BULLETIN OF MARINE SCIENCE, 49(3): 741-747,1991 SOME QUESTIONS CONCERNING THE SYNGNATHIDAE BROOD POUCH Marie Y. Azzarello ABSTRACT For more than a century the physiological role of the Syngnathidae brood pouch has been the subject of scientific interest and debate. Some of the earliest investigators purported that the highly vascular brood pouch was physiologically adapted for the reception of fertilized eggs and for the sustenance of the embryos (i.e., a "pseudo-placenta"). Others posited that the brood pouch served as an osmoregulatory organ for the developing embryos. To determine whether the primary physiological role of the brood pouch is one of nutrition or osmoreg- ulation, Syngnathus scove/li embryos were removed from the brood pouch at different de- velopmental stages (4.0-13.0 mm TL), placed in sterilized, aerated, artificial seawater hyper- or iso-osmotic to the blood and pouch fluid, to which no nutritive substances were added. In hyperosmotic media 25.7% of the in vitro embryos completed their normal gestation versus 18.7% in iso-osmotic media. These results appear to indicate that the male Syng- nathidae brood pouch serves neither as the primary nutritional source nor as an osmotic buffer for the developing embryos after a length of 4.0 mm TL is achieved. Members of the Syngnathidae manifest atypical reproductive behavior and parental care. Reproduction is ovoviviparous with a complete reversal of the usual maternal and paternal brooding roles. Large telolecithal eggs produced by the female are fertilized by the male the moment they are deposited in his brood pouch. Embryos are then incubated throughout their entire gestation period, which varies depending upon genus and species, in the paternal brood pouch (Gill, 1905; Hubbs, 1943; Breder and Rosen, 1966). The physiological function of the brood pouch has been an area of much spec- ulation and investigation having involved the work of physiologists, embryolo- gists, anatomists, and histologists, for more than a century; its intriguing history is briefly described. Inasmuch as eggs and embryos of seahorses would not develop in seawater after their removal from the brood pouch, some of the earliest investigators concluded that the male provided a source of sustenance for the charges in his marsupial pouch. Lockwood (1867) observed that upon the male's reception of the eggs, the wall of the pouch became enriched internally with fat, and that upon birth the pouch hung flaccid becoming a thin membrane. Utilizing a sectioning technique, Huot (1902) demonstrated that each eggwas encompassed by epithelium perfused with a rich network of blood vessels supplying nourishment presumably by os- mOSIS. Gill's observations (1905) of the thickened, vascular, ovigerous pouch, led him to concur with earlier investigators that the male was not only physiologically adapted for the reception of eggs, but for their sustenance as well. Based solely on histological evidence, Gudger (1905) believed he had established evidence confirming the transfer of nutrients from the male pipefish's marsupium to its developing embryos. Thevenin (1936) noted considerable changes in the con- junctive tissue lining the pouch at the moment of conception. Capillaries multi- plied and enlarged causing the tissue to swell and become sponge-like. Within this "pseudo-placental" tissue the embryos remained quiescent until all their yolk was absorbed. Hudson and Hardy (1975) purported that the narrow end of the seahorse egg embeds in the wall of the brood pouch and that the transfer of nutrients takes place through this part of the egg. 741 742 BULLETIN OF MARINE SCIENCE, YOLo 49, NO.3, 1991 More recently, Haresign and Shumway (1980) introduced the use of a radio- active tracer to demonstrate the transfer of low molecular weight compounds across the epithelium of the male's brood pouch. They injected a non-metabolized radioactively labeled amino acid (alpha-amino isobutyric acid) into the marsu- pium of Syngnathus scovelli. Although their data indicated embryonic incorpo- ration of the labeled amino acid, these investigators were entremely cautious in drawing conclusions as to the permeability of the marsupium to nutrient exchange, having noted the likelihood of contamination with the methods they employed. The nutritive role was first questioned by Leiner (1934) who examined the osmotic pressure of the pouch fluid of Hippocampus brevirostris and H. guttulatus. He found that during early incubation, this pressure was similar to that of the male's blood while it approached that of seawater late in gestation. Leiner was also successful in getting a few embryos to develop partially in dilute seawater. An implication of these findings was that the male provided an optimum osmotic chamber for the developing young. The completely fused brood pouch of the seahorse, with its internal walls densely perfused with blood vessels, according to Nikolsky (1963), undoubtedly provided a means to supply oxygen to the developing embryos during gestation. Thirty years after Leiner's study, Linton