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.     

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.     

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     

Transport of naturally occurring amino acids and alpha-amino isobutyric acid by normal and diapausing mouse blastocysts

Uptake of ¹⁴C-labelled α-amino isobutyric acid and a mixture of naturally occurring amino acids by normal mouse preimplantation blastocysts was found to be twice that of delayed implantation blastocysts. With both groups of embryos concentration of the amino acids against a gradient occurred. The results may explain, in part, the lower amino acid incorporation rates found with diapausing vs. normal blastocysts and open the question of “metabolic dormancy” of the diapausing embryo.     

The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

The failure of a nutritionally balanced diet to ameliorate the impact of symbiont disruption in the pea aphid Acyrthosiphon pisum (Harris) was investigated using two approaches. The assimilation of dietary nutrients by aphids was investigated using chemically-defined diets containing ³ H-labelled inulin and ¹⁴C-labelled sucrose or amino acids. Symbiotic aphids (i.e., aphids containing their bacteria) had a high sucrose demand and assimilated 72% of sucrose ingested in the diet, whereas the assimilation of sucrose by aposymbiotic aphids (in which the bacteria had been disrupted), was significantly reduced to 47%. The assimilation of individual dietary amino acids by symbiotic aphids varied between 61 and 92%, and there was no impact on the feeding or assimilation rate when the aphids were fed a phloem sap-like diet containing a reduced amount of essential amino acids. Consequently, the absolute amount of each essential amino acid assimilated by symbiotic aphids feeding on a phloem sap-like diet was reduced by 36–59%. Aposymbiotic aphids consistently assimilated a lower proportion of ingested amino acids, and lysine in particular was poorly assimilated from the diet. In a second experiment, the allocation of free amino acids in the haemocoel to aphid embryos was investigated following microinjection of ¹⁴C-labelled amino acids. After 2 h, radiolabel could be detected at varying levels from the embryo complement of both symbiotic and aposymbiotic aphids, indicating rapid but selective uptake by the embryos. The essential amino acids phenylalanine and lysine were incorporated into the protein fraction of embryo tissues, but the rate of incorporation per unit biomass was approximately 4-fold higher in the embryos of aposymbiotic aphids, possibly reflecting increased demand due to the lack of amino acid provisioning from the symbiotic bacteria.     

Metabolism of l[U-14C]-arginine and l[U-14C]-ornithine in maturing and vernalised embryos and megagametophytes of Picea abies

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these 14 C-labelled amino acids into liberated CO₂, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.     

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.     

Ca2+-dependence of the evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Spontaneous and electrically evoked release of exogenous labelled amino acids and endogenous amino acids labelled from D-[U-14C]glucose were compared in control and Ca2+-free medium using guinea pig cerebral cortex slices. Spontaneous release of all labelled amino acids, except that of endogenous 14C-labelled threonine-serine-glutamine (unseparated) and exogenous [14C]aspartate, was doubled in Ca2+-free medium. The major portion of the electrically evoked release of endogenous [14C]glutamate, [14C]aspartate, gamma-amino[14C]butyrate (14C-labelled GABA) and exogenous 3H-labelled GABA was Ca2+-inpendent. More than half of the evoked release of the other labelled amino acids was Ca2+-independent. As the pattern of Ca2+-dependence of the evoked release concurred with the selectivity of the evoked release for endogenous [14C]-glutamate, [14C]aspartate, and 14C-labelled GABA, it was concluded that these labelled amino acids were probably released from the amino acid 'transmitter pool'.     

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.     

Effects of maternal and embryo characteristics on post-fertilization provisioning in fishes of the genus <Emphasis Type="Italic">Gambusia</Emphasis>

Maternal provisioning of embryos in Gambusia (Poeciliidae) entails both production of large, yolky eggs and mother-to-embryo transfer of nutrients, the latter of which is readily quantified using injection of radiolabeled nutrients. We assayed patterns of nutrient transfer in broods of 26 Gambusia geiseri and 23 Gambusia affinis females, using injection of tritiated leucine. We examined maternal and embryo characteristics affecting the instantaneous rate of transfer and characterized the pattern of transfer to individual embryos within broods. Maternal (female size and condition) and brood characteristics (mean embryo size, developmental stage, brood size) did not predict the mean level of nutrient transfer to embryos in a brood for either species. Within broods, individual provisioning of embryos was not related to developmental stage, but was related to embryo mass in G. affinis with nutrient transfer higher to larger embryos. In addition, overall within-brood variation in nutrient transfer, measured as coefficient of variation in embryo radioactivity, was higher in G. affinis than in G. geiseri.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

