Parent-offspring conflict and motivational control of brooding in an amphipod (Crustacea)

Models of parent-offspring conflict concerning levels of caregiving centre on conflict resolution by offspring control, compromise or offspring 'honest signalling' that parents use to maximize their own fitness. Recent empirical studies on motivational control of parental feeding of offspring are interpreted as supporting the latter model. Here, we examine parental care in an amphipod, Crangonyx pseudogracilis, which directs care to embryos in a brood pouch. Embryo removal and transplantation elucidated causal factors that determine levels of caregiving. In the short-term, females with all embryos removed reduced care activities, but partial embryo removal did not affect caregiving, evidence of 'unshared' parental care. In the long-term, females with all embryos removed ceased care. Thus, females have a maternal state that is maintained by stimuli from offspring. Transplantation of early/late stage embryos among females originally carrying early/late stage embryos revealed that stimuli from embryos indicate their age-dependent needs, but only modify caregiving within the constraints of a changing endogenous maternal state. Thus, we demonstrate that mothers and offspring share motivational control of care. However, we highlight the inappropriate use of motivational data in reaching conclusions about the resolution of parent-offspring conflict.     

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.     

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 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.     

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.     

Life history of the talitrid amphipod Macarorchestia remyi (Schellenberg, 1950) on a Tyrrhenian sandy beach, Italy

In this study, we analysed the life cycle and some aspects of the reproductive biology of the talitrid amphipod Macarorchestia remyi (Schellenberg, 1950) on a sandy beach in central Italy (Tyrrhenian Sea). The species is strongly associated with rotting wood carried by the sea. The animals were sampled once a month by hand over 1 year (May 2006–April 2007) and a total of 3,484 individuals were collected. Results showed the occurrence of intersexes in addition to males and females (n = 268) and a sex ratio always in favour of females. Breeding peaked twice during the year and the number of eggs in the ventral brood chamber ranged from 3 to 19. Linear regression analysis showed a positive correlation between total length of ovigerous females and the number of eggs they carried. The mean length of both mature males and mature females was 6 mm, while the maximum length was 13 mm. The presence of two cohorts during the year was also identified. Our results suggest that, despite the ephemeral habitats where the species lives, the population is stable over time and its life cycle does not differ in relation to other talitrid species that live on beaches.     

Is fatter fitter? Body storage and reproduction in ten populations of the freshwater amphipod Gammarus minus

Relationships between body storage (estimated as fat content and residuals of body mass regressed against body length) and offspring investment [brood mass, brood size (number of embryos per brood) and embryo mass] were examined within and among populations of the amphipod Gammarus minus in ten cold springs in central Pennsylvania, USA. Two major hypotheses and six corollary hypotheses were tested. Total reproductive investment (brood mass and brood size) was usually strongly positively correlated with maternal body length and body storage both within and among populations. These positive associations between reproductive and somatic investments are expected if individual variation in resource acquisition exceeds that of resource allocation. That is, individuals or populations that are able to acquire more resources should also be able to allocate more resources to both reproduction and somatic reserves than those acquiring fewer resources. This hypothesis is consistent with evidence showing that individual differences in body storage in G. minus and other amphipods are related to differences in resource acquisition. Positive associations between reproductive and somatic investments do not mean that energy costs of reproduction do not exist in G. minus. Evidence for reproductive energy costs included the lower body-fat contents of brooding versus nonbrooding females and the relatively low body mass per length of females who had just deposited eggs in their brood pouch. Unlike brood mass and brood size, individual embryo mass was usually unrelated to maternal body length and body storage. This pattern is largely consistent with optimal offspring investment theory, which predicts that offspring size should be insensitive to variation in parental resource status. However, in contrast to theory, embryo mass increased in winter when brooding females were significantly ”fatter”, presumably due to the availability of autumn-shed leaf food. This seasonal change in offspring size may be a maternally mediated effect of increased resource availability, though other explanations are possible. Overall, this study suggests that ”fatter” female amphipods are fitter than ”thinner” ones, though both the costs and benefits of increased body storage and brood size require investigation to substantiate this claim. This study also suggests that effects of individual variation in resource acquisition on life-history patterns deserve more theoretical and empirical attention by ecologists than they have received. It should be recognized that positive and/or nonsignificant correlations between life-history traits are just as interesting and important as are the negative correlations predicted by many theoretical models. Received: 20 January 1999 / Accepted: 26 September 1999     

Effects of egg removal on maternal aggression,biogenic amines,and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one’s offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48?h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

Reproductive behaviour of female Chorthippus biguttulus grasshoppers

Female grasshoppers of acoustically communicating species assume series of reproductive states that are associated with particular behaviours. Studies on laboratory populations of Chorthippus biguttulus (L.) revealed that females of this species lack the period of ‘passive copulatory readiness’, increase their attractiveness to males by sound production and mate multiple times before their first oviposition. In particular, female Ch. biguttulus display a period of ‘primary rejection’ after their imaginal moult during which they reject male mating attempts followed by a period of ‘active copulatory readiness’ in which they produce acoustic signals and may copulate with courting males. Female stridulation generally stimulated male mating activity and stridulating females attracted more male mating attempts than mute females in the same cage, indicating that males preferentially court females that signal ‘active copulatory readiness’. After receipt of a spermatophore, Ch. biguttulus females displayed periods of ‘secondary rejection’ followed by re-establishment of ‘active copulatory readiness’. Acoustic responses of females to male songs, an indicator of reproductive readiness, were significantly reduced until 2 days after mating and remained slightly reduced in comparison to pre-mating levels. Some females mated multiple times before their first oviposition and cycled between ‘secondary rejection’ and ‘active copulatory readiness’.     

