hox gene expression predicts tetrapod-like axial regionalization in the skate, Leucoraja erinacea.

The axial skeleton of tetrapods is organized into distinct anteroposterior regions of the vertebral column (cervical, trunk, sacral, and caudal), and transitions between these regions are determined by colinear anterior expression boundaries of Hox5/6, -9, -10, and -11 paralogy group genes within embryonic paraxial mesoderm. Fishes, conversely, exhibit little in the way of discrete axial regionalization, and this has led to scenarios of an origin of Hox-mediated axial skeletal complexity with the evolutionary transition to land in tetrapods. Here, combining geometric morphometric analysis of vertebral column morphology with cell lineage tracing of hox gene expression boundaries in developing embryos, we recover evidence of at least five distinct regions in the vertebral skeleton of a cartilaginous fish, the little skate (Leucoraja erinacea). We find that skate embryos exhibit tetrapod-like anteroposterior nesting of hox gene expression in their paraxial mesoderm, and we show that anterior expression boundaries of hox5/6, hox9, hox10, and hox11 paralogy group genes predict regional transitions in the differentiated skate axial skeleton. Our findings suggest that hox-based axial skeletal regionalization did not originate with tetrapods but rather has a much deeper evolutionary history than was previously appreciated.

Husbandry conditions of spotted ratfish (Hydrolagus colliei, Chimaeriformes) in aquaria for successful embryonic development and long-term survival of juveniles.

The spotted ratfish Hydrolagus colliei is the most common holocephalan species exhibited in aquaria worldwide for introducing deep-sea environments and raising awareness of their conservation. However, little is known about the biology of H. colliei. Current practices in aquaria allow long-term survival of sexually mature H. colliei specimens; however, this species struggles to complete a reproductive cycle in captivity mostly because embryos do not reach the hatchling stage. The aquarists of Planet Ocean Montpellier (POM, France) have bred H. colliei for 15 years and recorded parameters suitable for this species' successful embryonic and post-embryonic development. POM aquarists now regularly record egg-laying events of H. colliei and use four tanks to incubate eggs and raise neonates, late hatchlings, early and intermediate juveniles, subadults, and sexually mature specimens. In this work we provide the first long-term biometric data on H. colliei from the hatchling to the subadult stage. We also report the biotic and abiotic parameters sufficient to breed H. colliei in aquaria. We finally describe the methods used to facilitate individual monitoring of specimens along the ontogeny and several pathologies identified in this species, their putative causes, and the corresponding treatments. This work highlights the importance of ex situ research and points to the valuable outcomes of collaborative efforts between aquaria and academia in deciphering the biology of species whose study in the wild remains challenging.

Increasing the data on elasmobranch plasma protein electrophoresis: electrophoretogram reference values determination in the undulate skate (Raja Undulata) and the nursehound shark (Scyliorhinus stellaris) maintained under human care.

BACKGROUND: This study determined plasma protein electrophoresis (PPE) reference intervals in two elasmobranch species: the undulate skate (Raja undulata) and the nursehound shark (Scyliorhinus stellaris), using a reference population of 48 undulate skates (27 males, 21 females) and 62 nursehounds (32 males, 30 females), considered to be clinically healthy. Plasma samples were analyzed using capillary zone electrophoresis (CZE). RESULTS: The undulate skate electrophoretogram resembled those previously reported in other batoids and could be divided into seven consistent fractions. No statistically significant differences were detected between sexes and developmental stages. The nursehound electrophoretogram was similar to that previously described in other shark species and could be divided into eight consistent fractions. Fraction 5% was significantly higher in juvenile nursehounds when compared to adults, while fraction 6 concentration and percentage were significantly higher in adults. Fraction 4% was higher in males than in females. Albumin band was not detected, and pre-albumin was negligible in both studied species. Alpha-globulins were predominant in the undulate skate, while beta-globulins were predominant in nursehounds. Statistically significant differences were found in all electrophoretogram fraction percentages and concentrations between the two species. CONCLUSION: To the authors knowledge, this is the first study reporting PPE values in undulate skates and nursehounds, and the first study using CZE in elasmobranch plasma. These findings can serve as a primary reference for health monitoring in both species and will add to the limited data available on PPE in elasmobranchs.

Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates.

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

First record of albinism in the lanternshark family, Etmopteridae.

A single albino specimen of the lanternshark, Lucifer's dogfish (Etmopterus lucifer), is reported here. The specimen was found among museum collections, having been captured near Cape Palliser, off the North Island of New Zealand in 1984. Morphometrics are reported, and with no retainment of pigmentation, the specimen is considered a complete albino. This is the first record of albinism for the family Etmopteridae and one of a handful of records for any deep-sea chondrichthyans.

Development and evolution of tooth renewal in neoselachian sharks as a model for transformation in chondrichthyan dentitions.

A defining feature of dentitions in modern sharks and rays is the regulated pattern order that generates multiple replacement teeth. These are arranged in labio-lingual files of replacement teeth that form in sequential time order both along the jaw and within successively initiated teeth in a deep dental lamina. Two distinct adult dentitions have been described: alternate, in which timing of new teeth alternates between two adjacent files, each erupting separately, and the other arranged as single files, where teeth of each file are timed to erupt together, in some taxa facilitating similarly timed teeth to join to form a cutting blade. Both are dependent on spatiotemporally regulated formation of new teeth. The adult Angel shark Squatina (Squalomorphii) exemplifies a single file dentition, but we obtained new data on the developmental order of teeth in the files of Squatina embryos, showing alternate timing of tooth initiation. This was based on micro-CT scans revealing that the earliest mineralised teeth at the jaw margin and their replacements in file pairs (odd and even jaw positions) alternate in their initiation timing. Along with Squatina, new observations from other squalomorphs such as Hexanchus and Chlamydoselachus, together with representatives of the sister group Galeomorphii, have established that the alternate tooth pattern (initiation time and replacement order) characterises the embryonic dentition of extant sharks; however, this can change in adults. These character states were plotted onto a recent phylogeny, demonstrating that the Squalomorphii show considerable plasticity of dental development. We propose a developmental-evolutionary model to allow change from the alternate to a single file alignment of replacement teeth. This establishes new dental morphologies in adult sharks from inherited alternate order.

Introduction to Northeast Pacific Shark Biology, Ecology, and Conservation.

Sharks are iconic, sometimes apex, predators found in every ocean and, as a result, they have featured prominently in the mythology, history, and fisheries of diverse human cultures around the world. Because of their regional significance to fisheries and ecological role as predators, and as a result of concern over long-term stability of their populations, there has been an increasing amount of work focused on shark conservation in recent decades. This volume highlights the biodiversity and biological attributes of, and conservation efforts targeted at, populations of sharks that reside in the Northeast Pacific Ocean bordering the west coast of the United States and Canada, one of the most economically and ecologically important oceanic regions in the world. A companion volume addresses details of fisheries and ecotourism in the same region, as well as delving into the relationship between captive husbandry of sharks and education/outreach efforts aimed at fostering a conservation mindset in the public at large. Together, these volumes provide readers a detailed backdrop against which to consider their own actions, and those of resource managers, academics, and educators, as they relate to the long-term conservation of sharks and their relatives.

Some observations on the biology of two rarely seen deep-sea chimaerids, Chimaera carophila and Hydrolagus homonycteris.

