Brood pouch evolution in pipefish and seahorse based on histological observation.

INTRODUCTION: Fishes of the Syngnathidae family are rare in having male pregnancy: males receive eggs from females and egg development occurs in the male brood pouch that diverged during evolution. The family is divided into two subfamilies: Nerophinae and Syngnathinae. METHODS: We compared histologically five types of the brood pouch in Syngnathinae: an open pouch without skinfolds (alligator pipefish); an open pouch with skinfolds (messmate pipefish); a closed pouch with skinfolds (seaweed pipefish); and closed pouches with a sac-like pouch on the tail (pot-bellied seahorse) or within a body cavity (Japanese pygmy seahorse). RESULTS: Histological observations revealed that all the examined species possess vascular egg compartments during the brooding period. The present immunohistochemical study revealed that the pregnant egg compartment epithelium grows thin in both open and closed pouches. The placenta of open and closed pouches is composed of dermis and reticulin fibers, respectively. The closed pouch placenta is a flexible and moist tissue, suitable for substance transport between the father and embryos through the epithelium and blood vessels and responsible for supplying nutrition and removing waste. DISCUSSION: These results suggest that the basic egg incubation structures were established at an early stage of Syngnathinae evolution. On the other hand, it is likely that the innovation of tissue structure, where dermis was replaced with reticular fibers, occurred in closed brood pouches to regulate the pregnant pouch environment. The present study presents the morphological evolutionary pathway of the brood pouch in Syngnathinae, providing a basis for further molecular-level evolutionary studies.

Evolved Bmp6 enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks.

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences in enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here, we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish, the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish, these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.

Structural changes to the brood pouch of male pregnant seahorses (Hippocampus abdominalis) facilitate exchange between father and embryos.

INTRODUCTION: Embryonic growth and development require efficient respiratory gas exchange. Internal incubation of developing young thus presents a significant physiological challenge, because respiratory gas diffusion to embryos is impeded by the additional barrier of parental tissue between the embryo and the environment. Therefore, live-bearing species exhibit a variety of adaptations facilitating respiratory gas exchange between the parent (usually the mother) and embryos. Syngnathid fishes are the only vertebrates to exhibit male pregnancy, allowing comparative studies of the biology and evolution of internal incubation of embryos, independent of the female reproductive tract. Here, we examine the fleshy, sealed, seahorse brood pouch, and provide the first quantification of structural changes to this gestational organ across pregnancy. METHODS: We used histological analysis and morphometrics to quantify the surface area for exchange across the brood pouch epithelium, and the structure of the vascular bed of the brood pouch. RESULTS: We show dramatic remodelling of gestational tissues as pregnancy progresses, including an increase in tortuosity of the gestational epithelium, an increase in capillary density, and a decrease in diffusion distance between capillaries and the pouch lumen. DISCUSSION: These changes produce an increased surface area and expansion of the vascular bed of the placenta that likely facilitates respiratory gas exchange. These changes mirror the remodelling of gestational tissue in viviparous amniotes and elasmobranchs, and provide further evidence of the convergence of adaptations to support pregnancy in live-bearing animals.

Subtle effects of radiation on embryo development of the 3-spined stickleback.

The Chernobyl and Fukushima nuclear power plant (NPP) accidents that occurred in 1986 and 2011 respectively have led to many years of chronic radiation exposure of wildlife. However, controversies remain on the dose threshold above which an impact on animal health occurs. Fish have been highly exposed immediately after both accidents in freshwater systems around Chernobyl and in freshwater and marine systems around Fukushima. The dose levels decreased during the years after the accidents, however, little is known about the effects of environmental low doses of radiation on fish health. The present laboratory study assesses the effects of an environmentally relevant dose range of radiation (0.1, 1 and 10 mGy/day) on early life stages of the 3-spined stickleback, Gasterosteus aculeatus. The cardiac physiology and developmental features (head width, diameter, area) of high exposed embryos (10 mGy/day) showed no significant change when compared to controls. Embryos exposed to the medium and high dose were slower to hatch than the controls (between 166 and 195 h post-fertilization). After 10 days of exposure (at 240 h post-fertilization), larvae exposed to the high dose displayed comparable growth to controls. High-throughput sequence analysis of transcriptional changes at this time point revealed no significant changes in gene regulation compared to controls regardless of exposure conditions. Our results suggest that exposure of fish embryos to environmental radiation elicits subtle delays in hatching times, but does not impair the overall growth and physiology, nor the gene expression patterns in the recently hatched larvae.

