Shark Embryos Research Articles

One-year extra-uterine life support for viviparous shark embryos: first technological application to mid-term embryos

Shark-specific artificial uterine fluid is a recently developed material suitable for maintaining viviparous shark embryos outside the maternal body. Previous studies confirmed that this fluid greatly extends the captive maintenance period of the embryonic slendertail lantern shark (Etmopterus molleri) from <1 week to several months. Its application, however, has been limited to late-term embryos. This study reports the first application of this fluid on mid-term embryos with long external gill filaments. The incubation was found to be successful, with the embryos growing to their natural birth size after 1 year. A high mortality of mid-term embryos was observed during incubation due to pressure ulcers. This indicates that not only appropriate chemical environment but also appropriate physical environment is critical for the stable maintenance of mid-term embryos.

Successful delivery of viviparous lantern shark from an artificial uterus and the self-production of lantern shark luciferin.

Our recent success in the long-term maintenance of lantern shark embryos in artificial uterine systems has provided a novel option for the medical treatment of premature embryos for captive viviparous elasmobranchs. The remaining issue with this system is that the embryos cannot survive the abrupt change in the chemical environment from artificial uterine fluid (AUF) to seawater during delivery. To overcome this issue, the present study developed a new protocol for seawater adaptation, which is characterized by a long-term and stepwise shift from AUF to seawater prior to delivery. This protocol was employed successfully, and the specimen survived for more than seven months after delivery, the longest captive record of the species. During the experiment, we unexpectedly detected bioluminescence of the embryonic lantern shark in the artificial uterus. This observation indicates that lantern sharks can produce luciferin, a substance for bioluminescence. This contradicts the recent hypothesis that lantern sharks lack the ability to produce luciferin and use luciferin obtained from food sources.

Mercury Concentrations in Whale Shark (Rhincodon typus) Embryo Muscle Tissue.

Mercury (Hg) is known to be maternally transferred during embryonic development in sharks; however, Hg concentrations in embryos of filter feeding shark species have not previously been reported. This study measured the total Hg (THg) concentration in muscle tissue of 27 embryos taken from a pregnant whale shark (Rhincodon typus) landed in Taiwan in 1995 and the mean THg concentration compared to the mean muscle THg concentration in embryos from other shark species. The mean (± standard deviation) THg concentration in whale shark embryos was 0.0762 ± 0.0163µg/g dry weight (0.0224 ± 0.0054µg/g wet weight). There was no relationship between muscle THg concentration and body length and no significant difference in THg concentration between male and female embryos (p > 0.05). Whale shark embryos have the lowest reported muscle THg concentrations compared to literature values for muscle THg concentrations for embryos from other shark species.

Impacts of Deoxygenation and Hypoxia on Shark Embryos Anti-Predator Behavior and Oxidative Stress.

Climate change is leading to the loss of oxygen content in the oceans and endangering the survival of many marine species. Due to sea surface temperature warming and changing circulation, the ocean has become more stratified and is consequently losing its oxygen content. Oviparous elasmobranchs are particularly vulnerable as they lay their eggs in coastal and shallow areas, where they experience significant oscillations in oxygen levels. Here, we investigated the effects of deoxygenation (93% air saturation) and hypoxia (26% air saturation) during a short-term period (six days) on the anti-predator avoidance behavior and physiology (oxidative stress) of small-spotted catshark (Scyliorhinus canicula) embryos. Their survival rate decreased to 88% and 56% under deoxygenation and hypoxia, respectively. The tail beat rates were significantly enhanced in the embryos under hypoxia compared to those exposed to deoxygenation and control conditions, and the freeze response duration showed a significant opposite trend. Yet, at the physiological level, through the analyses of key biomarkers (SOD, CAT, GPx, and GST activities as well as HSP70, Ubiquitin, and MDA levels), we found no evidence of increased oxidative stress and cell damage under hypoxia. Thus, the present findings show that the projected end-of-the-century deoxygenation levels elicit neglectable biological effects on shark embryos. On the other hand, hypoxia causes a high embryo mortality rate. Additionally, hypoxia makes embryos more vulnerable to predators, because the increased tail beat frequency will enhance the release of chemical and physical cues that can be detected by predators. The shortening of the shark freeze response under hypoxia also makes the embryos more prone to predation.

