Osmoregulatory role of the brood pouch in the euryhaline Gulf pipefish, Syngnathus scovelli

Many species of pipefish exhibit a reversal of parental roles, in which females insert eggs into the brood pouch of the male where they are incubated until the end of embryonic development. While the significance of the male brood pouch has been examined for over a century, the role of the pouch is still unclear. One possible function is to aid in osmoregulation by buffering embryos from the external environment. To investigate this role, the euryhaline Gulf pipefish, Syngnathus scovelli, was collected and maintained in either a low salinity or a saltwater environment. Changes in plasma and pouch fluid osmolality and morphological changes of the pouch were examined. Brood pouch fluid was similar to male plasma during the early and late stages of the brooding period for low salinity males, but was significantly hyperosmotic during the middle of the brooding period. In saltwater males, brood pouch fluid was similar to plasma during early brooding, but became hyperosmotic as brood time progressed. The brood pouch epithelium of both low salinity and saltwater males contained mitochondria-rich cells. In early brooding saltwater males these cells contained an apical opening into the pouch lumen. Osmotic and morphological differences observed suggest that the brood pouch plays an active role in regulating osmotic concentration of the pouch fluid. Additionally, pouch fluid concentration may be regulated more during early stages of embryonic development.     

Brooding season, sex ratio, and brood pouch development in the seaweed pipefish, Syngnathus schlegeli, in Otsuchi Bay, Japan

Males of the seaweed pipefish, Syngnathus schlegeli, take care of their eggs in the brood pouch. These pipefish were periodically collected from the shallow seagrass beds in Otsuchi Bay on the Pacific coast of northern Honshu, Japan, from spring to autumn to investigate the basic reproductive ecology. Appearance of the pipefish in the coastal seagrass beds coincided with the initiation of reproduction. The reproductive season was from May to at least October, with its peak in July. A rearing experiment revealed that the brooding period of the male had a negative correlation with water temperature, and it was estimated to last about 1 month in the bay. Almost all males were brooding during the peak of the reproductive season. Although, the brood pouch of most males was either full or devoid of eggs, 6.2% of the males had a partially filled (20%–90%) brood pouch, and multiple clutches were identified in the brood pouch of some males, indicating that the mating system of the pipefish is polygamous, perhaps polygynous. Sex ratio fluctuated among months, and the overall sex ratio tended to be biased to male. Body size of males with an immature brood pouch had a wide range, from 133 to 215 mm standard length (SL). The smallest brooding male was 134 mm SL. Mean SL of brooding males was significantly larger than that of nonbrooding mature males. The number of males with an immature brood pouch was greater at the beginning than later in the reproductive season. The results seem to collectively indicate that the occurrence of a larger proportion of immature males at the onset of the reproductive season may be ascribed to both new recruitment and larger body size at maturation, resulting from the males trading the reproductive effort to somatic growth, perhaps to increase future reproductive success. Received: April 4, 2000 / Revised: September 21, 2000 / Accepted: January 16, 2001     

Maternal care in the rockpool amphipod Apherusa jurinei: developmental and environmental cues

There is evidence that active, pre-emergence maternal brood care in amphipod crustaceans may be associated with ‘harsh’ environmental conditions. We examined, in the rockpool amphipod Apherusa jurinei, behavioural activities that may function as a form of active brood care. Only ovigerous females showed ‘curl’ and ‘stretch’ activities, with consequent flushing of the brood pouch and cycling of the eggs therein. There was a significant decline in these activities as embryonic development advanced and brood care almost ceased when well-developed embryos showed a heart pulse and self-ventilation. We propose that this pattern of brood care reflects changes in the physiological requirements of embryos as they develop within the egg membrane. In addition, ovigerous females showed significantly higher levels of brood care under lowered oxygen conditions. They achieved this by increasing the average duration of the ‘stretch’ component, with other brood care components remaining constant. Thus, developmental and environmental cues alter the components of active brood care in distinct ways. Experimental removal showed that the physical presence of eggs in the brood pouch is important in controlling the expression of brood care activities. However, females with all of their eggs removed continued to brood at low levels, suggesting that a maternal state also controls brood care. The sophisticated expression of active maternal brood care in amphipods under ‘harsh’ environmental conditions such as rockpools has implications both for individual reproductive success and the distribution and abundance of brooding versus nonbrooding species. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Structural and functional features of gill epithelium and the brood pouch of pipefish <Emphasis Type="Italic">Sygnathus acusimilis</Emphasis> (Syngnathidae,Gasterosteiformes) during adaptation to dilute sea water

Study of the dilate sea-water tolerance of the pipefish Syngnathus acusimilis indicated that it successfully acclimatized to an abrupt change in water salinity from 5 to 32‰. Larvae in the brood pouch also successfully acclimatized to salinity change, and, after several days, juveniles left the brood pouch and continued to live. In freshwater, adult and fry perished during several minutes. In the gill epithelium of S. acusimilis, chloride cells of only a marine type were found. A comparison of the ultrastructure of chloride cells of gill epithelium and mitochondrion-rich cells of the brood pouch of S. acusimilis demonstrated their high similarity. The ultrastructure of cells of the brood pouch indisputably attests to their involvement in maintaining an osmotic and ionic homeostasis in the cavity of the brood pouch.     