Visualizing primordial germ cell migration in embryos of rice field eel (Monopterus albus) using fluorescent protein tagged 3' untranslated regions of nanos3, dead end and vasa.

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.

Unravelling paralogous gene expression dynamics during three-spined stickleback embryogenesis.

Development requires the implementation of a plethora of molecular mechanisms, involving a large set of genes to ensure proper cell differentiation, morphogenesis of tissues and organs as well as the growth of the organism. Genome duplication and resulting paralogs are considered to provide the raw genetic materials important for new adaptation opportunities and boosting evolutionary innovation. The present study investigated paralogous genes, involved in three-spined stickleback (Gasterosteus aculeatus) development. Therefore, the transcriptomes of five early stages comprising developmental leaps were explored. Obtained expression profiles reflected the embryo's needs at different stages. Early stages, such as the morula stage comprised transcripts mainly involved in energy requirements while later stages were mostly associated with GO terms relevant to organ development and morphogenesis. The generated transcriptome profiles were further explored for differential expression of known and new paralogous genes. Special attention was given to hox genes, with hoxa13a being of particular interest and to pigmentation genes where itgb1, involved in the melanophore development, displayed a complementary expression pattern throughout studied stages. Knowledge obtained by untangling specific paralogous gene functions during development might not only significantly contribute to the understanding of teleost ontogenesis but might also shed light on paralogous gene evolution.

Cryptic haplotype-specific gamete selection yields offspring with optimal MHC immune genes.

Females choose specific mates in order to produce fitter offspring. However, several factors interfere with females' control over fertilization of their eggs, including sneaker males and phenotypically unpredictable allele segregation during meiosis. Mate choice at the individual level thus provides only a poor approximation for obtaining the best genetic match. Consequently, postcopulatory sperm selection by female oocytes has been proposed as a mechanism to achieve complementary combinations of parental haplotypes. Here, using controlled in vitro fertilization of three-spined stickleback eggs, we find haplotype-specific fertilization bias toward gametes with complementary major histocompatibility complex (MHC) immunogenes. The resulting zygote (and thus offspring) genotypes exhibit an intermediate level of individual MHC diversity that was previously shown to confer highest pathogen resistance. Our finding of haplotype-specific gamete selection thus represents an intriguing mechanism for fine-tuned optimization of the offspring's immune gene composition and an evolutionary advantage in the Red Queen dynamics of host-parasite coevolution.

Early development of the longsnout seahorse Hippocampus reidi (Syngnathidae) within the male brood pouch.

Fertilized and unfertilized eggs and embryos of the longsnout seahorse Hippocampus reidi were collected at different stages of development and provided the basis for a description of morphological development from fertilization until release from the paternal pouch. Images of fertilized eggs, as well as their rupture after a few minutes in seawater are reported for the first time. The yolk sac transitioned from ovoid to spherical shape and was reabsorbed progressively until release. The tail began rising from the surface of the deuteroplasm while embryos were in the egg envelope. Embryos lacked a primordial fin fold and developed some species characteristics, such as rays in the dorsal fin, before resorption of the yolk sac. At release, juvenile seahorses were in an advanced stage of development even if they lacked important adult characteristics, such as ring plates and coronet. The tail was not prehensile in juveniles at release; a small caudal fin was present, although this fin is lost in adults.

Heterochronic development of lateral plates in the three-spined stickleback induced by thyroid hormone level alterations.