First report of synophthalmia and leucism in a bull shark (Carcharhinus leucas) embryo from the southeastern Gulf of Mexico

An abnormal embryo (male of 465 mm total length) and 13 other embryos were removed from a pregnant female bull shark (Carcharhinus leucas) caught in coastal waters between the Grijalva River and Terminos Lagoon in the southwestern Gulf of Mexico. The first record of synophthalmia and leucism in the bull shark is presented. The shark cyclopia case is reviewed.

Aquatic Walking and Swimming Kinematics of Neonate and Juvenile Epaulette Sharks.

The epaulette shark, Hemiscyllium ocellatum, is a small, reef-dwelling, benthic shark that-using its paired fins-can walk, both in and out of water. Within the reef flats, this species experiences short periods of elevated CO2 and hypoxia as well as fluctuating temperatures as reef flats become isolated with the outgoing tide. Past studies have shown that this species is robust (i.e., respiratory and metabolic performance, behavior) to climate change-relevant elevated CO2 levels as well as hypoxia and anoxia tolerant. However, epaulette shark embryos reared under ocean warming conditions hatch earlier and smaller, with altered patterns and coloration, and with higher metabolic costs than their current-day counterparts. Findings to date suggest that this species has adaptations to tolerate some, but perhaps not all, of the challenging conditions predicted for the 21st century. As such, the epaulette shark is emerging as a model system to understand vertebrate physiology in changing oceans. Yet, few studies have investigated the kinematics of walking and swimming, which may be vital to their biological fitness, considering their habitat and propensity for challenging environmental conditions. Given that neonates retain embryonic nutrition via an internalized yolk sac, resulting in a bulbous abdomen, while juveniles actively forage for worms, crustaceans, and small fishes, we hypothesized that difference in body shape over early ontogeny would affect locomotor performance. To test this, we examined neonate and juvenile locomotor kinematics during the three aquatic gaits they utilize-slow-to-medium walking, fast walking, and swimming-using 13 anatomical landmarks along the fins, girdles, and body midline. We found that differences in body shape did not alter kinematics between neonates and juveniles. Overall velocity, fin rotation, axial bending, and tail beat frequency and amplitude were consistent between early life stages. Data suggest that the locomotor kinematics are maintained between neonate and juvenile epaulette sharks, even as their feeding strategy changes. Studying epaulette shark locomotion allows us to understand this-and perhaps related-species' ability to move within and away from challenging conditions in their habitats. Such locomotor traits may not only be key to survival, in general, as a small, benthic mesopredator (i.e., movements required to maneuver into small reef crevices to avoid aerial and aquatic predators), but also be related to their sustained physiological performance under challenging environmental conditions, including those associated with climate change-a topic worthy of future investigation.

How the Skate Embryo Develops: A Fine Developmental Timeline of Kong Skate Okamejei kenojei (Müller & Henle, 1841)

Cartilaginous fishes, as an important group of jawed vertebrates, hold great promise for improving our understanding of vertebrate evolution. Previous studies have established a good staging scheme for shark embryos. However, to date, little attention has been paid to the development of skate embryos. In this study, we established a fine developmental timeline for the Kong skate. We hatched eggs of the Kong skate (Okamejei kenojei) at fixed temperatures (16.5°C ± 0.5°C) while observing three samples every day and recorded the complete embryonic development. The embryo weight and development time were found to have a quadratic relationship. The weight-at-day curve was fitted by y = 0.0005x2 - 0.0109x + 0.0882 (R² = 0.9406). In addition, complete embryonic development from gastrula to skate larvae took 92 ± 5 days. Here, we divided the entire development process into 39 stages. This shows that the jaw of O. kenojei is developed from the first pair of pharyngeal arches, and its body disc is formed by the fusion of the pectoral fin and head. The eyespots began to form from stage 36 and then became thicker and bigger until the young skate broke out of the egg. Notably, we found that males and females develop differently from stage 31; the end of the male pelvic fins expands inward, which develop into copulatory organ-claspers, whereas in the females, they do not expand. To the best of our knowledge, this is the first study to record the embryonic development of eyes, body discs, eyespots, and other organs of O. kenojei in detail. We hope that our work will provide valuable information for researchers to identify specific time points for developing skate embryos.