Immunocytochemical detection of mitochondria-rich cells in the brood pouch epithelium of the pipefish, Syngnathus schlegeli : structural comparison with mitochondria-rich cells in the gills and larval epidermis

The brood pouch of the male pipefish (Syngnathus schlegeli) is a ventral organ located on the tail, with the anterior region closely associated with the genital pore. The embryos in the pouch are attached to highly vascularized placenta-like tissue which seals the pouch folds from inside during incubation. The epithelium of the placenta-like tissue consists of mitochondria-rich cells (MRCs) and pavement cells. Differences in MRC morphology in the brood pouch epithelium, the gills and the larval epidermis of the pipefish were examined by light and electron microscopy. Transmission electron microscopy revealed that the MRCs in the brood pouch and the gills shared common characteristics: the presence of numerous mitochondria packed among a well-developed tubular system and the close association of the basal parts with the capillaries running underneath the epithelia. The size of the apical opening of the elongate, flask-shaped brood pouch MRC was about one-tenth that of the apical pit of the gill MRC. The gill and larval epidermal MRCs formed a multicellular complex, in contrast to solitary brood pouch MRCs. The brood pouch MRCs were intensively stained by immunocytochemistry with an antiserum specific for Na⁺,K⁺-ATPase. The Na⁺ concentrations in the brood pouch were maintained near those in the serum rather than seawater during incubation. We conclude that the brood pouch MRCs function as an ion-transporting cell, absorbing ions from the brood pouch lumen, perhaps to protect the embryos from the hyperosmotic environment.     

Evolutionary ecology of pipefish brooding structures: embryo survival and growth do not improve with a pouch

For animals that reproduce in water, many adaptations in life‐history traits such as egg size, parental care, and behaviors that relate to embryo oxygenation are still poorly understood. In pipefishes, seahorses and seadragons, males care for the embryos either in some sort of brood pouch, or attached ventrally to the skin on their belly or tail. Typically, egg size is larger in the brood pouch group and it has been suggested that oxygen supplied via the pouch buffers the developing embryos against hypoxia and as such is an adaptation that has facilitated the evolution of larger eggs. Here, using four pipefish species, we tested whether the presence or absence of brood pouch relates to how male behavior, embryo size, and survival are affected by hypoxia, with normoxia as control. Two of our studied species Entelurus aequoreus and Nerophis ophidion (both having small eggs) have simple ventral attachment of eggs onto the male trunk, and the other two, Syngnathus typhle (large eggs) and S. rostellatus (small eggs), have fully enclosed brood pouches on the tail. Under hypoxia, all species showed lower embryo survival, while species with brood pouches suffered greater embryo mortality compared to pouchless species, irrespective of oxygen treatment. Behaviorally, species without pouches spent more time closer to the surface, possibly to improve oxygenation. Overall, we found no significant benefits of brood pouches in terms of embryo survival and size under hypoxia. Instead, our results suggest negative effects of large egg size, despite the protection of brood pouches.     

A low rate of multiple maternity for pregnant male northern pipefish Syngnathus fuscus

Microsatellite parentage analysis was applied to 22 broods of the northern pipefish Syngnathus fuscus for the first time. The majority of males mated singly, 23% of males mated with two females, and no males mated with more than two females. The arrangement of embryos within the brood pouch of multiply mated males reflects a previously undocumented fill pattern where full‐sib groups are segregated within the pouch by both right and left sides as well as anterior and posterior ends.     

Testes investment and spawning mode in pipefishes and seahorses (Syngnathidae)

Externally fertilizing fishes are predicted to invest heavily in testes, because large numbers of sperm should be favoured by the high risk of sperm competition from sneaker males, and/or the dilution of ejaculates when shed into open water. Using museum specimens, we measured testes mass and body mass of 83 mature males, belonging to 21 genera of the family Syngnathidae (pipefishes and seahorses). In this family all species show paternal care, ranging in degree from eggs being attached to the skin of the male, to eggs being completely enclosed and nurtured within a brood pouch. The former, 'unprotected' group, is thought to have external fertilization, whereas in the latter, 'protected' group, males fertilize the eggs internally in their brood pouch. Smaller relative testes investment was thus predicted for genera that have protected compared with unprotected brood care. However, we found this not to be the case. Instead, all genera showed the same relationship between testes and body mass, regardless of brooding type. The possible implications of this surprising result are discussed, including the possibility that the mode of fertilization might have been misjudged for the pouchless syngnathids.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 369–376.     