Chimaera carophila (n = 45) and Hydrolagus homonycteris (n = 11), two deep-sea chimaerids rarely caught in the waters off New Zealand, were collected from research trawl catches and commercial fishery catches around New Zealand at depths between 400 and 1300 m, between 2014 and 2016. Additional preserved specimens of both species (n = 58) from museum collections were analysed for size, sex and maturity. External assessment of male claspers and a combination of internal assessments of female gonad mass and oviducal gland width, were used to determine maturity. For both species, length at first maturity was 0·70-0·82 of their maximum observed chimaera length (LC ), with females maturing at a larger size. Length at maturity for C. carophila (LC range: 28·7-103·9 cm) was estimated at 72·5 cm LC for males (n = 163) and 82·5 LC for females (n = 58). In H. homonycteris, length at maturity (length range: 78·6-99·8 cm LC ) was estimated at 79·1 cm LC for males (n = 51) and 80·1 cm LC for females (n = 17). Ovarian fecundity was up to 31 for C. carophila and sperm storage was confirmed in the oviducal gland of this species. Both species preyed on benthic invertebrates. Some C. carophila and H. homonycteris inhabit depths beyond most current fisheries, but both species appear to be relatively rare and have reproductive parameters characteristic of low productivity, which may make these species vulnerable to population decline if mortality was to increase in the future.

Population biology of an endangered species: the common guitarfish Rhinobatos rhinobatos in Lebanese marine waters of the eastern Mediterranean Sea.

This study focuses on the population biology of the common guitarfish Rhinobatos rhinobatos, a cartilaginous fish listed as Endangered in the International Union for the Conservation of Nature (IUCN) Red List. Between December 2012 and January 2014, 67 individuals were collected by bottom longlining in coastal Lebanese marine waters at different ports at depths ranging from 10 to 110 m. The total length (L(T)) of the specimens ranged from 50 to 143 cm, and the mean ± s.d. was 76.2 ± 19.7 cm. The most common L(T) classes were between 60 and 70 cm. The total mass of the specimens ranged from 410 to 10,000 g, and the mean ± s.d. was 1841 ± 1987 g. A total of 34 males and 33 females were collected, and the sex ratio was not significantly different from 1:1. The mass and L(T) relationship showed positive allometric growth (b = 3.096 and r(2) = 0.99), and the mean ± s.d. L(T) at which 50% of the individuals were sexually mature was 84.73 ± 5.81 cm for females and 78.57 ± 4.88 cm for males. The gonado-somatic and hepato-somatic indices were determined along with a condition factor, and parturition appeared to occur in winter. The primary prey items found in the fish stomachs during the autumn and winter seasons were Penaeidae. The results of this study will help to parameterize models of the population dynamics for this exploited fish stock to ensure the long-term sustainability of its fishery.

Age, growth and maturity of the pelagic thresher Alopias pelagicus and the scalloped hammerhead Sphyrna lewini.

Indonesia has the greatest reported chondrichthyan catches worldwide, with c.110,000 t caught annually. The pelagic thresher (Alopias pelagicus) and scalloped hammerhead (Sphryna lewini) together comprise about 25% of the total catches of sharks landed in Indonesia. Age and growth parameters were estimated for A. pelagicus and S. lewini from growth-band counts of thin-cut vertebral sections. Alopias pelagicus (n = 158) and S. lewini (n = 157) vertebrae were collected from three Indonesian fish markets over a 5 year period. A multi-model analysis was used to estimate growth parameters for both species. The models of best fit for males and females for A. pelagicus was the three-parameter logistic (L∞ = 3169 mm LT , k = 0·2) and the two-parameter von Bertalanffy models (L∞ = 3281 mm LT , k = 0·12). Age at maturity was calculated to be 10·4 and 13·2 years for males and females, respectively, and these are the oldest estimated for this species. The samples of S. lewini were heavily biased towards females, and the model of best fit for males and females was the three-parameter Gompertz (L∞ = 2598 mm LT , k = 0·15) and the two-parameter Gompertz (L∞ = 2896 mm LT , k= 0·16). Age at maturity was calculated to be 8·9 and 13·2 years for males and females, respectively. Although numerous age and growth studies have previously been undertaken on S. lewini, few studies have been able to obtain adequate samples from all components of the population because adult females, adult males and juveniles often reside in different areas. For the first time, sex bias in this study was towards sexually mature females, which are commonly lacking in previous biological studies on S. lewini. Additionally, some of the oldest aged specimens and highest age at maturity for both species were observed in this study. Both species exhibit slow rates of growth and late age at maturity, highlighting the need for a re-assessment of the relative resilience of these two globally threatened sharks at current high levels of fishing mortality throughout the eastern Indian Ocean.