The three-spined stickleback Gasterosteus aculeatus is an important model for studying microevolution and parallel adaptation to freshwater environments. Marine and freshwater forms differ markedly in their phenotype, especially in the number of lateral plates, which are serially repeated elements of the exoskeleton. In fishes, thyroid hormones are involved in adaptation to salinity, as well as the developmental regulation of serially repeated elements. To study how thyroid hormones influence lateral plate development, we manipulated levels of triiodothyronine and thiourea during early ontogeny in a marine and freshwater population with complete and low plate phenotypes, respectively. The development of lateral plates along the body and keel was heterochronic among experimental groups. Fish with a low dosage of exogenous triiodothyronine and those treated with thiourea exhibited retarded development of bony plates compared to both control fish and those treated with higher a triiodothyronine dosage. Several triiodothyronine-treated individuals of the marine form expressed the partial lateral plate phenotype. Some individuals with delayed development of lateral plates manifested 1-2 extra bony plates located above the main row of lateral plates.

Effects of a glyphosate-based herbicide in pejerrey Odontesthes humensis embryonic development.

Glyphosate-based herbicides (GBH) are the major pesticides used worldwide. Among them, the Roundup formulations are the most popular. Some aspects of GBH toxicity are well known, such as induction of oxidative stress. However, embryotoxicity is scarcely known. Therefore, the aim of the present study was to evaluate the effect of exposure to different Roundup Transorb R concentrations (0.36, 1.80, 3.62 and 5.43 mg glyphosate a.e./L) on Odontesthes humensis embryonic development. Embryos were sampled at three exposure times (48, 72 and 96 h). After 48 h, the stage of embryonic development and the number of somite pairs were analyzed; after 72 h, the percentage of pigmented embryos were evaluated and after 96 h, the eye diameter (ED) and the distance between eyes (DE) were measured. Mortality rates were daily calculated. Results show that Roundup exposure to all concentrations did not alter the endpoints evaluated at 48 and 72 h. On the other hand, exposure for 96 h to all concentrations induced a concentration-dependent reduction in ED and DE. Additionally, exposure to 5.43 mg a.e./L increased mortality. These findings indicate that Roundup has the potential to produce morphological alterations in fish embryos even at the lower and ecologically relevant concentration tested (0.36 mg a.e./L). This result corroborates the hypothesis that glyphosate alters the retinoic acid signaling pathway. Additionally, our findings indicate that exposure to high concentrations of Roundup (5.43 mg a.e./L) for 96 h causes high mortality rates of fish embryos. This is the first report of GBH embryotoxicity in an endemic fish of southern areas in South America.

Contrasting effects of hypoxia on copper toxicity during development in the three-spined stickleback (Gasterosteus aculeatus).

Hypoxia is a global problem in aquatic systems and often co-occurs with pollutants. Despite this, little is known about the combined effects of these stressors on aquatic organisms. The objective of this study was to investigate the combined effects of hypoxia and copper, a toxic metal widespread in the aquatic environment. We used the three-spined stickleback (Gasterosteus aculeatus) as a model because of its environmental relevance and amenability for environmental toxicology studies. We focused on embryonic development as this is considered to be a sensitive life stage to environmental pollution. We first investigated the effects of hypoxia alone on stickleback development to generate the information required to design subsequent studies. Our data showed that exposure to low oxygen concentrations (24.7 ± 0.9% air saturation; AS) resulted in strong developmental delays and increased mortalities, whereas a small decrease in oxygen (75.0 ± 0.5%AS) resulted in premature hatching. Stickleback embryos were then exposed to a range of copper concentrations under hypoxia (56.1 ± 0.2%AS) or normoxia (97.6 ± 0.1%AS), continuously, from fertilisation to free swimming larvae. Hypoxia caused significant changes in copper toxicity throughout embryonic development. Prior to hatching, hypoxia suppressed the occurrence of mortalities, but after hatching hypoxia significantly increased copper toxicity. Interestingly, when exposures were conducted only after hatching, the onset of copper-induced mortalities was delayed under hypoxia compared to normoxia, but after 48 h, copper was more toxic to hatched embryos under hypoxia. This is the second species for which the protective effect of hypoxia on copper toxicity prior to hatching, followed by its exacerbating effect after hatching is demonstrated, suggesting the hypothesis that this pattern may be common for teleost species. Our research highlights the importance of considering the interactions between multiple stressors, as understanding these interactions is essential to facilitate the accurate prediction of the consequences of exposure to complex stressors in a rapidly changing environment.

