Life-history traits of the spiny dogfish Squalus acanthias in the Adriatic Sea.

Pivotal life history traits concerning age structure and reproduction of the spiny dogfish (Squalus acanthias, Linnaeus 1758) were investigated in the Adriatic Sea from mid February 2012 to mid July 2013 and in 2016. The whole sample consisted of 176 females and 150 males, ranging between 217-1025 mm and 219-875 mm, respectively. The individual age, which was estimated using a cross-sectioning technique of the second dorsal-fin spine, ranged from 0 to 13+ years for females and from 0 to 9+ years for males. Based on the length-at-age estimates, the Gompertz growth parameters were L∞ = 1130 mm, k = 0.18 and L∞ = 920 mm, k = 0.24 for females and males, respectively. The size at sexual maturity (L50) was 659 mm for females and 575 mm for males, corresponding to 7.5 and 5.5 years of age (A50), respectively. Mean biennial fecundity was approximately 11 embryos/female and 12 ripe oocytes/female. Mature males occurred during much of the sampling period, while mature females with nearly full-term embryos were exclusively recorded in May 2013 and July 2016. Monitoring of catches conducted in a sample port of the north Adriatic (Chioggia) over the past 20 years has shown fluctuating trends in landings, with peaks during the summer reproductive season.

Functional analysis of the musculo-skeletal system of the gill apparatus in Heptranchias perlo (Chondrichthyes: Hexanchidae).

Musculo-skeletal morphology is an indispensable source for understanding functional adaptations. Analysis of morphology of the branchial apparatus of Hexanchiform sharks can provide insight into aspects of their respiration that are difficult to observe directly. In this study, I compare the structure of the musculo-skeletal system of the gill apparatus of Heptranchias perlo and Squalus acanthias in respect to their adaptation for one of two respiratory mechanisms known in sharks, namely, the active two-pump (oropharyngeal and parabranchial) ventilation and the ram-jet ventilation. In both species, the oropharyngeal pump possesses two sets of muscles, one for compression and the other for expansion. The parabranchial pump only has constrictors. Expansion of this pump occurs only due to passive elastic recoil of the extrabranchial cartilages. In Squalus acanthias the parabranchial chambers are large and equipped by powerful superficial constrictors. These muscles and the outer walls of the parabranchial chambers are much reduced in Heptranchias perlo, and thus it likely cannot use this pump. However, this reduction allows for vertical elongation of outer gill slits which, along with greater number of gill pouches, likely decreases branchial resistance and, at the same time, increases the gill surface area, and can be regarded as an adaptation for ram ventilation at lower speeds.

3D scanning and printing skeletal tissues for anatomy education.

Detailed anatomical models can be produced with consumer-level 3D scanning and printing systems. 3D replication techniques are significant advances for anatomical education as they allow practitioners to more easily introduce diverse or numerous specimens into classrooms. Here we present a methodology for producing anatomical models in-house, with the chondrocranium cartilage from a spiny dogfish (Squalus acanthias) and the skeleton of a cane toad (Rhinella marina) as case studies. 3D digital replicas were produced using two consumer-level scanners and specimens were 3D-printed with selective laser sintering. The fidelity of the two case study models was determined with respect to key anatomical features. Larger-scale features of the dogfish chondrocranium and frog skeleton were all well-resolved and distinct in the 3D digital models, and many finer-scale features were also well-resolved, but some more subtle features were absent from the digital models (e.g. endolymphatic foramina in chondrocranium). All characters identified in the digital chondrocranium could be identified in the subsequent 3D print; however, three characters in the 3D-printed frog skeleton could not be clearly delimited (palatines, parasphenoid and pubis). Characters that were absent in the digital models or 3D prints had low-relief in the original scanned specimen and represent a minor loss of fidelity. Our method description and case studies show that minimal equipment and training is needed to produce durable skeletal specimens. These technologies support the tailored production of models for specific classes or research aims.

Reproductive and population parameters of spiny dogfish Squalus acanthias in the south-western Atlantic Ocean.