Interspecific differences of parental polychlorinated biphenyl exposure on nutrient availability, egg production and brooding in two Syngnathus species

Parental nutrient contribution of two closely related, sympatric pipefish species was investigated following exposure to mid-range environmental concentrations of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (mean ± s . e . = 52·89 ± 2·12 μg l⁻¹). This study tested the hypothesis that differential nutrient supplementation during development predicts interspecific sensitivity to environmental stressors. Specifically, paternal PCB exposure was predicted to affect Syngnathus fuscus development because embryos depend on paternally derived nutrient sources in the brood pouch. Conversely, changes in Syngnathus floridae egg production were expected following maternal PCB treatment as a nurse-egg system generates the nutrient-rich pouch fluid. Lipid concentrations in adult blood plasma were significantly elevated following Aroclor 1254 treatment in S. floridae gravid females and post-brooding S. fuscus males ( P < 0·001) which suggested mobilization from body tissues in response to metabolic demands. Aroclor 1254 treatment also resulted in decreased protein concentrations in S. fuscus pouch fluid ( P < 0·05). These changes were not translated into significant exposure-related effects in the nutrient reserves of eggs and fry or fry size. Overall S. fuscus demonstrated a general sensitivity to disturbances from laboratory exposure conditions. Whereas a previous investigation of field-collected pipefishes showed comparable interspecific pouch fluid and fry nutrient concentrations and egg size, here, nutrient levels and egg diameter decreased in both laboratory-mated control and PCB-exposed S. fuscus . This study demonstrates the potential physiological effects of PCB exposure on parental nutrient allocation in syngnathids and the general sensitivity of S. fuscus to environmental disturbances.     

Effects of egg removal on maternal aggression, biogenic amines, and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one's offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48 h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

POLYGYNANDRY IN THE DUSKY PIPEFISH SYNGNATHUS FLORIDAE REVEALED BY MICROSATELLITE DNA MARKERS

In the dusky pipefish Syngnathus floridae, like other species in the family Syngnathidae, ‘pregnant’ males provide all post-zygotic care. Male pregnancy has interesting implications for sexual selection theory and the evolution of mating systems. Here, we employ microsatellite markers to describe the genetic mating system of S. floridae, compare the outcome with a previous report of genetic polyandry for the Gulf pipefish S. scovelli, and consider possible associations between the mating system and degree of sexual dimorphism in these species. Twenty-two pregnant male dusky pipefish from one locale in the northern Gulf of Mexico were analyzed genetically, together with subsamples of 42 embryos from each male's brood pouch. Adult females also were assayed. The genotypes observed in these samples document that cuckoldry by males did not occur; males often receive eggs from multiple females during the course of a pregnancy (six males had one mate each, 13 had two mates, and three had three mates); embryos from different females are segregated spatially within a male's brood pouch; and a female's clutch of eggs often is divided among more than one male. Thus, the genetic mating system of the dusky pipefish is best described as polygynandrous. The genetic results for S. floridae and S. scovelli are consistent with a simple model of sexual selection which predicts that for sex role-reversed organisms, species with greater degrees of sexual dimorphism are more highly polyandrous.     

Effect of Acanthocephala infection on the reproductive potential of crustacean intermediate hosts

The effect of a naturally acquired infection by three acanthocephalan parasites Dentitruncus truttae, Echinorhynchus truttae, and Polymorphus minutus on the reproductive potential of their intermediate host, Echinogammarus tibaldii (Amphipoda) from Lake Piediluco (Centre of Italy) was assessed. During May 2007, 1135 amphipods were collected from two different samplings and examined for larval helminths. Forty-five amphipods were infected and of those, 16 were infected with D. truttae (intensity = 1-3 larvae), 15 with E. truttae (intensity = 1-2 larvae), and 14 with P. minutus (intensity = 1 larva). The sex ratio was nearly 1:1 in all examined amphipods. One female infected with D. truttae contained six eggs in the brood pouch and another female infected with E. truttae contained five eggs. However, none of the eight female amphipods harbouring P. minutus larva contained eggs in their brood pouch. Uninfected females of the same size and body length as that of the infected females contained between 20 and 32 eggs. No acanthocephalan species were found to co-occur.     

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.     