Foxl3, a Target of miR-9, Stimulates Spermatogenesis in Spermatogonia During Natural Sex Change in Monopterus albus.

Two foxl2 paralogs, foxl2 (also named foxl2a) and foxl3 (also named foxl2b), were considered as fish-specific duplicates. Both belong to the Forkhead box family of transcription factors, which play important roles in regulating reproduction involved in sexual differentiation and regulation of primordial germ cell and gonadal somatic cell development. We isolated the complete foxl3 cDNA from the rice field eel (Monopterus albus), which undergoes a natural female-to-male sex change via an intersex stage during its life cycle. The deduced amino acid sequence of M albus Foxl3 exhibited high identity with that of the European sea bass (Dicentrarchus labrax, 82.9%). The foxl3 expression levels in gonads were increased during the natural sex change process in M albus. The relative expression level in the testis was greater than 40-fold greater than in the ovary (P < .05). A dual-luciferase assay confirmed that the miR-9, but not the miR-430 family, bound to the foxl3 3' untranslated region of M albus. Foxl3 was primarily expressed in granulosa cells and previtellogenic follicles in the ovary and in spermatogonia and Sertoli cells in the testis. In conclusion, Foxl3 and miR-9 may be involved in physiological processes that promote oocyte degeneration in the ovotestis and stimulating spermatogenesis in spermatogonia in M albus.

Costs and Benefits to Pregnant Male Pipefish Caring for Broods of Different Sizes.

Trade-offs between brood size and offspring size, offspring survival, parental condition or parental survival are classic assumptions in life history biology. A reduction in brood size may lessen these costs of care, but offspring mortality can also result in an energetic gain, if parents are able to utilize the nutrients from the demised young. Males of the broad-nosed pipefish (Syngnathus typhle) care for the offspring by brooding embryos in a brood pouch. Brooding males can absorb nutrients that emanate from embryos, and there is often a reduction in offspring number over the brooding period. In this study, using two experimentally determined brood sizes (partially and fully filled brood pouches), we found that full broods resulted in larger number of developing offspring, despite significantly higher absolute and relative embryo mortality, compared to partial broods. Male survival was also affected by brood size, with males caring for full broods having poorer survival, an effect that together with the reduced embryo survival was found to negate the benefit of large broods. We found that embryo mortality was lower when the brooding males were in good initial condition, that embryos in broods with low embryo mortality weighed more, and surprisingly, that males in higher initial condition had embryos of lower weight. Brood size, however, did not affect embryo weight. Male final condition, but not initial condition, correlated with higher male survival. Taken together, our results show costs and benefits of caring for large brood sizes, where the numerical benefits come with costs in terms of both embryo survival and survival of the brooding father, effects that are often mediated via male condition.

Early development and replacement of the stickleback dentition.

Teeth have long served as a model system to study basic questions about vertebrate organogenesis, morphogenesis, and evolution. In nonmammalian vertebrates, teeth typically regenerate throughout adult life. Fish have evolved a tremendous diversity in dental patterning in both their oral and pharyngeal dentitions, offering numerous opportunities to study how morphology develops, regenerates, and evolves in different lineages. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a new system to study how morphology evolves, and provide a particularly powerful system to study the development and evolution of dental morphology. Here, we describe the oral and pharyngeal dentitions of stickleback fish, providing additional morphological, histological, and molecular evidence for homology of oral and pharyngeal teeth. Focusing on the ventral pharyngeal dentition in a dense developmental time course of lab-reared fish, we describe the temporal and spatial consensus sequence of early tooth formation. Early in development, this sequence is highly stereotypical and consists of seventeen primary teeth forming the early tooth field, followed by the first tooth replacement event. Comparing this detailed morphological and ontogenetic sequence to that described in other fish reveals that major changes to how dental morphology arises and regenerates have evolved across different fish lineages. J. Morphol. 277:1072-1083, 2016. © 2016 Wiley Periodicals, Inc.

An evolutionary insight into the hatching strategies of pipefish and seahorse embryos.

