The Ultrastructure of Brood Pouch Formation in Tokophrya infusionum

SYNOPSIS Asexual reproduction in Tokophrya infusionum is by internal budding, whereby a ciliated, motile embryo is formed inside the sessile, non-ciliated parent in a specialized structure, the brood pouch. The process of embryogenesis and brood pouch formation was studied with the electron microscope using synchronized cultures. Reproduction begins with invagination of the pellicle and plasma membrane in the apical region of the adult. Early invagination is characterized by the presence of numerous microtubules beneath the plasma membrane or epiplasmic layer of the invaginating membranes. These microtubules apparently are important in formation of the brood pouch for colchicine blocks embryogenesis during the early stages. When the embryo is completed, it is ejected from the brood pouch thru the birth pore, an opening which is the site of the initial invagination and is present thruout embryogenesis. Theories of brood pouch formation are reviewed and discussed in light of the present investigation.     

Viviparous development in Botrylloides (Compound Ascidians)

The mode of sexual reproduction and embryogenesis was compared in 3 species of Botrylloides: B. simodensis, B. lenis, and B. violaceus. In all species, a testis and an egg (occasionally 2 eggs), the former being anterior to the latter, mature in the mantle on either side of a zooid. The egg is surrounded by 2 follicular layers and is attached by a vesicular follicle stalk (oviduct) to the atrial brood pouch. The egg is ovulated into the brood pouch, where it is fertilized and undergoes embryogenesis. The egg of B. simodensis is heavily yolked and measures about 180 μm in diameter. The course of embryogenesis in this species is that typical of ascidians. A mature tadpole larva is produced and shed in about 5 days; then, the mother zooid degenerates. The larva is smallest of the three species and has 8 ampullae. The metamorphosed oozooid bears a single bud on the right side only. Extraembryonic nutrition seems to be very limited. Both Botrylloides lenis and B. violaceus are species which display extreme examples of viviparity. Their eggs are devoid of yolk granules, measuring about 90 μm in diameter in the former species and 60 μm in the latter. The course of embryogenesis is similar in these 2 species. The neurula stage is characterized by a spherical vesicular shape owing to precocious differentiation of the embryonic pharynx, whose ectoderm becomes vacuolated. At the posterior end of the neurula, the mesodermal cells are located in a mass, from which the tail is extended later. In B. lenis, embryogenesis takes about 20 days. At the neurula stage of the embryo, the mother zooid becomes a mantle sac as a result of visceral disintegration. During further embryogenesis, the growth of buds of successive generations in the colony is characteristically arrested. A swimming larva of this species is somewhat larger than that of B. simodensis. It has 14–24 ampullae, and the oozooid carries a single bud on its right side. In B. violaceus, the gestation period lasts for more than a month. At the early gastrula stage of the embryo, the body of the mother zooid fully disintegrates. Only the brood pouch bearing the embryo survives and remains connected with the colonial vascular system. In this species, sexual reproduction does not affect the growth of buds in the colony. The swimming larva is gigantic, being furnished with 24–34 ampullae, and the oozooid always bears 3 buds, 2 on the right side and one on the left side.     

Ovoviviparity and the structure of the brood pouch in Melanoides tuberculata (Gastropoda: Prosobranchia: Thiaridae)

The freshwater gastropod Melanoides tuberculata broods its young in a pouch located in the anterodorsal region of the head-foot. The wall of the brood pouch is composed of smooth muscle surrounded by connective tissue. The lumen of the brood pouch is incompletely partitioned by trabeculae, formed by extensions or folds in the chamber wall that are composed of smooth muscle, connective tissue, nonciliated squamous epithelial cells, and some storage cells containing lipid and glycogen. The lumen of the chamber also contains a few cells with storage products. The general absence of secretory cells suggests that embryos derive little nutrition from the mother, and therefore embryonic development is probably ovoviviparous. Embryos in various stages of development were found within brood pouches, with later stage embryos varying in size. There was a negative relationship between embryo size and number of embryos in the brood pouch.     

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.     