Five-Month Incubation of Viviparous Deep-Water Shark Embryos in Artificial Uterine Fluid

In this study, we developed an extra-uterine life-support system (artificial uterus) for viviparous elasmobranchs. Using this system, we maintained two embryonic specimens of the slendertail lantern shark (Etmopterus molleri) for approximately 5 months, the longest published record of the captive maintenance of the embryos of this species. This system is characterized by the use of a urea-containing artificial incubation fluid, of which the salinity and osmotic pressure are approximately equivalent to those of shark blood plasma. We hypothesized that this fluid reduces the salinity gradient between embryonic blood plasma and the environmental fluid, which thereby contributes to countering the low osmoregulatory capacity of viviparous shark embryos. However, the incubated specimens subsequently died during the process of seawater adaptation following “artificial birth.” Accordingly, information pertaining to uterine chemical dynamics throughout the gestation period is necessary for the design of a more effective incubation system. This technique will be relevant and may find application in conservation breeding at public aquaria in the future.

Mode of uterine milk secretion in the white shark.

Examination of the uterus of a dead female white shark (Carcharodon carcharias), which contained the earliest known white shark embryos, revealed that the uterine wall produces lipid-rich secretion (histotroph or "uterine milk") for embryonic nutrition. Uterine tissue was processed for light and electron microscopy, and immunohistochemical techniques to identify its secretory mechanism. Our results indicate that the white shark uterus secretes lipids via holocrine secretion. This type of secretion is characterized by the release of large lipid droplets accumulated in the epithelial cells into the uterine lumen through cell disintegration. The secretory epithelium of the uterus is stratified, and new surface epithelial cells are continuously supplied from deeper epithelial layers to replace the dead secretory cells at the surface. This vertical replacement possibly facilitates the active renewal of the surface epithelium, which is necessary for maintaining holocrine secretory mechanisms. These secretory mechanisms are different from those of myliobatiform stingrays, another elasmobranch taxon that exhibits lipid histotrophy. This may reflect the different origins of lipid histotrophy between these taxa.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Hot shark embryos freeze less to stay safe

Have you ever seen a scary movie where the murderer is stalking a potential victim, and then it cuts to a close-up of their prey frozen still, holding their breath for dear life? Believe it or not, shark embryos have a remarkably similar freeze response when predators are nearby. Small-spotted catshark (Scyliorhinus canicula) embryos develop for 21 weeks in egg cases called mermaid's purses. After the 10th week, these shark embryos produce secretions that open slits into the egg case, and for the remainder of their development, the tiny sharks have to move to exchange seawater to ensure enough oxygen is available to continue their development. However, these movements attract shark predators. Fortunately, the youngsters are able to detect the presence of the threat and stop moving and breathing – a behavior known as the freeze response – although they are only able to do so for a limited amount of time as they have to resume respiration and moving once again. As shark embryos are particularly vulnerable to predation, any factor that affects the freeze response is a risk for shark populations in the wild. As temperatures are expected to increase and affect the metabolic rate of marine animals, Daniel Ripley and researchers from the University of Manchester, UK, investigated the effect of increasing temperatures on the freeze response of small-spotted catshark youngsters.First, the team transported ∼3-week-old small-spotted catshark embryos from Germany to the UK and placed the developing youngsters in either 15 or 20°C water until they were 15 weeks old. Then, the researchers took the shark embryos and gently flicked them underwater, to imitate a predator inspecting an egg case, while measuring how long the youngsters froze and the amount of oxygen that they consumed at their respective temperatures. Finally, the team measured how fast the embryos fanned their gills to inhale oxygen at the different temperatures.Despite the difference in the embryos’ temperatures, the team did not find a difference in youngsters’ resting metabolic rates. However, they did discover that the embryos fan their gills faster at 20°C than at 15°C. Additionally, as the resting metabolic rate or incubation temperature of embryos increased, the duration of their freezing response decreased. This means that the length of time that the embryos hold themselves still does not depend solely on their metabolic rate. Most alarmingly, the researchers also found that the warmest embryos only froze for a seventh of the time that the cooler embryos remained stock still. This decrease in the length of the freeze response has significant implications as temperatures increase as a result of climate change. The freeze response is a behavior that helps embryos avoid predators and improves their chances of survival, but small-spotted catshark embryos will likely be at increasing risk from passing predators as temperatures rise and this response declines.For these shark embryos, the duration of this freeze response can be the difference between life and death. The research team also advises that predator avoidance behavior is another factor that needs to be taken into account when thinking about the conservation of sharks and the impact of rising temperatures owing to climate change.