The objective of this study was to estimate reproductive and population parameters of the spiny dogfish Squalus acanthias for the south-western Atlantic Ocean. In total, 2714 specimens (1616 males and 1098 females) were collected from surveys carried out using research vessels. Males ranged from 225 to 861 mm total length (LT ) and females from 235 to 925 mm LT . The size at maturity of females (651 mm) was significantly greater than that of males (565 mm). The maximum proportion of mature individuals (Pmax ) of the gestation ogive was <1, which indicates that a proportion of mature females was not in gestation. This inactivity may be explained by the occurrence of resting periods between cycles or by the asynchrony of the reproductive cycle. The estimated Pmax for the maternity ogive suggested that about one third of mature females were in the maternity stage (i.e. with embryos >156 mm). The temporal and spatial co-occurrence of non-gravid adult females at different stages of ovarian development, as well as gravid females at all embryonic development stages would indicate that the female reproductive cycle in the south-western Atlantic Ocean is asynchronous. The results indicate that S. acanthias is susceptible to fishing pressure on account of its length at maturity, extended reproductive cycles and low fecundity.

Muscles of chondrichthyan paired appendages: comparison with osteichthyans, deconstruction of the fore-hindlimb serial homology dogma, and new insights on the evolution of the vertebrate neck.

Here we present the first study comparing all the paired appendages muscles of representatives of each major extant gnathostome group. We address a crucial and enigmatic question in evolutionary and comparative anatomy: Why are the pelvic and pectoral appendages of gnathostomes, and particularly of tetrapods, in general so similar to each other? We argue that an integrative analysis of the new myological data and the information from the literature contradicts the idea that the forelimbs and hindlimbs are serial homologues. The data show that many of the strikingly similar fore- and hindlimb muscles of extant tetrapods evolved independently in each appendage because the ancestors of extant gnathostomes and osteichthyans only had an adductor and an abductor in each fin. Therefore, these data contradict the idea that at least some muscles present in the tetrapod fore- and hindlimbs were already present in some form in the first fishes with pectoral and pelvic appendages, as the result of an ancestral duplication of the paired appendages leading to a true serial homology. The origin of the pectoral girdle was instead likely related to head evolution, as illustrated by the cucullaris of gnathostomes such as chondrichthyans inserting onto both the branchial arches and pectoral girdle. Only later in evolution the cucullaris became differentiated into the levatores arcuum branchialium and protractor pectoralis, which gave rise to the amniote neck muscles trapezius and sternocleidomastoideus. These changes therefore contributed to an evolutionary trend toward a greater anatomical and functional independence of the pectoral girdle from head movements.

Comparative squamation of the lateral line canal pores in sharks.

The current study collected the first quantitative data on lateral line pore squamation patterns in sharks and assessed whether divergent squamation patterns are similar to experimental models that cause reduction in boundary layer turbulence. In addition, the hypothesis that divergent orientation angles are exclusively found in fast-swimming shark species was tested. The posterior lateral line and supraorbital lateral line pore squamation of the fast-swimming pelagic shortfin mako shark Isurus oxyrinchus and the slow-swimming epi-benthic spiny dogfish shark Squalus acanthias was examined. Pore scale morphology and pore coverage were qualitatively analysed and compared. In addition, pore squamation orientation patterns were quantified for four regions along the posterior lateral line and compared for both species. Isurus oxyrinchus possessed consistent pore scale coverage among sampled regions and had a divergent squamation pattern with multiple scale rows directed dorsally and ventrally away from the anterior margin of the pore with an average divergent angle of 13° for the first row of scales. Squalus acanthias possessed variable amounts of scale coverage among the sampled regions and had a divergent squamation pattern with multiple scale rows directed ventrally away from the anterior margin of the pore with an average angle of 19° for the first row of scales. Overall, the squamation pattern measured in I. oxyrinchus fell within the parameters used in the fluid flow analysis, which suggests that this pattern may reduce boundary layer turbulence and affect lateral line sensitivity. The exclusively ventral oriented scale pattern seen in S. acanthias possessed a high degree of divergence but the pattern did not match that of the fluid flow models. Given current knowledge, it is unclear how this would affect boundary layer flow. By studying the relationship between squamation patterns and the lateral line, new insights are provided into sensory biology that warrant future investigation due to the implications for the ecology, morphology and sensory evolution of sharks.

Anatomy and muscle activity of the dorsal fins in bamboo sharks and spiny dogfish during turning maneuvers.