The Permanence of the Infraciliature in Suctoria: An Electronmicroscopic Study of Pattern Formation in Tokophrya infusionum*

SYNOPSIS. The adult Tokophrya infusionum does not possess cilia, but has 20–30 barren basal bodies arranged in 6 short rows adjacent to the contractile vacuole pore. During reproduction, which is by internal budding, the contractile vacuole sinks into the parent along with the invaginating membranes that form the embryo and the wall of the brood pouch. The 6 rows of basal bodies radiate away from the pore and elongate to form 5 long ciliary rows, that encircle the anterior half of the embryo, and 1 short row at the posterior end. The contractile vacuole pore, along with several barren basal bodies, remains in the parent when the embryo is completed. The pore rises to the surface when the embryo is born. New basal bodies are then formed in the parent to replace those which were incorporated into the embryo, and formation of another embryo may begin. The cilia of the embryo are partially resorbed 10 min after the start of metamorphosis, with depolymerization of the ciliary microtubules. Later, the cilia and most of the basal bodies disappear completely, except for a group of barren basal bodies near the embryo's contractile vacuole pore, which form 6 rows and serve as an anlage for the basal bodies and cilia that arise during embryogenesis. There is, therefore, an organized infraciliature in Suctoria throughout their life cycle, and a distinct continuity of basal bodies across the generations.     

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.     

Study of the late embryogenesis of Daphnia (Anomopoda, ‘Cladocera’, Branchiopoda) and a comparison of development in Anomopoda and Ctenopoda

The embryonic development of Daphnia galeata and D. hyalina (`Cladocera', Anomopoda, Daphniidae) has been investigated by observing living embryos removed from female brood pouches. The sequence of morphological changes was analysed, as was the time at which the activity of certain organs began. The timing of these events at 22 °C is documented for both species.These data were compared with similar information, previously obtained for two representatives of the Ctenopoda (Kotov & Boikova, 1998). The sequence of events is basically similar in the two groups during early and late phases of their development, but the time of shedding of the embryonic membranes is different in the Anomopoda and Ctenopoda. The ctenopod embryo hatching from the second egg membrane is covered by the third membrane, which will be cast some hours later. The anomopod embryo hatches from the second egg membrane approximately simultaneously with the shedding of the third membrane, and it is covered already by the fourth membrane after the shedding of the second egg membrane.Earlier (Kotov & Boikova, 1998), we determined four embryonic instars in the course of the development of the Ctenopoda. Two of them are passed within the egg membranes, the next two instars occur after the shedding the egg membranes within the mother's brood pouch. However, in anomopods, one of the latter (the third) occurs within the second egg membrane, one is incorporated into the egg. Thus, the development of the Anomopoda is more embryonized in comparison with that of the Ctenopoda.     

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.     

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     

Brooding and embryonic development in the crustacean Paragnathia formica (Hesse, 1864) (Peracarida: Isopoda: Gnathiidae)

The crustacean family Gnathiidae Leach, 1814 (Peracarida: Isopoda) comprises 12 genera known mostly from marine environments. Juvenile gnathiid isopods are fish ectoparasites, feeding on blood and tissue fluids in order to complete their life cycles. Gnathiid juvenile development generally includes three moults, the last involving metamorphosis to non-feeding, adult stages. The blood meal ingested by juveniles provides resources for adult survival, reproduction and embryological development. Reproductive biology in the brackish water gnathiid, Paragnathia formica (Hesse, 1864), is unusual amongst crustaceans, since brooding females have paired internal uterine sacs, rather than an external brood pouch. Known embryological development for P. formica includes three post gastrulation stages. In the current study, brooding and embryological development in this gnathiid were reexamined using histological and fluorescence methods, and by scanning electron microscopy. Novel observations were made of the blastodisc and germ cell migration within developing eggs, release of Stage 2 embryos by rupture of embryonic membranes, the in utero moult of Stage 2 to Stage 3 embryos, and the asynchronous development of the brood within the paired uterine sacs. These findings highlight the remarkable nature of brooding in P. formica and expand the paucity of knowledge of embryological development in gnathiids in general.     