Future thermal regimes for epaulette sharks (Hemiscyllium ocellatum): growth and metabolic performance cease to be optimal

Climate change is affecting thermal regimes globally, and organisms relying on their environment to regulate biological processes face unknown consequences. In ectotherms, temperature affects development rates, body condition, and performance. Embryonic stages may be the most vulnerable life history stages, especially for oviparous species already living at the warm edge of their distribution, as embryos cannot relocate during this developmental window. We reared 27 epaulette shark (Hemiscyllium ocellatum) embryos under average summer conditions (27 °C) or temperatures predicted for the middle and end of the twenty-first century with climate change (i.e., 29 and 31 °C) and tracked growth, development, and metabolic costs both in ovo and upon hatch. Rearing sharks at 31 °C impacted embryonic growth, yolk consumption, and metabolic rates. Upon hatch, 31 °C-reared sharks weighed significantly less than their 27 °C-reared counterparts and exhibited reduced metabolic performance. Many important growth and development traits in this species may peak after 27 °C and start to become negatively impacted nearing 31 °C. We hypothesize that 31 °C approximates the pejus temperature (i.e., temperatures at which performance of a trait begin to decline) for this species, which is alarming, given that this temperature range is well within ocean warming scenarios predicted for this species’ distribution over the next century.

Friend or foe? Development of odour detection, differentiation and antipredator response in an embryonic elasmobranch

Young animals, especially those developing within eggs, are extremely vulnerable to predation risk given their immobility, small size and limited functionality of developing sensory systems. Embryos from a range of taxa can detect predator cues and use antipredator responses to reduce risk; however, little is known about this capacity in elasmobranchs, especially regarding olfactory detection. Embryonic Port Jackson sharks (Heterodontus portusjacksoni) were exposed to elasmobranch and teleost odours across key developmental stages to investigate their capacity to detect and discern between cues. Oxygen uptake rates were measured as a proxy for antipredator response and to estimate their energetic costs. Earlier developmental stages exhibited limited responses, regardless of the odour, likely associated with an undeveloped sensory system. However, older shark embryos depressed oxygen uptake rates (i.e. crypsis responses) when exposed to teleost cues, but showed little response to elasmobranch cues. In contrast, hatchlings exhibited minimal responses to teleost cues but a significant increase in oxygen uptake rates when exposed to elasmobranch odours, indicative of a general stress response in preparation for escape. Collectively, our data suggest that embryonic sharks can differentiate between odour cues and elicit various responses, but this ability is limited by their developing sensory capacity.

Mineralization of the Callorhinchus Vertebral Column (Holocephali; Chondrichthyes).

Members of the Chondrichthyes (Elasmobranchii and Holocephali) are distinguished by their largely cartilaginous endoskeletons, which comprise an uncalcified core overlain by a mineralized layer; in the Elasmobranchii (sharks, skates, rays) most of this mineralization takes the form of calcified polygonal tiles known as tesserae. In recent years, these skeletal tissues have been described in ever increasing detail in sharks and rays, but those of Holocephali (chimaeroids) have been less well-studied, with conflicting accounts as to whether or not tesserae are present. During embryonic ontogeny in holocephalans, cervical vertebrae fuse to form a structure called the synarcual. The synarcual mineralizes early and progressively, anteroposteriorly and dorsoventrally, and therefore presents a good skeletal structure in which to observe mineralized tissues in this group. Here, we describe the development and mineralization of the synarcual in an adult and stage 36 elephant shark embryo (Callorhinchus milii). Small, discrete, but irregular blocks of cortical mineralization are present in stage 36, similar to what has been described recently in embryos of other chimaeroid taxa such as Hydrolagus, while in Callorhinchus adults, the blocks of mineralization are more irregular, but remain small. This differs from fossil members of the holocephalan crown group (Edaphodon), as well as from stem group holocephalans (e.g., Symmorida, Helodus, Iniopterygiformes), where tesserae are notably larger than in Callorhinchus and show similarities to elasmobranch tesserae, for example with respect to polygonal shape.