Syngnathiform fishes carry their eggs in a brood structure found in males. The brood structure differs from species to species: seahorses carry eggs within enclosed brood pouch, messmate pipefish carry eggs in the semi-brood pouch, and alligator pipefish carry eggs in the egg compartment on abdomen. These egg protection strategies were established during syngnathiform evolution. In the present study, we compared the hatching mode of protected embryos of three species. Electron microscopic observations revealed that alligator pipefish and messmate pipefish egg envelopes were thicker than those of seahorses, suggesting that the seahorse produces a weaker envelope. Furthermore, molecular genetic analysis revealed that these two pipefishes possessed the egg envelope-digesting enzymes, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE), as do many euteleosts. In seahorses, however, only HCE gene expression was detected. When searching the entire seahorse genome by high-throughput DNA sequencing, we did not find a functional LCE gene and only a trace of the LCE gene exon was found, confirming that the seahorse LCE gene was pseudogenized during evolution. Finally, we estimated the size and number of hatching gland cells expressing hatching enzyme genes by whole-mount in situ hybridization. The seahorse cells were the smallest of the three species, while they had the greatest number. These results suggest that the isolation of eggs from the external environment by paternal bearing might bring the egg envelope thin, and then, the hatching enzyme genes became pseudogenized. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc.

Effect of temperature on embryonic development of Melanotaenia boesemani (Allen and Cross, 1982).

The present study aimed to provide data on the time required for Melanotaenia boesemani to complete embryonic development, and to investigate the influence that incubation at different temperatures caused in this species. The effects of temperature on the time and hatching rate are presented, as well as information related to embryonic development stages. After fertilization, the eggs were kept in incubators at 23, 26, 29 or 32°C and observed at predetermined times until the moment of hatching. Stages of development were identified and classified according to morphological and physiological characteristics. Oil droplets were visualized inside the eggs as well as filament adhesion present at the chorion. Embryonic development was similar to that observed in other species of the genus Melanotaenia with hatching and faster development in higher temperatures.

Embryonic developmental plasticity in the long-snouted seahorse (Hippocampus reidi, Ginsburg 1933) in relation to parental preconception diet.

The aim of the present study was to investigate the hypothesis that parental periconception nutrition in adult seahorses affects the development and growth of their offspring. We tested the hypothesis that because seahorse embryos develop inside the male's brood pouch, manipulation of the male's diet would affect offspring growth and development independently of the female's diet. Adult males and females were fed separately with either wild-caught crustaceans or commercial aquarium diet for 1 month before conception to influence the periconception environment. Approximately 10000 offspring were obtained from four different treatment groups (Male/Wild or Male/Commercial×Female/Wild or Female/Commercial). Weights, physical dimensions and fatty acid profiles of the newborns were determined. Offspring produced when the males receiving commercial diet were mated with wild-fed females were larger (P<0.05) than those produced by wild-fed males. When both males and females were fed with commercial diet, their offspring were significantly smaller than those from the other treatment groups. When commercial diet-fed females were mated with wild-fed males, the offspring showed distortion of the snout:head length ratio. These results support the view that the preconception diet received by males and females differentially affects embryonic development.

Developmental abnormalities and differential expression of genes induced in oil and dispersant exposed Menidia beryllina embryos.

