as 2.0 mg l^–1 of dissolved oxygen, although this level varies widely across studies and agency regulations. Such definitions may be too conservative, as ecologically‐relevant non‐lethal effects (e.g. consumption and growth) of hypoxia on important aquatic species, such as fish, often occur at oxygen levels much higher than 2.0 mg l^–1. In addition, many mechanisms that regulate hypoxia tolerance in fish have been proposed, including temperature, habitat, location in the water column, and body size, but there is ongoing debate over which mechanisms are most important. Using a structured meta‐analysis of published studies, we showed consistent, significant negative effects on fish growth and consumption below 4.5 mg l^–1. While the total amount of variation explained was generally low, below 4.5 mg l^–1 of dissolved oxygen, phylogenetic relationships accounted for most of the explained variation in fish growth. Ecological factors including body size, location in the water column (pelagic, demersal, or benthopelagic), habitat (freshwater, marine, or diadromous), and temperature explained very little of the effect of hypoxia on fish growth and explained only a moderate level of variation in consumption. Our results suggest a dramatically higher threshold for sub‐lethal effects of hypoxia on fish than oxygen levels generally set for regulation purposes, and provide little support for accepted ecological mechanisms thought to influence hypoxia tolerance.     

Morphometric changes associated with the reproductive cycle and behaviour of the intertidal-nesting, male plainfin midshipman Porichthys notatus

Morphometric changes in body condition, liver, sonic muscle and gonadal development associated with the annual reproductive cycle and behaviour of the intertidal-nesting male plainfin midshipman Porichthys notatus were investigated. Body condition of type I males rapidly increased during the pre-nesting (PN) period, peaked at the beginning of the summer nesting cycle and then gradually declined to lowest levels during the non-reproductive (NR) period. The gonado-somatic index of type I males peaked during PN and then declined during the summer nesting cycle to lowest levels at the end of the nest cycle and during NR. Indices of sonic muscle and liver of type I males were lowest during NR, gradually increased during PN and then peaked during the summer nesting cycle. Results indicate that body condition and fecundity of type I males were positively correlated with body mass at the end of the nest cycle. These findings as they relate to the annual reproductive cycle and behaviour of the type I male P. notatus are discussed.     

The response of the tidepool sculpin, Oligocottus maculosus, to hypoxia in laboratory, mesocosm and field environments

Animals living in the intertidal zone experience regular, predictable fluctuations in physical parameters including temperature, oxygen and salinity and rely on behavioural, physiological and biochemical mechanisms to cope with environmental variation. In the present study, behavioural strategies induced by aquatic hypoxia (e.g. emergence) were performed at similar oxygen tensions across laboratory, mesocosm and field environments; the number of individuals performing these behaviours at any one time was similar in mesocosms and the field. The use of aquatic surface respiration (ASR) was more plastic than emergence behaviour, occurring at a lower oxygen tension in juveniles than adults and being influenced by the addition of alarm substance. Oxygen uptake was lower in air than in water in adults but, in contrast, oxygen uptake was not influenced by the respiratory medium in juveniles. In the laboratory, 72 h of forced emergence did not affect whole body concentrations of lactate but when ASR and emergence were prevented within mesocosm environments there was a significant elevation of lactate. The present study highlights the benefits of transcending traditional laboratory/field boundaries allowing the responses of laboratory-held animals to environmental fluctuation to be integrated with how these animals perform in their natural environment.     

Flow increases tolerance of heat and hypoxia of an aquatic insect

Recent experiments support the idea that upper thermal limits of aquatic insects arise, at least in part, from a lack of sufficient oxygen: rising temperatures typically stimulate metabolic demand for oxygen more than they increase rates of oxygen supply from the environment. Consequently, factors influencing oxygen supply, like water flow, should also affect thermal and hypoxia tolerance. We tested this hypothesis by measuring the effects of experimentally manipulated flows on the heat and hypoxia tolerance of aquatic nymphs of the giant salmonfly (Plecoptera: Pteronarcys californica), a common stonefly in western North America. As predicted, stoneflies in flowing water (10 cm s⁻¹) tolerated water that was approximately 4°C warmer and that contained approximately 15% less oxygen than did those in standing water. Our results imply that the impacts of climate change on streamflow, such as changes in patterns of precipitation and decreased snowpack, will magnify the threats to aquatic insects from warmer water temperatures and lower oxygen levels.     

Diet and cannibalism in plainfin midshipman Porichthys notatus

The macroscopic and microscopic diversity of potential food items available in the nests of plainfin midshipman Porichthys notatus were quantified and compared with items that were found in the stomach and intestine (digestive tract) of the guarding males. In this species, males occur as one of two possible reproductive morphs: guarder males that care for young and sneaker males that parasitize the courtship and care of guarder males. Although it was predicted that guarder males would have fewer feeding opportunities due to their confinement to the nest, they in fact had more food items in their digestive tracts than did sneaker males and females. Date in the breeding season (a proxy of care duration) and body condition were not correlated with the amount of food consumed by guarder males. The main type of food consumed was P. notatus embryos; 69% of all guarder males sampled had cannibalized offspring. By comparing the diet of both sexes and tactics, this study sheds light on some of the strategies designed to cope with the costs of providing parental care.     

Reproductive ecology of the jacky dragon (Amphibolurus muricatus): an agamid lizard with temperature‐dependent sex determination

Abstract The jacky dragon, Amphibolurus muricatus (White, ex Shaw 1790) is a medium sized agamid lizard from the southeast of Australia. Laboratory incubation trials show that this species possesses temperature‐dependent sex determination. Both high and low incubation temperatures produced all female offspring, while varying proportions of males hatched at intermediate temperatures. Females may lay several clutches containing from three to nine eggs during the spring and summer. We report the first field nest temperature recordings for a squamate reptile with temperature‐dependent sex determination. Hatchling sex is determined by nest temperatures that are due to the combination of daily and seasonal weather conditions, together with maternal nest site selection. Over the prolonged egg‐laying season, mean nest temperatures steadily increase. This suggests that hatchling sex is best predicted by the date of egg laying, and that sex ratios from field nests will vary over the course of the breeding season. Lizards hatching from eggs laid in the spring (October) experience a longer growing season and should reach a larger body size by the beginning of their first reproductive season, compared to lizards from eggs laid in late summer (February). Adult male A. muricatus attain a greater maximum body size and have relatively larger heads than females, possibly as a consequence of sexual selection due to male‐male competition for territories and mates. If reproductive success in males increases with larger body size, then early hatching males may obtain a greater fitness benefit as adults, compared to males that hatch in late summer. We hypothesize that early season nests should produce male‐biased sex ratios, and that this provides an adaptive explanation for temperature‐dependent sex determination in A. muricatus.     

Male care in primates: does it ever reflect paternity?

Female mammals have internal fertilization, long gestation, and lactation. These basic facts of reproductive biology have important social consequences. Internal fertilization (accompanied by sperm competition) forces males either to face considerable uncertainty about paternity or to invest heavily in mate guarding. Long gestation increases the benefits of mate desertion for males. And because only females lactate, males have relatively less to contribute to rearing the young. Hence, it is not surprising that male mammals rarely assist their mates in caring for their young: Direct infant care by males occurs in fewer than 5 percent of all mammalian species.^1,2 In fact, many mammalian mothers attack males, even possible father of their offspring.³