Same species, different prerequisites: investigating body condition and foraging success in young reef sharks between an atoll and an island system

Acquiring and storing energy is vital to sharks of all age-classes. Viviparous shark embryos receive endogenous maternal energy reserves to sustain the first weeks after birth. Then, in order to maintain body condition, sharks must start foraging. Our goal was to understand whether maternal energy investments vary between blacktip reef sharks (Carcharhinus melanopterus) from two populations and to what extent body condition and the initiation of foraging might be affected by presumably variable maternal investments. A total of 546 young sharks were captured at St. Joseph atoll (Seychelles) and Moorea (French Polynesia) between 2014 and 2018, and indices of body condition and percentage of stomachs containing prey were measured. Maternal investment was found to be site-specific, with significantly larger, heavier, and better conditioned individuals in Moorea. Despite these advantages, as time progressed, Moorea sharks exhibited significant decreases in body condition and were slower to initiate foraging. We suggest that the young sharks’ foraging success is independent of the quality of maternal energy resources, and that other factors, such as prey availability, prey quality, and/or anthropogenic stressors are likely responsible for the observed differences across sites. Insights into intraspecific variations in early life-stages may further support site-specific management strategies for young sharks from nearshore habitats.

Locomotion is not a privilege after birth: Ultrasound images of viviparous shark embryos swimming from one uterus to the other

AbstractUnderwater ultrasound, a new tool for observing the internal body parts of aquatic animals by scuba divers, allowed us long‐term and frequent observations of the embryos of captive aquatic vertebrates. New ultrasound data of captive tawny nurse sharks (Nebrius ferrugineus) revealed that their embryos frequently migrate between the right and left uteri during gestation. This report is the first reliable evidence of active embryonic locomotion in live‐bearing vertebrates and is contradictory to the concept of “sedentary embryo” which has mainly arisen from studies of mammals. The tawny nurse shark is unique among orectolobiform sharks, in which the embryo develops by feeding on sibling eggs in utero. Thus, we hypothesized that swimming aids in an efficient search and capture of these eggs in the uterine environment.

Study of pallial neurogenesis in shark embryos and the evolutionary origin of the subventricular zone

The dorsal part of the developing telencephalon is one of the brain areas that has suffered most drastic changes throughout vertebrate evolution. Its evolutionary increase in complexity was thought to be partly achieved by the appearance of a new neurogenic niche in the embryonic subventricular zone (SVZ). Here, a new kind of amplifying progenitors (basal progenitors) expressing Tbr2, undergo a second round of divisions, which is believed to have contributed to the expansion of the neocortex. Accordingly, the existence of a pallial SVZ has been classically considered exclusive of mammals. However, the lack of studies in ancient vertebrates precludes any clear conclusion about the evolutionary origin of the SVZ and the neurogenic mechanisms that rule pallial development. In this work, we explore pallial neurogenesis in a basal vertebrate, the shark Scyliorhinus canicula, through the study of the expression patterns of several neurogenic markers. We found that apical progenitors and radial migration are present in sharks, and therefore, their presence must be highly conserved throughout evolution. Surprisingly, we detected a subventricular band of ScTbr2-expressing cells, some of which also expressed mitotic markers, indicating that the existence of basal progenitors should be considered an ancestral condition rather than a novelty of mammals or amniotes. Finally, we report that the transcriptional program for the specification of glutamatergic pallial cells (Pax6, Tbr2, NeuroD, Tbr1) is also present in sharks. However, the segregation of these markers into different cell types is not clear yet, which may be linked to the lack of layering in anamniotes.