Alternative mating strategies in a desert grasshopper: a transitional analysis

We describe between- and within-individual variation in signalling behaviour for a population of male Ligurotettix coquilletti in a North American desert. For each observation date individual males were categorized as actively-signalling or inactive by the amount and regularity of their stridulation and their signalling location. Few males were active or inactive on all dates they were observed. By comparing the behaviour of individuals on consecutive observation dates, we identified the primary ways that individuals changed between active signalling and inactive behaviour. Behavioural transitions were frequently associated with movement between bushes. Inactive males became active upon moving to a vacant bush, and lone active males often became inactive after moving to a bush containing other males. Male-male aggression (primarily chases involving no contact between the participants, or only very brief contact) had a pronounced, short-term effect upon the signalling behaviour of males that received an attack. Approximately 33% of these recipients that had been stridulating became silent immediately following the chase. However, chases did not markedly alter the behaviour of recipients on the following observation date. Recipients were no more likely to change from inactive to active behaviour than vice versa between the observation dates immediately preceding and following the day of the chase. Although the transition analysis failed to detect marked behavioural changes among recipients, analyses based on longer periods of time revealed that males observed to be recipients were more likely to display inactive behaviour and less likely to mate than males observed to be initiators of aggression.     

Relationship Between Male Size and Newborn Size in the Seaweed Pipefish, Syngnathus Schlegeli

Relationships between male size, number, and weight of newborn were determined in the seaweed pipefish, Syngnathus schlegeli, with paternal care of embryos in the brood pouch. While the number of newborn increased linearly with the increase in male size, the pouch volume increased exponentially with the male size. This resulted in embryo density in the pouch negatively correlating with the male size. Newborn dry weight was negatively correlated with the embryo density in the pouch. Thus, larger males were considered to brood embryos at lower densities and give birth to heavier newborn. Neither paternal size nor embryo density seemed to have an effect upon developmental stage and total length of the newborn. Substantial weight loss of embryos during incubation indicated that paternal nutritional contribution to embryos is insignificant; therefore, the tendency of larger males to produce heavier newborn may not relate to allocation of paternal nourishment. Because egg weight increased with the female size, larger males are considered to receive the eggs of larger females and give birth to heavier newborn that developed from heavier eggs. Lower embryo density in larger males is attributable to the larger size of the heavier eggs. Assuming the pouch to be a cylindrical tube and the egg to be a sphere, geometrical calculations revealed that the relationship between the volumetric fraction of the pouch used by eggs (i.e. functional volume) and the male size was negatively quadric and had a peak. Increase in egg diameter increased the male size at which the functional volume of the pouch is maximized. Thus it appears that the size relationship of mates in pipefish is related to effective use of the pouch space.     

Alternative mating strategies in a desert grasshopper: evidence of density-dependence

Field observations revealed that, on a given day, male mating behaviour in a population of the grasshopper Ligurotettix coquilletti ranged from little or no stridulation (inactive) to relatively persistent singing (active signalling). Inactive males were usually located in the territories of active signallers. Actively signalling male achieved more matings, and also more frequently approached and mounted females in incidents terminating in the male departing the female without copulating. Individuals switched between inactive and actively-signalling behaviour during their adult lives, and males that were usually active signallers achieved greater lifetime mating success. Thus, we obtained no evidence to support the hypothesis that variation in mating behaviour was maintained by negative frequency-dependent selection. By comparing the behaviour of males in two field plots maintained at different population densities, however, we found that high density was directly related to a higher incidence of inactive behaviour. Males perched on creosote (Larrea tridentata) bushes (their host plant), and certain bushes. Mating behaviour was independent of body size and, while young (<7 days old) males tended to be inactive, neither age nor the time of adult maturation during the season could fully account for the incidence of inactive behaviour among males. Instead, we suggest that the adoption of inactive behaviour resulted partly from aggressive encounters between males.     