Stability and procured instability characterize two opposing types of swimming, steady and maneuvering, respectively. Fins can be used to manipulate flow to adjust stability during swimming maneuvers either actively using muscle control or passively by structural control. The function of the dorsal fins during turning maneuvering in two shark species with different swimming modes is investigated here using musculoskeletal anatomy and muscle function. White-spotted bamboo sharks are a benthic species that inhabits complex reef habitats and thus have high requirements for maneuverability. Spiny dogfish occupy a variety of coastal and continental shelf habitats and spend relatively more time cruising in open water. These species differ in dorsal fin morphology and fin position along the body. Bamboo sharks have a larger second dorsal fin area and proportionally more muscle insertion into both dorsal fins. The basal and radial pterygiophores are plate-like structures in spiny dogfish and are nearly indistinguishable from one another. In contrast, bamboo sharks lack basal pterygiophores, while the radial pterygiophores form two rows of elongated rectangular elements that articulate with one another. The dorsal fin muscles are composed of a large muscle mass that extends over the ceratotrichia overlying the radials in spiny dogfish. However, in bamboo sharks, the muscle mass is divided into multiple distinct muscles that insert onto the ceratotrichia. During turning maneuvers, the dorsal fin muscles are active in both species with no differences in onset between fin sides. Spiny dogfish have longer burst durations on the outer fin side, which is consistent with opposing resistance to the medium. In bamboo sharks, bilateral activation of the dorsal in muscles could also be stiffening the fin throughout the turn. Thus, dogfish sharks passively stiffen the dorsal fin structurally and functionally, while bamboo sharks have more flexible dorsal fins, which result from a steady swimming trade off.

Reassessment of spiny dogfish Squalus acanthias age and growth using vertebrae and dorsal-fin spines.

Male and female spiny dogfish Squalus acanthias were collected in the western North Atlantic Ocean in the Gulf of Maine between July 2006 and June 2009. Squalus acanthias ranged from 25 to 102 cm stretch total length and were caught during all months of the year except January. Age estimates derived from banding patterns visible in both the vertebrae and second dorsal-fin spines were compared. Vertebral growth increments were visualized using a modified histological staining technique, which was verified as appropriate for obtaining age estimates. Marginal increment analysis of vertebrae verified the increment periodicity, suggesting annual band deposition. Based on increased precision and accuracy of age estimates, as well as more biologically realistic parameters generated in growth models, the current study found that vertebrae provided a more reliable and accurate means of estimating age in S. acanthias than the second dorsal-fin spine. Age estimates obtained from vertebrae ranged from <1 year-old to 17 years for male and 24 years for female S. acanthias. The two-parameter von Bertalanffy growth model fit to vertebrae-derived age estimates produced parameters of L∞ = 94·23 cm and k = 0·11 for males and L∞ = 100·76 cm and k = 0·12 for females. While these growth parameters differed from those previously reported for S. acanthias in the western North Atlantic Ocean, the causes of such differences were beyond the scope of the current study and remain to be determined.

Testicular degeneration during spermatogenesis in the blue shark, Prionace glauca: nonconformity with expression as seen in the diametric testes of other carcharhinids.

The elasmobranch testis consists of spherical spermatocysts, each housing a single germ cell stage and its own clone of Sertoli cells. Because of the simple diametrical arrangement of cysts in maturational order, the testes of Squalus acanthias, Scyliorhinus canicula, and Prionace glauca are classified as the diametric shark testis type. The aim of this study was to document histologically the spermatocyst composition in the blue shark stage-by-stage and to establish whether the diametric testis type confers any uniformity regarding the expression of spermatogenesis in all sharks with this testis type. Analysis of the testes of blue sharks breeding in summer revealed extensive cyst degeneration of various forms and degrees, cyst shrinkage, and cyst disorganization with or without evidence of cell death, initially at the spermatogonia-spermatocyte transition but predominantly in spermatocyte and spermatid cysts. Animals could be grouped into two categories based on the major degenerative phenomena observed, namely those with extensive multinucleate cell (MNC) formation, and those with pronounced vacuolation in cysts. A major finding was the significant (P < 0.001) predominance of MNC formation and vacuolation in late-stage spermatogonial cysts in the respective categories of sharks. Spermatocyte cysts showed varying degrees of germ cell depletion, with or without evidence of degeneration. Normal-looking, but clearly subnormal-sized primary and secondary spermatocyte cysts with no evidence of degeneration were significantly the dominant spermatocyte cyst types in both categories. It is proposed that these subnormal-sized spermatocyte cysts could proceed into spermiogenesis. Because neighboring spermatid cysts lacked ordered bundling of spermatid heads (disorganized), a morphology significantly correlated with the vacuolation category of sharks, these results suggest that further progression into spermiogenesis was halted in such cysts. Thus, testicular degeneration in the diametric testis type is species specific in quantity and quality.