The genetic mating system and tests for cuckoldry in a pipefish species in which males fertilize eggs and brood offspring externally

Highly variable microsatellite loci were used to study the mating system of Nerophis ophidion, a species of pipefish in which pregnant males carry embryos on the outside of their body rather than in an enclosed brood pouch. Despite this mode of external fertilization and brooding, otherwise rare in the family Syngnathidae, the genotypes of all embryos proved to be consistent with paternity by the tending male, thus indicating that cuckoldry by sneaker males is rare or nonexistent in this species. N. ophidion is a phylogenetic outlier within the Syngnathidae and its reproductive morphology is thought to be close to the presumed ancestral condition for pipefishes and seahorses. Thus, our genetic results suggest that the evolutionary elaboration of the enclosed brood pouch elsewhere in the family was probably not in response to selection pressures on pregnant males to avoid fertilization thievery. With regard to maternity assignments, our genotypic data are consistent with behavioural observations indicating that females sometimes mate with more than one male during a breeding episode, and that each male carries eggs from a single female. Thus, the polyandrous genetic mating system in this species parallels the social mating system, and both are consistent with a more intense sexual selection operating on females, and the elaboration of secondary sexual characters in that gender.     

Direct evidence for embryonic uptake of paternally-derived nutrients in two pipefishes (Syngnathidae: <Emphasis Type="Italic">Syngnathus</Emphasis> spp.)

Seahorses, sea dragons and pipefishes of the teleost family Syngnathidae are unique in that embryos develop within specialized brooding structures of the male. We enriched brooding Syngnathus fuscus and Syngnathus floridae males with injections of L-lysine-[¹⁵N₂] and 16:0-palmitic acid 1-[¹³C] to demonstrate embryonic uptake of paternally-derived nutrients. While all embryos demonstrated amino acid enrichment, late stages showed significantly higher [¹⁵N], indicating greater utilization of paternal resources as yolk reserves diminished and embryonic energy demands increased. Limited embryonic [¹³C] uptake, defined as less than 10% of adult enrichment, in 75 and 81% of S. floridae and S. fuscus respectively signified rapid lipid metabolism and thus the need for greater enrichment. Interspecific differences in embryonic uptake of paternally-derived nutrients were not demonstrated. However, interspecific differences in egg nutrient reserves and fry size but comparable fry nutrient levels along with data from a published paternal exposure study indicate paternal transfer in S. fuscus most likely compensates for the comparative egg nutrient deficiency. This study is the first to our knowledge to provide direct evidence for the functional significance of the brood pouch in nutrient provisioning. These results add comparative information on the diversity of Syngnathid paternal care and further our understanding of paternal influence on development.     

Effects of mating order and male size on embryo survival in a pipefish

In species that provide parental care, individuals should invest adaptively in their offspring in relation to the pre‐ and post‐zygotic care provided by their partners. In the broad‐nosed pipefish, Syngnathus typhle L., females transfer large, nutrient‐rich eggs into the male brood pouch during mating. The male broods and nourishes the embryos for several weeks before independent juveniles emerge at parturition. Given a choice, females clearly prefer large partners. Yet, females provide protein‐richer eggs when the same individual mates with a smaller than a larger male. In the present study, we allowed each female to mate with one small and one large male, in alternated order. We found a strong effect of female mating order, with larger clutches and higher embryo mortality in first‐ than second‐laid broods, which may suggest that eggs over‐ripen in the ovaries or reflect the negative effects of high embryo density in the brood pouch. In either case, this effect should put constraints on the possibility of a female being selective in mate choice. We also found that small and large males produced embryos of similar size and survival, consistent with the reproductive compensation hypothesis, suggesting that, in this species, larger males provide better nourishment to the embryos than smaller males. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 639–645.     

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.     