Geographical variability in life-history traits of a midslope dogfish: the brier shark Deania calcea.

Deania calcea (n = 420) were collected from the catch of deep-water trawlers in the southern and eastern scalefish and shark fishery in southern Australia during the years 2008-2011. The total length (LT ) range varied between sexes, females being larger (n = 264; 280-1530 mm) than males (n = 156; 310-921 mm). The reproductive cycle in this population is non-continuous and asynchronous. The estimated LT at which 50% of males are mature is 807 mm and is 914 mm for females. Populations of D. calcea in higher latitudes appear to mature at a larger size than conspecifics in lower latitudes, in both the northern and southern hemispheres. Litters ranged from three to 10 embryos with a 1:1 sex ratio, but litter size does not increase with maternal LT . Deania calcea shows geographical variability in its biological parameters and gathering information on life-history traits of populations is vital to understand the trade-offs made by this species in response to environmental conditions and to predict intraspecific spatial differences. Such information is a basis for specific spatial management to protect populations from excessive fishing.

Slow growth of the overexploited milk shark Rhizoprionodon acutus affects its sustainability in West Africa.

Age and growth of Rhizoprionodon acutus were estimated from vertebrae age bands. From December 2009 to November 2010, 423 R. acutus between 37 and 112 cm total length (LT ) were sampled along the Senegalese coast. Marginal increment ratio was used to check annual band deposition. Three growth models were adjusted to the length at age and compared using Akaike's information criterion. The Gompertz growth model with estimated size at birth appeared to be the best and resulted in growth parameters of L∞ = 139.55 (LT ) and K = 0.17 year(-1) for females and L∞ = 126.52 (LT ) and K = 0.18 year(-1) for males. The largest female and male examined were 8 and 9 years old, but the majority was between 1 and 3 years old. Ages at maturity estimated were 5.8 and 4.8 years for females and males, respectively. These results suggest that R. acutus is a slow-growing species, which render the species particularly vulnerable to heavy fishery exploitation. The growth parameters estimated in this study are crucial for stock assessments and for demographic analyses to evaluate the sustainability of commercial harvests.

From egg to hatchling: preferential retention of fatty acid biomarkers in young-of-the-year Port Jackson sharks Heterodontus portusjacksoni.

The muscle and liver fatty acid composition of young-of-the-year (YOY) Port Jackson sharks Heterodontus portusjacksoni were investigated to determine the effects of a known dietary lipid source v. maternal input as demonstrated by egg yolk fatty acid profiles. Ten Heterodontus portusjacksoni egg yolks were collected in situ and compared with four hatched H. portusjacksoni fed a known diet in a controlled feeding experiment of 185 days. This study demonstrated that fatty acids are probably conservatively transferred from egg yolks to YOY H. portusjacksoni, while diet did not have a large effect on the fatty acid composition of the liver or muscle.

Bioaccumulation of Essential and Potentially Toxic Elements in the Muscle and Liver of the Spotted Ratfish (Hydrolagus colliei) From Deep-Sea Waters off the Northern Gulf of California.

This study aimed to establish the distribution of As, Cd, Cu, Pb, and Zn, in the muscle and liver of the spotted ratfish Hydrolagus colliei from the northern Gulf of California to establish the bioaccumulation background data in this species. The individuals (n = 110) were obtained by bycatch from the Gulf of California hake fisheries, and the metals and metalloid were measured by atomic absorption spectrometry. The element with the highest concentration in the muscle (15.19 ± 5.40 mg kg-1) and the liver (20.98 ± 10.30 mg kg-1) was As, followed by essential elements (Zn > Cu), and the lowest were the non-essential Pb (0.029 ± 0.014 and 0.048 ± 0.038 mg kg-1, muscle and liver, respectively) and Cd (0.022 ± 0.014 and 0.796 ± 0.495 mg kg-1, muscle and liver, respectively). The liver showed higher bioaccumulation than the muscle in all the studied elements. The sex was not a factor that influenced the bioaccumulation. The concentrations of As in the muscle did not exceed the maximum permissible limits of Mexican legislation, and < 50% of the samples exceed Cd and Pb limits of the Mexican, European Union, and WHO/FAO regulations. The differences found between the elements and tissues could be related to the different diets of the species, their migratory patterns, and their life conditions. Studies in the deep-sea water H. colliei are limited, and further investigations are needed regarding the feeding habits of H. colliei as well as the interactions of potentially toxic elements within the deep-sea water habitat.

