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.     

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.     

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.     

NF-κB Signaling Negatively Regulates Osteoblast Dedifferentiation during Zebrafish Bone Regeneration

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Ultraviolet absorbance of the mucus of a tropical damselfish: effects of ontogeny, captivity and disease

The ultraviolet (UV) absorbance of the mucus of a Great Barrier Reef damselfish Pomacentrus amboinensis was investigated with regard to ontogeny and time spent in captivity. The UV absorbance of P. amboinensis mucus increased with fish size and decreased with time spent in captivity. The wavelength of maximum absorbance of the mucus did not change with fish size, but shifted towards shorter wavelengths with increasing time spent in captivity. The UV absorbance of the mucus of fish with 'fin rot' was compared to that of similar healthy individuals, and a significant decrease in UV absorbance of unhealthy fish mucus was detected; no wavelength shifting occurred. Pomacentrus amboinensis appears to sequester mycosporine-like amino acids from the diet in order to protect epithelial tissues from UV damage, and decreases in UV absorbance in captive fish were probably due to insufficient dietary availability.     

Bluegill Lepomis macrochirus synchronize pectoral fin motion and opercular pumping

The relative timing between operculum and pectoral fin motion was examined in swimming bluegill Lepomis macrochirus to determine if respiratory fluid flows from the operculum might have an effect on flow over the pectoral fin. Five bluegill were filmed swimming at speeds from 0·5 to 1·5 body (total) lengths s⁻¹. The timing of opercular pumping and pectoral fin beating was noted and analysed using circular statistics. Fish tended to ventilate their gills every second or third pectoral fin beat. While locomotion and ventilation had different frequencies, however, they were synchronized: fish maintained a consistent phase relationship between them. Thus, within pectoral fin beats when the operculum pumps, the jet consistently occurred during pectoral fin abduction, ending just after the fin was fully abducted and beginning adduction. Based on the distance between the opercular slit and the pectoral fin base, the jet was estimated to reach the fin during maximum abduction. Dye flow visualization confirmed this estimate, revealing that the opercular flow wraps around the base of the fin during peak abduction, when it is likely to have little hydrodynamic effect.     

实验室条件下唐鱼两性异形及其与游泳能力关系

为了解唐鱼两性异形及其与游泳能力关系,检测了性成熟阶段唐鱼躯干部和鱼鳍形态特征以及爆发游泳速度(U_burst)和临界游泳速度(U_crit)在雌雄之间的差异,旨在从形态适应角度探究长期进化中雌雄唐鱼各自面对选择压力所产生的游泳能力差异及其机制,从而为野生唐鱼保护提供基础数据.结果表明: 雌性唐鱼的体长、头高、头宽、尾鳍面积以及吻端至枕骨后末端、腹鳍起点至背鳍末端等长度均与雄性无显著差异.而体高、体宽、腹鳍起点至背鳍起点等反映腹腔大小的形态参数以及吻端至背鳍起点、吻端至臀鳍起点、枕骨后末端至背鳍起点等反映躯干部大小的形态参数均显示为雌性显著大于雄性,但头长以及胸鳍面积、腹鳍面积、背鳍面积和臀鳍面积均显示为雄性显著大于雌性.对所有数据进行主成分分析,结果显示第1主成分贡献率为74.2%,负载量较大的是体长、头长、头高、体高、头宽、体宽以及各鳍之间距离等主要反映唐鱼躯干整体特征的参数;第2主成分贡献率为15.7%,负载量较大的是胸鳍面积、腹鳍面积、背鳍面积和臀鳍面积等主要反映鱼鳍特征的参数.唐鱼性别在第1主成分上无法区分,但在第2主成分却可以明显区分.根据体宽、胸鳍面积、腹鳍面积、背鳍面积和臀鳍面积等建立的性别判别方程对雌雄判断准确率达到91.8%～92.5%.唐鱼游泳能力测定结果显示,雌性U_burst与雄性无显著差异,但U_crit显著小于雄性.以上结果表明,雌雄唐鱼两性异形主要集中在与游泳能力相关的鱼鳍特征上.相比雄性,雌性唐鱼虽然胸鳍等鱼鳍面积较小导致其U_crit小于雄性,却具有更长的躯干部以保证其同样具有较高的爆发游泳能力,从而有利于在流速波动很大的溪流中躲避捕食和进行其他应急活动;相比雌性,雄性唐鱼则具有较大的鱼鳍面积保证其U_crit高于雌性,以利于日常活动及在繁殖过程中追逐雌性等相对持久性游泳运动.     

Mysticete migration revisited: are Mediterranean fin whales an anomaly?

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