In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Fin whale (Balaenoptera physalus) population identity in the western Mediterranean Sea

Archival bottom‐mounted audio recorders were deployed in nine different areas of the western Mediterranean Sea, Strait of Gibraltar, and adjacent North Atlantic waters during 2006–2009 to study fin whale (Balaenoptera physalus) seasonal presence and population structure. Analysis of 29,822 recording hours revealed typical long, patterned sequences of 20 Hz notes (here called “song”), back‐beats, 135–140 Hz notes, and downsweeps. Acoustic parameters (internote interval, note duration, frequency range, center and peak frequencies) were statistically compared among songs and song notes recorded in all areas. Fin whale singers producing songs attributable to the northeastern North Atlantic subpopulation were detected crossing the Strait of Gibraltar and wintering in the southwestern Mediterranean Sea (Alboran basin), while songs attributed to the Mediterranean were detected in the northwest Mediterranean basin. These results suggest that the northeastern North Atlantic fin whale distribution extends into the southwest Mediterranean basin, and spatial and temporal overlap may exist between this subpopulation and the Mediterranean subpopulation. This new interpretation of the fin whale population structure in the western Mediterranean Sea has important ecological and conservation implications. The conventionally accepted distribution ranges of northeastern North Atlantic and Mediterranean fin whale subpopulations should be reconsidered in light of the results from this study.     

Global-scale genetic identification of hammerhead sharks: Application to assessment of the international fin trade and law enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (“Bai Chun” with S. lewini, “Gui Chun” with S. zygaena and “Gu Pian” with S.␣mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified “ S. lewini” from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast.     

Genetic profiling reveals illegal international trade in fins of the great white shark, <Emphasis Type="Italic">Carcharodon carcharias</Emphasis>

Great white sharks are protected by national legislation in several countries, making this species the most widely protected elasmobranch in the world. Although the market demand for shark fins in general has continued to grow, the value and extent of utilization of white shark fins in trade has been controversial. We combine law enforcement with genetic profiling to demonstrate that illegal trade in fins of this species is occurring in the contemporary international market. Furthermore, we document the presence of fins from very young white sharks in the trade, suggesting a multiple-use market (food to trophies) exists for fins of this species. The presence of small fins in the trade contradicts the view that white shark fins have market value only as large display trophies, and not as food. Our findings indicate that effective conservation of protected shark species will require international management regimes that include monitoring of the shark fishery and trade on a species-specific basis.     

Developmental constraints on fin diversity

The fish fin is a breathtaking repository full of evolutionary diversity, novelty, and convergence. Over 500 million years, the adaptation to novel habitats has provided landscapes of fin diversity. Although comparative anatomy of evolutionarily divergent patterns over centuries has highlighted the fundamental architectures and evolutionary trends of fins, including convergent evolution, the developmental constraints on fin evolution, which bias the evolutionary trajectories of fin morphology, largely remain elusive. Here, we review the evolutionary history, developmental mechanisms, and evolutionary underpinnings of paired fins, illuminating possible developmental constraints on fin evolution. Our compilation of anatomical and genetic knowledge of fin development sheds light on the canalized and the unpredictable aspects of fin shape in evolution. Leveraged by an arsenal of genomic and genetic tools within the working arena of spectacular fin diversity, evolutionary developmental biology embarks on the establishment of conceptual framework for developmental constraints, previously enigmatic properties of evolution.     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose     

A new rheophilic South American darter (Crenuchidae: Characidium) from the rio Juruena basin,Brazil, with comments on morphological adaptations to life in fast-flowing waters

Characidium iaquira, a new species from the upper rio Juruena, rio Tapajós basin, Brazil, is described. The new species can be promptly distinguished from all congeners by having a unique v-shaped dark mark lying along the caudal-fin extension, in medium- and large-sized specimens, and a remarkable iridescent green colouration in life. Characidium iaquira is closely related to Characidium crandellii and Characidium declivirostre by sharing unambiguous synapomorphies such as branchiostegal membranes united to each other across the isthmus, a scaleless area extending from the isthmus to the pectoral girdle, and dermal flaps surrounding anterior and posterior naris independent, but touching each other distally. Morphological specializations of the paired fins in the three riffle-dwellers species are discussed, including the wing-like shape, robustness, and inclination of the pectoral fin.     