Unique properties of prothrombin in the bullhead shark Heterodontus japonicus: the first report of purification and characterization of a blood coagulation factor in Chondrichthyes.

Prothrombin is a serine protease precursor of the blood coagulation system. In this study, the primary structure of prothrombin of a cartilaginous fish, bullhead shark (Heterodontus japonicus), was determined using RNA-Seq and the protein was purified from the blood plasma. Bullhead shark prothrombin was found to be comprised of four domains, as in the case of reported mammalian homologues. Two arginine residues that should be cleaved by activated factor X were found in the amino acid sequence of the shark prothrombin, but only one of the two cleavage sites for thrombin or meizothrombin was conserved. The apparent molecular mass of the shark prothrombin on SDS-PAGE was 110 kDa, whereas that of its amino acid sequence was 65 kDa. Potential N-glycosylation sites were found at 79th, 108th, 121st, 179th, 199th, 507th, and 527th asparagine residues in the shark prothrombin, and treatment with N-glycosidase reduced the molecular mass to 65 kDa. This indicates that, in contrast to human prothrombin, which has only 7-kDa N-glycans, the prothrombin of the shark is highly N-glycosylated. This study is the first to report on the purification and characterization of blood coagulation factors in a cartilaginous fish.

Blood collection under anesthesia, peripheral blood cells, plasma biochemistry, and plasma protein electrophoresis in a living fossil: the Spotted Ratfish (Hydrolagus colliei).

This report describes the safe and effective handling under anesthesia, blood collection and main clinical pathology values determination for three adult Spotted Ratfish (Hydrolagus colliei) (two males and one female) successfully maintained under human care for more than 20 years. The anesthetic MS-222 diluted in tamponed salt water at 50 ppm provided deep narcosis with mean induction and recovery times of 5 and 20 min, allowing safe handling and blood collection in the three animals sampled. Major leukocyte types were similar to other teleost and elasmobranch species, identifying lymphocytes as the predominant leukocyte (75.5%), followed by the fine eosinophilic granulocytes (10.25%), the coarse eosinophilic granulocytes (9.75%), and a small percentage of monocytes (5.50%). Plasma biochemistry values in this species were like those seen in elasmobranchs, with the highest levels of blood urea nitrogen described in any Chondrichthyes (mean 679.7 mmol/l). Plasma protein electrophoresis analysis in the Spotted Ratfish showed five consistent fractions, like those previously described in other fish species, with a negligible quantity of proteins migrating in the region equivalent to albumin, and with fraction 4 (mean 53.20%) as the predominant fraction. Despite the limitations imposed by the small sample size and the challenging access to the studied species under human care, this study will shed light on and enhance clinical knowledge regarding Ratfish handling, anesthesia, blood collection, and analysis. It aims to deliver a comprehensive clinical pathology description, presenting valuable data for professionals engaged in the care and management of Chimaerans.

Erratum: Pharmacokinetic studies in elasmobranchs: Meloxicam administered at 0.5 mg/kg using intravenous, intramuscular, and oral routes to nursehound sharks (Scyliorhinus stellaris).

[This corrects the article DOI: 10.3389/fvets.2022.845555.].

Structure and permeability of the egg capsule of the placental Australian sharpnose shark, Rhizoprionodon taylori.