A non-invasive manual method for the assessment of pregnancy in female Scyliorhinus canicula catsharks

Enhanced survival of oviparous shark populations can rely on only one easily applicable method. We tested a powerful method to retrieve egg cases from dead oviparous females at the fish market which is non-invasive. We approved this method for Scyliorhinus canicula and Scyliorhinus stellaris (smallspotted catshark and Nursehound) in Valletta, Malta in 2014. Here, catsharks are landed in whole, whereas in many other Mediterranean countries sharks are already eviscerated when brought ashore. Saving egg cases bears the grateful opportunity to rescue shark embryos and this then helps again to restore fish stocks. The common work flow of fishermen is to throw away the egg cases when eviscerating the shark and so the embryos were left in the bin to die. To apply a non-invasive method is very important at fish markets where sharks are landed in one piece as fish brokers do not tolerate many dissections. So, our goal was to detect pregnant females with maximum accuracy and dissect only females with egg cases inside. Formerly, the common method used by members of Sharklab-Malta was to dissect females more or less randomly based on only two parameters (i) a total length above 40cm and (ii) a relatively corpulent body shape. This was not effective as many females were dissected without containing egg cases. This led to the more problematic fact that pregnant individuals were not found and so their offspring died in the end. The basic concept in our newly tested method is based on using a fixed touching pattern which allows to screen the belly of all females available simply by hand. To screen one female only 2 seconds are needed, and this works surprisingly well. We called this method “gotIt” simply because we got all available egg cases and could save all embryos. A validation underpinned the success and showed that gotIt works with 100% accuracy. After rescuing the egg cases they were transported to an aquarium. Egg cases were attached to a lanyard by their tendrils and left for development in the tank for 3-6 months (depending on their initial developmental stage at the fish market) at a water temperature of 15-19°C. After hatching, sharks were fed with squid and released into the sea. The number of rescued embryos arose significantly from five to above hundreds of shark pups which were saved and released by applying gotIt. Additionally, fishermen can be encouraged to use this method, as awareness programs can be promoted to encourage consumption of this sustainable fish, which can therefore increase the marketable prices. Widely applied, gotIt can help to preserve oviparous shark populations at fish markets where sharks are landed whole.

New report of malformations in blue shark embryos (Prionace glauca) from the western coast of Baja California Sur, Mexico

Four new malformations observed in blue shark embryos in the western coast of Baja California Sur, Mexico are reported in this study. The embryos of blue shark samples were obtained on board the industrial fishing boat ‘Damasta’; the organisms were frozen and transferred to CIBNOR Fish Ecology Laboratory where the corresponding measurements were taken and malformations observed. The first malformation was the presence of one nostril in two female embryos that showed short and crushed snouts. The second one was observed in a female with two jaws and the middle portion of the column trunk in screw-shape. The third one was an embryo with dicephaly, two separate vertebral columns, two first dorsal fin pairs, absence of the second dorsal fin, five pelvic fins and two joint caudal fins. The fourth malformation observed was an embryo with incomplete development and undifferentiated sex. Malformations are still a mystery and also an object of study because they affect different organisms, not only sharks but also fish. It is highly relevant to know the causes and perform further studies to understand if the origin is either genetic or anthropogenic.

A staging table for the embryonic development of the brownbanded bamboo shark (Chiloscyllium punctatum).

Background: Studying cartilaginous fishes (chondrichthyans) has helped us understand vertebrate evolution and diversity. However, resources such as genome sequences, embryos, and detailed staging tables are limited for species within this clade. To overcome these limitations, we have focused on a species, the brownbanded bamboo shark (Chiloscyllium punctatum), which is a relatively common aquarium species that lays eggs continuously throughout the year. In addition, because of its relatively small genome size, this species is promising for molecular studies. Results: To enhance biological studies of cartilaginous fishes, we establish a normal staging table for the embryonic development of the brownbanded bamboo shark. Bamboo shark embryos take around 118 days to reach the hatching period at 25°C, which is approximately 1.5 times as fast as the small‐spotted catshark (Scyliorhinus canicula) takes. Our staging table divides the embryonic period into 38 stages. Furthermore, we found culture conditions that allow early embryos to grow in partially opened egg cases. Conclusions: In addition to the embryonic staging table, we show that bamboo shark embryos exhibit relatively fast embryonic growth and are amenable to culture, key characteristics that enhance their experimental utility. Therefore, the present study is a foundation for cartilaginous fish research. Developmental Dynamics 247:712–723, 2018. © 2017 Wiley Periodicals, Inc.