Exposure of fish embryos to relatively low concentrations of oil has been implicated in sub-lethal toxicity. The objective of this study was to determine the effects of the exposure of Menidia beryllina embryos at 30-48h post-fertilization to the water accommodated fractions of oil (WAF, 200ppm, v/v), dispersants (20ppm, v/v, Corexit 9500 or 9527), and mixtures of oil and each of the dispersants to produce chemically enhanced water accommodated fractions (CEWAFs) over a 72-hour period. The polyaromatic hydrocarbon (PAH) and benzene, toluene, ethylene and xylene (BTEX) constituents of the 5X concentrated exposure solutions (control, WAF, dispersants and CEWAFs) were determined and those of the 1× exposures were derived using a dilution factor. PAH, BTEX and low molecular weight PAH constituents greater than 1ppb were observed in WAF and the dispersants, but at much higher levels in CEWAFs. The WAF and CEWAFs post-weathering were diluted at 1:5 (200ml WAF/CEWAF: 800ml 25ppt saltwater) for embryo exposures. Mortality, heartbeat, embryo normalcy, abnormality types and severities were recorded. The qPCR assay was used to quantify abundances of transcripts of target genes for sexual differentiation and sex determination (StAR, dmrt-1, amh, cyp19b, vtg and chg-L,), growth regulation (ghr) and stress response (cyp1a and Hsp90); and gapdh served as the housekeeping gene. Temperature was 21±1.5°C throughout the experimental period, while mortality was low and not significantly different (p=0.68) among treatments. Heartbeat was significantly different (0.0034) with the lowest heartbeats recorded in Corexit 9500 (67.5beats/min) and 9527 (67.1beats/min) exposed embryos compared with controls (82.7beats/min). Significantly more treated embryos were in a state of deterioration, with significantly more embryos presenting arrested tissue differentiation compared with controls (p=0.021). Exposure to WAF, dispersants and CEWAF induced aberrant expression of all the genes, with star, dmrt-1, ghr and hsp90 being significantly down-regulated in CEWAF and cyp19b in Corexit 9527. The cyp1a and cyp19b were significantly up-regulated in CEWAFs and WAF, respectively. The molecular endpoints were most sensitive, especially the expression of star, cyp19b, cyp1a, hsp90 and could therefore be used as early indicators of long term effects of Corexit 9500 and 9527 usage in oil spill management on M. beryllina, a valid sentinel for oil pollution events.

The evolutionary puzzle of egg size, oxygenation and parental care in aquatic environments.

Offspring fitness generally improves with increasing egg size. Yet, eggs of most aquatic organisms are small. A common but largely untested assumption is that larger embryos require more oxygen than they can acquire through diffusion via the egg surface, constraining egg size evolution. However, we found no detrimental effects of large egg size on embryo growth and survival under hypoxic conditions. We tested this in the broad-nosed pipefish, Syngnathus typhle, whose males provide extensive care (nourishment, osmoregulation and oxygenation) to their young in a brood pouch on their bodies. We took advantage of this species' pronounced variation in egg size, correlating positively with female size, and tested the effect of hypoxia (40% dissolved oxygen) versus fully oxygenated (100%) water on embryo size and survival of large versus small eggs after 18 days of paternal brooding. Egg size did not affect embryo survival, regardless of O2 treatment. While hypoxia affected embryo size negatively, both large and small eggs showed similar reductions in growth. Males in hypoxia ventilated more and males with large eggs swam more, but neither treatment affected their position in the water column. Overall, our results call into question the most common explanation for constrained egg size evolution in aquatic environments.

Embryo oxygenation in pipefish brood pouches: novel insights.

The pipefish brood pouch presents a unique mode of parental care that enables males to protect, osmoregulate, nourish and oxygenate the developing young. Using a very fine O2 probe, we assessed the extent to which males of the broad-nosed pipefish (Syngnathus typhle) oxygenate the developing embryos and are able to maintain pouch fluid O2 levels when brooding in normoxia (100% O2 saturation) and hypoxia (40% O2 saturation) for 24 days. In both treatments, pouch fluid O2 saturation levels were lower compared with the surrounding water and decreased throughout the brooding period, reflecting greater offspring demand for O2 during development and/or decreasing paternal ability to provide O2 to the embryos. Male condition (hepatosomatic index) was negatively affected by hypoxia. Larger males had higher pouch fluid O2 saturation levels compared with smaller males, and levels were higher in the bottom section of the pouch compared with other sections. Embryo size was positively correlated with O2 availability, irrespective of their position in the pouch. Two important conclusions can be drawn from our findings. First, our results highlight a potential limitation to brooding within the pouch and dismiss the notion of closed brood pouches as well-oxygenated structures promoting the evolution of larger eggs in syngnathids. Second, we provide direct evidence that paternal care improves with male size in this species. This finding offers an explanation for the documented strong female preference for larger partners because, in terms of oxygenation, the brood pouch can restrict embryo growth.