Shark placentae are derived from modifications to the fetal yolk sac and the maternal uterine mucosa. In almost all placental sharks, embryonic development occurs in an egg capsule that remains intact for the entire pregnancy, separating the fetal tissues from the maternal tissues at the placental interface. Here, we investigate the structure and permeability of the egg capsules that surround developing embryos of the placental Australian sharpnose shark (Rhizoprionodon taylori) during late pregnancy. The egg capsule is an acellular fibrous structure that is 0.42 ± 0.04 µm thick at the placental interface between the yolk sac and uterine tissues, and 0.67 ± 0.08 µm thick in the paraplacental regions. This is the thinnest egg capsule of any placental shark measured so far, which may increase the diffusion rate of respiratory gases, fetal wastes, water and nutrients between maternal and fetal tissues. Molecules smaller than or equal to ~ 1000 Da can diffuse through the egg capsule, but larger proteins (~ 3000-26,000 Da) cannot. Similar permeability characteristics between the egg capsule of R. taylori and other placental sharks suggest that molecular size is an important determinant of the molecules that can be exchanged between the mother and her embryos during pregnancy.

Pharmacokinetics of the Anti-Inflammatory Drug Meloxicam after Single 1.5 mg/kg Intramuscular Administration to Undulate Skates (Raja undulata).

The therapy database currently used in elasmobranchs is still mostly based on empirical data, and there are few efficacy and safety studies supporting clinical practice. In this study, meloxicam pharmacokinetics (PK) were evaluated after a single 1.5 mg/kg IM administration to a group of seven clinically healthy adult undulate skates (Raja undulata Lacepède, 1802). Blood samples were collected before administration and at 15, 30, 60 and 90 min and 2, 4, 8, 12, 24 and 48 h after the IM injection. The meloxicam concentrations in plasma were determined using high-performance liquid chromatography, and PK parameters were calculated using a non-compartmental model approach. The mean ± SEM values of the main PK values were 1.84 ± 0.31 µg/mL for peak plasma concentration, 1.5 ± 0.24 h for time to maximum plasma concentration, 11.43 ± 2.04 h·µg/mL for area under the plasma concentration vs. time curve, 3.55 ± 0.65 h for elimination half-life, and 5.37 ± 0.94 h for mean residency time. No adverse reactions were detected. The relatively high plasma concentration and short time to maximum plasma concentration suggest that meloxicam could turn into an efficient analgesic and anti-inflammatory candidate drug to be used in skates. Further efficacy, pharmacodynamic, and multiple-dose studies with meloxicam are needed in elasmobranchs.

Pharmacokinetic Studies in Elasmobranchs: Meloxicam Administered at 0.5 mg/kg Using Intravenous, Intramuscular, and Oral Routes to Nusehound Sharks (Scyliorhinus stellaris).

Infectious and inflammatory diseases are the most frequently diagnosed pathologies in elasmobranchs maintained under human care. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in veterinary medicine for their anti-inflammatory, analgesic, and antipyretic properties. Meloxicam is a commonly prescribed NSAID in elasmobranchs, but there are still no published pharmacokinetic (PK) studies supporting its use in this group of animals. In this study, meloxicam was administered at a single dose of 0.5 mg/kg to eight healthy adult nursehound sharks (Scyliorhinus stellaris) intravenously (IV), intramuscularly (IM), and orally (PO), with a minimum 4-week washout period between administrations. Blood samples were obtained both beforehand and at predetermined times after each administration. Plasma concentrations were measured using a validated high performance liquid chromatography method, and PK data was obtained using a non-compartmental analysis. Meloxicam administered orally did not produce detectable concentrations in blood plasma, while mean peak plasma concentration was 0.38 ± 0.08 µg/ml after IM administration. The mean terminal half-life was 10.71 ± 2.77 h and 11.27 ± 3.96 h for IV and IM injections, respectively. The area under the curve extrapolated to infinity was 11.37 ± 2.29 h·µg/ml after IV injections and 5.98 ± 0.90 h·µg/ml after IM injections. Meloxicam administered IM had a mean absolute bioavailability of 56.22 ± 13.29%. These numbers support meloxicam as a promising drug to be used IM in nursehounds, questions the efficacy of its single PO use in elasmobranchs, elucidate the need for higher dosage regimes, and evidence the need for further PK studies in sharks and rays.