Ontogenetic scaling of caudal fin shape in Squalus acanthias (Chondrichthyes, Elasmobranchii): a geometric morphometric analysis with implications for caudal fin functional morphology.

The shark heterocercal caudal fin and its contribution to locomotion are of interest to biologists and paleontologists. Current hydrodynamic data show that the stiff dorsal lobe leads the ventral lobe, both lobes of the tail are synchronized during propulsion, and tail shape reflects its overall locomotor function. Given the difficulties surrounding the analysis of shark caudal fins in vivo, little is known about changes in tail shape related to ontogeny and sex in sharks. A quantifiable analysis of caudal fin shape may provide an acceptable proxy for inferring gross functional morphology where direct testing is difficult or impossible. We examined ontogenetic and sex-related shape changes in the caudal fins of 115 Squalus acanthias museum specimens, to test the hypothesis that significant shape changes in the caudal fin shape occur with increasing size and between the sexes. Using linear and geometric morphometrics, we examined caudal shape changes within the context of current hydrodynamic models. We found no statistically significant linear or shape difference between sexes, and near-isometric scaling trends for caudal dimensions. These results suggest that lift and thrust increase linearly with size and caudal span. Thin-plate splines results showed a significant allometric shape change associated with size and caudal span: the dorsal lobe elongates and narrows, whereas the ventral lobe broadens and expands ventrally. Our data suggest a combination of caudal fin morphology with other body morphology aspects, would refine, and better elucidate the hydrodynamic factors (if any) that underlie the significant shape changes we report here for S. acanthias.

An evolutionary view on tooth development and replacement in wild Atlantic salmon (Salmo salar L.).

To gain an insight into the evolution of tooth replacement mechanisms, we studied the development of first-generation and replacement teeth on the dentary of wild Atlantic salmon (Salmo salar L.), a protacanthopterygian teleost, using serially sectioned heads of early posthatching stages as well as adults. First-generation teeth develop within the oral epithelium. The anlage of the replacement tooth is first seen as a placode-like thickening of the outer dental epithelium of the predecessor, at its lingual and caudal side. Ongoing development of the replacement tooth germ is characterized by the elaboration of a population of epithelial cells, termed here the middle dental epithelium, apposed to the inner dental epithelium on the lingual side of the tooth germ. Before the formation of the new successor, a single-layered outer dental epithelium segregates from the middle dental epithelium. The dental organs of the predecessor and the successor remain broadly interconnected. The absence of a discrete successional dental lamina in salmon stands in sharp contrast to what is observed in other teleosts, even those that share with salmon the extraosseous formation of replacement teeth. The mode of tooth replacement in Atlantic salmon displays several characters similar to those observed in the shark Squalus acanthias. To interpret similarities in tooth replacement between Atlantic salmon and chondrichthyans as a case of convergence, or to see them as a result of a heterochronic shift, requires knowledge on the replacement process in more basal actinopterygian lineages. The possibility that the middle dental epithelium functionally substitutes for a successional lamina, and could be a source of stem cells, whose descendants subsequently contribute to the placode of the new replacement tooth, needs to be explored.

Regulation of the Na+2Cl­K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.

Previously it has been shown that the Na+2Cl­K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl­K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl­ solution (6 mmol/l, low Cl­) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl­ had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl­ (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na+2Cl­K+ cotransporter probably independently: (1) lowering of cytosolic Cl­ concentration; (2) cell shrinkage; and (3) activation by cAMP.

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl­ channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl­ channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl­ channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl­K+ uptake and to reduce cell volume but not membrane trafficking of Cl­ channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl­ concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an "unfolding" of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl­ channels in these cells.