Advantages of a Biomimetic Stiffness Profile in Pitching Flexible Fin Propulsion

<正> The use of oscillating flexible fins in propulsion has been the subject of several studies in recent years, but attention israrely paid to the specific role of stiffness profile in thrust production.Stiffness profile is defined as the variation in localchordwise bending stiffness (EI) of a fin, from leading to trailing edge.In this study, flexible fins with a standard NACA0012shape were tested alongside fins with a stiffness profile mimicking that of a Pumpkinseed Sunfish (Lepomis gibbosus).The finswere oscillated with a pitching sinusoidal motion over a range of frequencies and amplitudes, while torque, lateral force andstatic thrust were measured.Over the range of oscillation parameters tested, it was shown that the fin with a biomimetic stiffness profile offered a significantimprovement in static thrust, compared to a fin of similar dimensions with a standard NACA0012 aerofoil profile.Thebiomimetic fin also produced thrust more consistently over each oscillation cycle.A comparison of fin materials of different stiffness showed that the improvement was due to the stiffness profile itself, andwas not simply an effect of altering the overall stiffness of the fin.Fins of the same stiffness profile were observed to follow thesame thrust-power curve, independent of the stiffness of the moulding material.Biomimetic fins were shown to produce up to26% greater thrust per watt of input power, within the experimental range.     

Internal morphology and function of paired fins in the epaulette shark,hemiscyllium ocellatum

Paired fins and associated internal structures of the epauletic sharkHemiscyllium ocellatum, were described on the basis of three specimens. A comparison with other genera showed the epaulette shark to be, characterized by two elongated basal cartilages articulating with a distally projecting articular condyle on the coracoid, a loosely separated radial series with an intermediate series, a levator pectoralis inferior muscle and an anterolaterally developed depressor pectoralis muscle in the pectoral fin, and an elongated anterior pelvic basal cartilage articulating with a distally projecting articular condyle and an anterolaterally developed depressor pelvicus muscle in the pelvic fin. In captivity, the sharks exhibited both upright and crawling behavior on the bottom by using the pectoral and pelvic fins and bending the body. The distinctive morphological characters are shared by otherHemiscyllium species and are suggested as important factors enabling their unique behavior associated with a complex coral reef habitat.     

Two Reproductive Life History Types of Kokanee, Onchorynchus nerka, Exhibit Multivariate Morphometric and Protein Genetic Differentiation

Two reproductive types of kokanee are found in Okanagan Lake, British Columbia: one form that spawns in streams, and another that spawns approximately 2–4 weeks later along beaches of the lake. We examined the levels and patterns of genetic and morphometric variation among three populations (1 beach and 2 stream populations) to better understand life history differentiation. We assayed allozyme variation at 74 loci and identified 15 P_0.095 loci. Average F_ST was 0.041 among the three samples; the two stream-spawning populations grouped together in genetic distance analyses. We examined multivariate morphometric variation and fin size/shape variation using 35 truss characters. Populations did not sort morphologically by spawning type in principal component and relative warp analyses. Instead both analyses indicated that one stream-spawning population (Peachland Creek) was significantly more robust and had shallower caudal fins in comparison to the other samples. Second, clear multivariate shape differences between males and females were identified involving jaw size, mid-body dimensions, and caudal fin shape. A simple bivariate plot of tail `forkness' against fork length demonstrated that males had more forked tails and females had flattened tails. Level of differentiation of secondary sexual characteristics did not vary between the spawning types, although the beach-spawning population and one stream-spawning population did not show the strong, gender-discriminating variability in tail forkness. Although these two reproductive life history types of kokanee form discrete genetic groups, environmental differences are apparently insufficient to effect consistent differences in body shape, or fin size and shape between stream and beach-spawning morphs.