and Soloff (1964) demonstrated that while the serum Na+ remained relatively constant, the Na+ ion concentration in the brood pouch fluid increased as the brood pouch epithelium actively trans- ported Na+. After parturition, the brood pouch Na+ concentration dropped back to blood Na+ levels. This low Na+ level represented the normal resting state signifying that the male was preparing for the reception of eggs. Calcium, on the other hand, being incorporated into the embryonic skeleton as well as the dermal exoskeleton, showed a decrease in concentration in the pouch fluid during ges- tation. This implied that the pouch epithelium may thus serve as a selective osmoregulatory organ protecting the embryos from a hyperosmotic environment by introducing them to higher salinities as their osmoregulatory systems (kidneys and gills) develop. Moreover, the fact that a small percentage of embryos survived to the stage of complete yolk absorption, after being removed from the brood pouch and introduced into 0.4 seawater (Linton and Soloff), seemed to preclude a dependence of the young on the male for nutrition. Spannhof and Bremer (1969), utilizing histophysiological techniques, noted functional changes in epithelial cells lining the brood pouch. They observed an increase in vascularization and that the epithelium consisted of thick cuboidal cells resembling cells having a secretory function. They concluded that the main function of the brood pouch was to ensure sufficient embryonal gas exchange. Kronester- Frei (1975) described the morphological changes in the brood pouch epithelium of Nerophis, a related genus, and concluded that the morphological prerequisites for transport of inorganic ions from the male's circulatory system to the embryos seemed to exist. In 1980, Quast and Howe investigated the osmotic role of the brood pouch in Syngnathus scovelli and determined that throughout incubation the pouch fluid osmolality was regulated near blood osmolality (370 (± 5) mOsm· kg-I). It was their surmise that the role of the brood pouch was to acclimate the embryos by regulating its osmolality during incubation to simulate that of the external en- vironment. In contrast to the proposed nutritional and osmoregulatory theories as de- scribed, Gross and Sargent (1985) and others examined the costs, benefits, and net advantage to both sexes during procreation. According to these two investi- gators, the one benefit incurred by parental care is increased survivorship of the AZZARELLO: SYNGNATHIDAE BROOD POUCH 743 young. However, three potential costs to care are incurred: a mating cost, a sur- vivorship cost, and a future fertility cost. Since only the male may lose spawnings by giving care, males alone have a potential mating cost. In general, the costs of care are probably less in males because male fertility is not a function of body growth. By contrast, fecundity accelerates with female size, which in turn increases the female's future fertility cost. Thus, male parental care is explained by these investigators, in terms of the male paying a smaller future cost relative to the female. Clearly, the scientific literature is divided as to the question of whether the role of the brood pouch is that of nutrition or ionic-osmotic regulation. The objectives of this study were two-fold: (I) to determine whether sustenance is the primary physiological function of the brood pouch, and (2) to determine whether hyper- and iso-osmotic incubating media have different effects upon the survival of embryos excised from the brood pouch. METHODOLOGY To test these objectives the following simplified procedure was devised: (I) gravid male Syngnathus scovelli were collected and kept in 38- and 76-liter aquaria filled with artificial seawater (30.60/00or 12.90/00at 24.5°C). Their diet consisted of newly hatched Anemia salina nauplii supplemented with zooplankton on alternate days; (2) pipe fishes were acclimated in their respective salinities for 24-96 h, depending upon the devel- opmental stage of the embryos at the time of collection; (3) a sterilized scalpel was used to separate the flaps and expose the fertilized ova (the membranous lateral flaps of the brood pouch meet medially but are unfused); (4) using a sterilized I" x 1/100 ml pipette, the embryos were suctioned out and transferred to a 400-ml jar containing a solution of sterilized, aerated, artificial seawater at the respective salinity of the incubating male; (5) the excised embryos were incubated at 24.5°C for the duration of the natural gestation as indicated by the embryos left intact in the male's marsupium; (6) the male was returned to the holding tank and observed until parturition took place. This procedure was repeated 22 times. When embryos were excised, those which did not have an intact yolk sac and whose survivability was highly improbable, were preserved in neutrally buffered formalin for the purpose of measuring the embryos at the time of removal. These embryos were not counted as part of the total number removed when determining percent survival.