How much should reproduction cost?

The ultimate goal of investigating costs of reproduction isto see whether evolutionary tradeoffs are important determinantsof observed variation in fecundity. However, the current empiricalapproach to studying costs of reproduction, manipulation ofbrood size, is only capable of demonstrating the existence ofa cost of reproduction. Little attention has been paid to thebiological significance of a cost of reproduction, when oneis found. In this paper, I outline an analytical framework thatcan be used in conjunction with brood manipulation experimentsto determine whether an observed cost of reproduction is capableof limiting clutch sizes at observed levels. In addition, thisframework can be used to determine whether patterns of variationin fecundity within a population are caused by evolutionarytrade-offs between present and future reproduction. Two patternsused as examples are increase in clutch size with female ageand intraseasonal decline in clutch size in birds. Because increasingbrood size can have several effects on adults (e.g., decreasedadult survival, decreased future fecundity of surviving adults,decreased care given to offspring in the enlarged brood), thereis a need to understand how all these effects are interrelated.The analytical framework outlined in this paper allows one toexpress a cost of reproduction (i.e., a decrease in future fecundityof parents) and decreases in the rate of survival of offspringfrom the current nesting attempt in a common "currency." Thispaper also suggests how brood manipulation experiments can distinguishbetween variation in clutch size resulting from life-historytrade-offs and variation that results from differences in parentalability or territory quality. The analytical methods can bereadily applied to other taxa.     

The effect of social context and reproductive status on the metabolic rates of dwarf seahorses (Hippocampus zosterae).

Although we are beginning to understand the conditions in which monogamy is favored over a more promiscuous lifestyle, little is known about the proximate effects of monogamous pair bonding, and subsequent reproduction, on general metabolism. I determined the effects of these factors on the metabolic rates of dwarf seahorses (Hippocampus zosterae), recognized for their monogamous lifestyle and unique male care of offspring, in a sealed brood pouch where embryos develop until birth. Resting routine oxygen consumption rates were measured in newly-paired and reproductive adults using a continuous flow respirometer, and then compared to metabolic rates of sexually-isolated fish. Sex differences were observed in the relationship between log(10)mass and log(10)oxygen consumption, with pair-bonded females exhibiting a significantly higher slope than either pair-bonded males, or sexually-isolated fish. Mass-specific metabolic rates in sexually-isolated fish were 15% higher than in pair-bonded fish, indicating that social conditions can strongly influence metabolic rate. Specific metabolic rates only differed by gender during male pregnancy, when male metabolic rate increased from 10 to 52% over pre-gravid levels. A male's developing brood only explained 4-31% of this increase, suggesting that increased metabolic demands on fathers accounts for most of the increase in metabolic rate during gestation. This study suggests that pair bonding can strongly affect the general metabolism of organisms, with potential differences between males and females that increase with age.     

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     

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.     

Avoidance of Filial Cannibalism in the Amphipod Gammarus pulex

Adult animals that cannibalise juvenile conspecifics may gain energy but also risk filial cannibalism, that is, consumption of their own offspring. However, individuals vary in the magnitude of the costs and benefits of cannibalism depending on factors such as their current energy reserves or the probability that they have offspring in the vicinity. They may therefore also vary in the extent to which they participate in cannibalism. This study investigated whether the sex or brooding status of adult amphipods (Gammarus pulex) influenced whether they participated in cannibalism of juveniles. For females carrying embryos within their brood pouch, we also investigated two hypotheses to explain the presence or absence of cannibalistic behaviour by determining whether cannibalism was correlated with factors that might reflect energy demands (body length, brood size), or that might reflect a temporal change in cannibalistic behaviour (corresponding to stage of brood development). All reproductive classes of adults participated in some level of juvenile cannibalism, but females carrying offspring at an advanced stage of development (close to emergence from the brood pouch) consumed significantly fewer juveniles than other groups. Females thus appear to significantly reduce cannibalism of juveniles concurrent with the time when their own eggs are hatching within the brood pouch, prior to the release of their offspring. Because the experiment tested female responses to unfamiliar juveniles, this reflects a temporal change in behaviour rather than a response to phenotypic recognition cues, although additional direct recognition cannot be ruled out. Brooding females with large brood sizes or large body lengths, which might have disproportionately greater energetic demands, were not more likely to cannibalise juveniles. We also noted that juveniles that survived in trials where cannibalism occurred were significantly more likely to be found at the water surface, suggesting a possible adaptation to escape cannibalistic adults. Overall, our results provide evidence that amphipods use indirect temporal cues to avoid filial cannibalism.     