Adjusting parental investment to changing environmental conditions: the effect of food ration on parental behaviour of the convict cichlid, Cichlasoma nigrofasciatum

Animals that care for their offspring may vary the amount of care provided for a particular brood in relation to environmental conditions. Food availablity is one factor that may affect the costs and benefits associated with parental investment. The convict cichlid, Cichlasoma nigrofasciatum, is a small, substrate-spawning cichlid from Central America. Both male and female provide parental care for eggs and fry. Paris were kept at one of three ration levels, high, medium or low. Time spent in parental fanning by females was positively related to ration. Males spent less time fanning than females and their parental behaviour varied considerably between individuals. Males on the high ration spent slightly more time fanning than those on the lower rations. The number of eggs produced per spawning and the post-spawning weight of both males and females were significantly and positively related to ration. Foraging frequency of both males and females was inversely related to ration. There was no significant effect of ration on the frequency of mouthing eggs and young or on intra-pair aggression. Eggs of low-ration fish hatched earlier than those of medium- and high-ration fish but there was no significant difference in the number of days that the young survived. These results suggest that the allocation of time and effort between parental and maintenance activities differs in relation to food supply. Parents may provide more care for the large brood produced when food is plentiful but place more emphasis on their own survival when food is short and broods are small.     

Reproductive state and care giving in Stegodyphus (Araneae: Eresidae) and the implications for the evolution of sociality

Cooperative brood care is a rare phenomenon in spiders and is restricted to a few social species, including three in the genus Stegodyphus. Brood care in Stegodyphus begins with regurgitation feeding followed by matriphagy: the young consume the body fluids of their mother causing her to die quickly. Whether such an extreme form of maternal care can become a communal task should depend on physiological or historical preconditions. I investigated whether femaleStegodyphus lineatus feed young or allow matriphagy according to their own reproductive state. Broods of young of two age classes (2 or 10 days after hatching) were isolated or fostered out to adult females that were unmated, had eggs or had young. Growth and survival of females and broods were followed over 21 days. The timing of matriphagy depended on the interaction between age of young and state of the foster mother. All broods that were fostered out to females with young grew and survived. Two-day-old young did not survive when isolated or fostered out to unmated females, but some survived and gained weight when placed with foster mothers that cared for egg sacs. Young of 10 days of age grew when fostered out to females with eggs but did not grow or lost weight when isolated or fostered out to unmated females. Survival among 10-day spiderlings was relatively high in all groups but differed significantly between treatments (young isolated or fostered out to unmated females or females with eggs) and control (left with the mother). The results show that these spiders will care for young from other females only when they are in the right developmental state. Such a constraint can have important consequences for the evolution of allomaternal care in social species: unless such a mechanism is overcome, nonreproductives cannot help in brood care. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Patterns in rotifer diapausing egg banks: Density and viability

Here we present a quantitative study on the density, age and viability of the diapausing egg banks of the rotifer species complex Brachionus plicatilis in the sediments of 15 water bodies from Eastern Spain. Sampled ponds, located in coastal and inland areas, varied in salinity and ranged in size, depth and permanence. By identifying ‘hatched’, ‘deteriorated’ and ‘viable’ diapausing eggs in the sediment samples, we estimated production, hatching and deterioration in relation to the habitat properties of each pond. Our results indicate the presence of large numbers of diapausing eggs in the sediments of almost all of the ponds studied (2-115 eggs cm^− 2). Inland ponds tended to have higher densities than coastal lagoons. The vertical distribution of eggs in the sediments frequently showed a non-decreasing pattern, which suggested a high among-year variation in egg production. Despite maximum age of eggs of 60-80 years, the median age (3-30 years) suggests that rotifer egg banks are young in the studied ponds. Egg senescence is suggested by the declining abundance of ‘healthy-looking’ eggs with depth. The proportion of ‘deteriorated’ eggs ranged 75-99% suggesting that deterioration rates in the sediments are high and vary among habitats. Hatching and deterioration rates, as estimated from the counts of ‘hatched’, ‘deteriorated’ and ‘healthy-looking’ eggs in the sediments, largely varied among ponds. An association between hatching and deterioration rates is suggested by our data. This is in agreement with the hypothesis that hatching rates of diapausing eggs depended not only on the risks associated with the water column, as initially expected by the general theory on diapause, but they are also related to the incidence of deterioration processes in the sediment.     

The postmarsupial development of Porcellio siculoccidentalis, with some data on reproductive biology (Crustacea, Isopoda, Oniscidea)

In the broader context of research on the Sicilian Porcellio imbutus-complex, the postmarsupial development of Porcellio siculoccidentalis Viglianisi, Lombardo & Caruso, 1992 was studied in detail. This research was conducted in the laboratory under controlled conditions, allowing us to follow the stages of development, from the formation of the marsupium in ovigerous females until the larval stages and development of the seventh pair of legs. The timing of developmental stages and the morphological modifications of appendages in the postmarsupial manca stages (M I-M III) are described. The manca stage M I had a duration of about one hour. Ovigerous females were collected and reared separately, and the number of parturial molts in the absence of males was counted. The results showed a maximum of four successive parturial molts. Fecundity and fertility were evaluated as the number of eggs and embryos, respectively, inside the marsupium of the ovigerous females. Both parameters were positively correlated with the size of the females. The maximum numbers of eggs and embryos in the marsupium were 113 and 141, respectively. Data describing the total number of postmarsupial mancas released per month indicated that the highest release occurred in April.     

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.