Female mate choice is not affected by mate condition in a fish with male care

Several studies have shown that mate choice based on condition leads to higher reproductive success of the choosing individual. Yet, a growing body of literature has failed to find support for mate choice based on mate condition, even when the choosing individual would clearly benefit from such a choice. This indicates that animals’ mate choice is often more complex than currently appreciated and that even well-founded expected preferences cannot be taken for granted. Using the broad-nosed pipefish, Syngnathus typhle, we manipulated male condition experimentally to explore whether it affects female mate choice. In this sex-role-reversed species, males care for the offspring in a specialised brood pouch. Males are the choosier sex, but given the opportunity, females are selective as well. During brooding, males can both provide embryos with nutrients and take up nutrients that originate from eggs deposited in the pouch, and embryo survival correlates positively with male condition. Together, this suggests that it would be beneficial for females to mate with males in high condition. However, we found no female preference for males in better condition. Thus, this study adds to the literature of mate choice that is unaffected by mate condition. Possible reasons for our result are discussed.     

Evidence of paternal nutrient provisioning to embryos in broad-nosed pipefish Syngnathus typhle

In two experiments, radioactively labelled nutrients (either (3)H-labelled amino-acid mixture or (14)C-labelled glucose) were tube-fed to brooding male Syngnathus typhle. Both nutrients were taken up by the males and radioactivity generally increased in the brood pouch tissue with time. Furthermore, a low but significant increase of (3)H-labelled amino acids in embryos was found over the experimental interval (48 h), whereas in the (14)C-glucose experiment the radioactivity was taken up by the embryos but did not increase over the experimental time (320 min). Uptake of radioisotopes per embryo did not differ with embryo size. A higher uptake mg(-1) tissue of both (3)H-labelled amino acids and (14)C-labelled glucose was found in smaller embryos, possibly due to a higher relative metabolic rate or to a higher surface-area-to-volume ratio compared to larger embryos. Uptake in embryos was not influenced by male size, embryonic developmental advancement or position in the brood pouch. It is concluded that brooding males provide amino acids, and probably also glucose, to the developing embryos in the brood pouch.     

Repeatable nest defense behavior in a wild population of Eastern bluebirds (Sialia sialis) as evidence of personality

Consistent individual differences in behavior, or personality, have been demonstrated in a variety of species other than humans, including mammals, birds, and invertebrates. Behavioral consistency has been shown to affect dispersal, foraging, exploration, and antipredator responses, which may have an impact on parental and offspring survival. Despite increasing research in behavioral consistency, the repeatability of nest defense behavior has rarely been assessed in wild bird populations. Furthermore, previous studies investigating nest defense behavior have utilized laboratory studies or mounted predators to elicit defensive behavior. It is important to assess personality in wild populations to fully understand the fitness consequences of behavioral consistency across natural contexts and to utilize live predators or competitors for accurate assessment of defensive behavior. We used an ecologically relevant, live, invasive, nest site competitor, the house sparrow (Passer domesticus), to elicit nest defense behavior in a wild population of Eastern bluebirds (Sialia sialis) to determine if nest defense behavior is repeatable within and between years for males and females (males: 2009, N?=?17; 2010, N?=?18; both years, N?=?9. Females: 2009, N?=?22; 2010, N?=?15; both years, N?=?11). We also determined if individual behavior changes as a function of season, parental age, brood size, or the number of house sparrows around the nest site. We found that females demonstrated repeatable behavior both within and between years. Male nest defense behavior was only repeatable in 1 year and was influenced by season in the other year. Parental age, brood size, and the number of house sparrows around the nest site did not affect nest defense behavior. We conclude that Eastern bluebirds demonstrate consistent nest defense behavior, or personality, although males are more plastic than females.     

The Effects of Food Limitation on Life History Tradeoffs in Pregnant Male Gulf Pipefish

Syngnathid fishes (pipefishes, seahorses and seadragons) are characterized by a unique mode of paternal care in which embryos develop on or in the male’s body, often within a structure known as a brood pouch. Evidence suggests that this pouch plays a role in mediating postcopulatory sexual selection and that males have some control over the events occurring within the pouch during the pregnancy. These observations lead to the prediction that males should invest differently in broods depending on the availability of food. Here, we use the Gulf pipefish to test this prediction by monitoring growth rate and offspring survivorship during the pregnancies of males under low- or high-food conditions. Our results show that pregnant males grow less rapidly on average than non-pregnant males, and pregnant males under low-food conditions grow less than pregnant males under high-food conditions. Offspring survivorship, on the other hand, does not differ between food treatments, suggesting that male Gulf pipefish sacrifice investment in somatic growth, and thus indirectly sacrifice future reproduction, in favor of current reproduction. However, a positive relationship between number of failed eggs and male growth rate in our low-food treatments suggests that undeveloped eggs reduce the pregnancy’s overall cost to the male compared to broods containing only viable offspring.     