The morphology of the sphaeromatid brood pouch (Crustacea: Isopoda: Sphaeromatidae)

The structure of the brood pouch and mouthparts of ovigerous female sphaeromatids is described. Taxonomically, these are shown to be important generic characters and some attempt is made to relate structure to function. Expanded maxillipeds (which produce a current of water through the marsupium) are shown to occur only in genera which have oostegites overlapping in the mid-line and reaching the mouthparts anteriorly. The reduction of the mouthparts of some genera cannot be explained, but it may conserve energy at the final moult. No platybranchiate genera (subfamily Cassidininae) have the mouthparts reduced but some have the maxilliped expanded. Some correlation between brood pouch structure and habitat is suggested. Genera with a brood pouch composed only of oostegites (and hence liable to disruption by turbulence and particle abrasion) occur sublittorally or, if intertidal, adopt a protected, crevice dwelling habit. Genera with a brood pouch composed of large opposing pockets (which are more able to resist disruption) occur intertidally or in shallow water, and may be found in wave-washed sand. Some wood-boring species of the genus Sphaeroma Latreille show a reduced number of oostegites when compared with non-boring species. This presumably relates to their burrowing habit.     

Both endogenous and environmental factors affect embryo proliferation in the polyembryonic wasp Copidosoma floridanum

Copidosoma floridanum is a polyembryonic, parasitic wasp of the moth Trichoplusia ni. Following oviposition into a host, the C. floridanum egg initially undergoes complete (holoblastic) cleavage to form a single morula stage embryo. This embryo then undergoes a proliferation phase in which multiple, secondary morulae develop. C. floridanum has also evolved a caste system whereby some secondary morulae develop into soldier larvae whose function is defense whereas others develop into reproductive larvae that become adult wasps. In the current study, we conducted manipulative and candidate gene studies to identify factors affecting the proliferation phase of C. floridanum development. Transplantation of morulae of different ages into different host stages indicated that both embryo age and host environment affected the total number of offspring produced per morula. Morula age and brood size also significantly affected whether offspring of one or both castes were produced in a brood. In contrast, the host environment did not significantly affect caste formation. A putative homolog of the gene hedgehog (Cf-hh) was partially cloned from C. floridanum. In situ hybridization studies indicated that Cf-hh was expressed in secondary morulae during the proliferation phase of development, suggesting a possible role for the Hh signaling pathway in the evolution of polyembryony.     

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.     

Evolution of single-chick broods in the Swallow-tailed Gull Creagrus furcatus

Swallow-tailed Gulls lay single-egg clutches, and so raise single-chick broods. As they are pelagic seabirds, this small brood size is expected to relate to proximate food limitation owing to infrequent food deliveries. However, a previous brood doubling experiment detected an 82% increase in fledging success from experimentally doubled broods compared to controls. We repeated the brood doubling experiment, and found that none of 50 enlarged broods produced more than one independent offspring. Control and experimental parents produced fledglings of similar body size, which also had indistinguishable rates of fledging and subsequent survival and reproduction. A variety of parameters estimating survival and breeding costs of reproduction showed no treatment effect. Since two-chick broods yield dramatically higher fledging rates at some times, apparently without excess costs of reproduction, selection on brood size appears to favour a two-chick brood. However, selection may not favour a two-egg clutch if egg production is very costly. Additionally, our estimates of reproductive success do not incorporate the performance of experimental and control offspring as adults, which could differ, since growth of chicks differed slightly by treatment.