Sexual size dimorphism predicts the frequency of multiple mating in the sex-role reversed pipefish Syngnathus typhle

The sex-role reversed pipefish Syngnathus typhle is a member of the Syngnathidae, a family of fishes in which males brood embryos on their body surface. As in most ectotherms, embryonic development is highly temperature dependent in syngnathids and male brooding periods are extended when water temperatures are reduced. The influence of temperature on reproduction is expected to effectively truncate the breeding season and reduce fecundity in cold waters, potentially enhancing the opportunity for both fecundity and sexual selection. We studied spatial variation in the morphology and reproductive biology of S. typhle in five European populations which vary in latitude and water temperature. Microsatellite analyses indicated that the average number of male mates per population ranged between 1.3 and 3.7. The frequency of multiple mating by males was negatively correlated with the degree of sexual size dimorphism in each population, suggesting that disproportionate increases in female fecundity may be able to compensate for increased male brood pouch capacity. Both sexes were larger and males had an increased brood size where water temperatures during the breeding season were lower. Morphological variation among populations may be mediated by differences in fecundity selection associated with different optimal reproductive strategies in cold and warm water environments.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Measurement of the rates of protein synthesis in rabbits. A method for the estimation of rates of change in the specific radioactivities of free amino acids during continuous infusions.

1. A method is described by which, from analysis of terminal samples, the rate constants that define the changes in specific radioactivity of free amino acids during continuous infusions can be estimated. The method involves the infusion of 3H-labelled and 14C-labelled forms of an amino acid for different, but overlapping, periods. 2. The procedure was developed for infusions of proline and tyrosine into New Zealand White rabbits and the rate constants were determined for blood and muscle. 3. The rate constant for equilibrium of radioactive free proline was much lower in muscle than in blood, and indicated that a plateau condition may not be attained in muscle by the end of a 6 h infusion. 4. Comparison of the ratio of areas under the curves of estimated specific radioactivity plotted versus time with the 3H/14C ratio of bound amino acid in muscle protein suggested that radioactive proline and tyrosine may be incorporated preferentially from an extracellular rather than an intracellular source.     

Evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Release of endogenous amino acids labelled via D-[U-14C]glucose was compared with that of several exogenous labelled amino acids using slices of guinea pig cerebral cortex. Electrical field stimulation evoked a selective release of endogenous [14C]glutamate, [14C]aspartate, and gamma-amino[14C]butyrate (14C-labelled GABA). The selectivity of release correlated well with 14C incorporation into endogenous amino acids. Calculations of the fraction of the tissue radioactivity released indicated that the selectivity was not an artifact due to differential incorporation. Because glucose in mammalian brain is metabolized almost entirely by the so-called 'large compartment', it is tentatively concluded that the releasable 'transmitter pool' of glutamate, aspartate, and GABA is located in this 'large compartment'.     

The morphology and independent origin of ovoviviparity in Tiphobia and Lavigeria (Caenogastropoda: Cerithioidea: Paludomidae) from Lake Tanganyika

To better understand the diversification of the endemic thalassoid (i.e. marine-like) cerithioidean gastropods of Lake Tanganyika, as well as the origin and significance of brooding among lake species, we here redescribe the anatomy and ontogeny of the ovoviviparous Tiphobia horei from Lake Tanganyika and compare it to that of Lavigeria sp. A, representing another ovoviviparous lake clade that has acquired a uterine brood pouch independently. Within the phylogenetic framework provided by recent molecular analyses, the distant relation of these two taxa is corroborated by many external and internal anatomical differences. Comparison of the brood pouches demonstrates that they each bear unique features consistent with their independent modification for brooding. Despite representing functionally analogous structures, they also share several similarities in organization likely representing symplesiomorphies of the Lake Tanganyika species flock. The ontogeny is characterized by the presence of a velum and by delayed calcification producing a characteristically wrinkled embryonic cap. Comparison with other brooding cerithioideans reveals that T. horei and Lavigeria sp. A retain many more embryos than other freshwater cerithioideans of comparable size with a uterine brood pouch, possibly facilitated by the presence of longitudinal lamellae. Compartmentalization of the oviduct and delayed calcification is strongly linked to the brooding of embryos.