Morphological and quantitative changes in paternal brood‐pouch vasculature during embryonic development in two Syngnathus pipefishes

Vascular corrosion casts of Syngnathus floridae and Syngnathus fuscus brood pouches were examined by scanning electron microscopy. Morphological and quantitative data on the vasculature of the paternal brood pouch during each stage of embryonic development were investigated to explore potential changes during brooding, to consider interspecific differences and to provide structural evidence for previously reported functional roles of the brood pouch. The brood pouches of both species are highly vascularized structures with cup‐like arrangements of brood‐pouch vasculature developing around each embryo shortly after fertilization and breaking down before fry release. The density and size of paternally derived blood vessels in contact with the embryos were found to be consistent for S. fuscus once this structure was established early in development. On the contrary, these vasculature measurements varied with early S. floridae brood stages when the embryo still relied heavily on the yolk sac. Diameter measurements of S. fuscus brood‐pouch blood vessels were also comparatively smaller during these early developmental stages, suggesting that the structural stability and opportunity for greater transport via slower blood flow may contribute to greater paternal allocation. This is the first study to document changes in brood‐pouch vasculature during specific stages of embryonic development, to show regression of this vasculature before fry release and to provide morphological data for two syngnathid species for which information on brood‐pouch physiology is available.     

Monogamous pair bonds and mate switching in the Western Australian seahorse Hippocampus subelongatus

Apparently monogamous animals often prove, upon genetic inspection, to mate polygamously. Seahorse males provide care in a brood pouch. An earlier genetic study of the Western Australian seahorse demonstrated that males mate with only one female for each particular brood. Here we investigate whether males remain monogamous in sequential pregnancies during a breeding season. In a natural population we tagged males and sampled young from two successive broods of 14 males. Microsatellite analyses of parentage revealed that eight males re‐mated with the same female, and six with a new female. Thus, in this first study to document long‐term genetic monogamy in a seahorse, we show that switches of mates still occur. Polygynous males moved greater distances between broods, and tended to have longer interbrood intervals, than monogamous males, suggesting substantial costs associated with the breaking of pair bonds which may explain the high degree of social monogamy in this fish genus.     

Altered brain metabolism of testosterone is correlated with reproductive decline in aging quail

In aging quail, an increasing proportion of males show no sexual behavior. A decrease in the mean size of the tests, cloacal gland, and sternotracheal muscles is also observed. In both sexually active and inactive males, plasma testosterone decreases with age but more so in inactive birds. The behavioral and morphological changes observed during aging are correlated with shifts in the intracellular testosterone metabolism resulting in a change in the ratio of active versus inactive metabolites. In the hypothalamus there is a steady decrease with age of 5 beta-reductase activity in all birds and an increase in 5 alpha-reductase activity only in the birds which remain sexually active. In the cloacal gland, the 5 beta-reductase activity markedly increases with age but more so in the birds which become sexually inactive. These data support the notion that the effects of testosterone are controlled by enzymatic shifts which could modulate the sensitivity to the hormone at the cellular level.     

Possible antimicrobial compounds from the pouch of the koala, Phascolarctos cinereus

The antimicrobial activity of secretions of the pouch of the koala, Phascolarctos cinereus has been documented in thepresence and absence of young. These secretions have been analysed by polyacrylamide gel electrophoresis, high performanceliquid chromatography and mass spectrometry (MS) including time of flight (TOF), MS-MS and Edman sequencing. Gel electrophoresis of reduced samples revealed the presence of a four polypeptides in active and inactive secretions. The sequence data obtained from all four do not appear to be either defensin or cathelicidin-like peptides and show little homology with knownantimicrobial protein/peptide sequence available on public accessdatabases. It is proposed that marsupials regulate microbialpopulations in the pouch via cleavage of large, inactive molecules to produce small active peptides.     

Possible antimicrobial compounds from the pouch of the koala,Phascolarctos cinereus

Summary The antimicrobial activity of secretions of the pouch of the koala,Phaseolarctos cinereus has been documented in the presence and absence of young. These secretions have been analysed by polyacrylamide gel electrophoresis, high performance liquid chromatography and mass spectrometry (MS) including time of flight (TOF), MS-MS and Edman sequencing. Gel electrophoresis of reduced samples revealed the presence of a four polypeptides in active and inactive secretions. The sequence data obtained from all four do not appear to be either defensin or cathelicidin-like peptides and show little homology with known antimicrobial protein/peptide sequence available on public access databases. It is proposed that marsupials regulate microbial populations in the pouch via cleavage of large, inactive molecules to produce small active peptides.