Musculoskeletal morphology of the pelvis and pelvic fins in the lungfish Protopterus annectens

The West African lungfish (Protopterus annectens) performs benthic, pelvic fin‐driven locomotion with gaits common to tetrapods, the sister group of the lungfishes. Features of P. annectens movement are similar to those of modern tetrapods and include use of the distal region of the pelvic fin as a “foot,” use of the fin to lift the body above the substrate and rotation of the fin around the joint with the pelvis. In contrast to these similarities in movement, the pelvic fins of P. annectens are long, slender structures that are superficially very different from tetrapod limbs. Here, we describe the musculoskeletal anatomy of the pelvis and pelvic fins of P. annectens with dissection, magnetic resonance imaging, histology and 3D‐reconstruction methods. We found that the pelvis is embedded in the hypaxial muscle by a median rostral and two dorsolateral skeletal projections. The protractor and retractor muscles at the base of the pelvic fin are fan‐shaped muscles that cup the femur. The skeletal elements of the fin are serially repeating cartilage cylinders. Along the length of the fin, repeating truncated cones of muscles, the musculus circumradialis pelvici, are separated by connective tissue sheets that connect the skeletal elements to the skin. The simplicity of the protractor and retractor muscles at the base of the fin is surprising, given the complex rotational movement those muscles generate. In contrast, the series of many repeating segmental muscles along the length of the fin is consistent with the dexterity of bending of the distal limb. P. annectens can provide a window into soft‐tissue anatomy and sarcopterygian fish fin function that complements the fossil data from related taxa. This work, combined with previous behavioral examination of P. annectens, illustrates that fin morphologies that do not appear to be capable of walking can accomplish that function, and may inform the interpretation of fossil anatomical evidence. J. Morphol. 275:431–441, 2014. © 2013 Wiley Periodicals, Inc.     

Immunohistochemical demonstration of calbindin-D 28K (CABP28K) in the spinal cord motoneurons of teleost fish

Summary The distribution and localization of the calciumbinding protein, calbindin-D 28K (CaBP28K), in the spinal cord motoneurons of larvae of the teleost fish, Apteronotus leptorhynchus (Gymnotidae) and Pollimyrus isidori (Mormyridae), and in the adult goldfish, Carassius auratus (Cyprinidae), were determined by means of immunohistochemistry. Sections of whole larvae and goldfish spinal cord were reacted with a polyclonal antibody to rat renal CaBP28K. CaBP28K was located by the PAP technique (Sternberger). It was found in the soma, dendrites, axons and axon terminals of spinal motoneurons but not in those of electromotoneurons of Apteronotus leptorhynchus, whereas it occurred in both motoneurons and electromotoneurons of the larval electric organ of Pollimyrus isidori. In these species CaBP28K was also present in the electromotoneuron axon terminals that make synaptic contacts with the pedicles of the electrocytes. In adult Carassius auratus, CaBP28K was found in the soma, dendrites and axons of certain spinal motoneurons. The results indicate that, in teleosts, the motoneurons containing CaBP28K may represent a well-defined population within the spinal cord; the role of this protein in these cells remains to be determined.     

Production of Tiger Puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis> Offspring from Triploid Grass Puffer <Emphasis Type="Italic">Takifugu niphobles</Emphasis> Parents

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2–5 kg) much larger than average market size (0.6–1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.     

Occurrence of partenites in mollusks and the influence that metacercaria of <Emphasis Type="Italic">Apophallus muehlingi</Emphasis> (Jagerskiold, 1898) and <Emphasis Type="Italic">Posthodiplostomum cuticola</Emphasis> (Nordmann, 1832) has on some biochemical parameters in fish

A heavy parasitic load from trematode partenites of Apophallus muehlingi (Jagerskiold, 1898) was observed in the mollusk population of Lithoglyphus naticoides (Pfeiffer) after its natural invasion into the Rybinsk Reservoir. An elevated concentration of glycogen was detected in the muscles of infected fish, both in the natural populations and under experimental conditions. Interspecific differences were found for the protein concentrations when elevated concentrations were observed in the muscles of the infected roach, and no differences were discovered for the goldfish (both infected and parasite-free). The ties between the biochemical parameters and motor activity are discussed for the infected fish.     

Cryopreservation of goldfish fins and optimization for field scale cryobanking

When gametes and embryos are not available, cryobanking of somatic tissues is one possibility to keep a genetic record of fish valuables in a context of biodiversity conservation and animal breeding management. Cryopreservation of whole fin pieces would be more advantageous than the commonly used cryopreservation of cells after fin culture, as it would allow extensive sampling without immediate need for laboratory facilities. The objective of this work was to assess the cryopreservation ability of fin pieces from goldfish (Carassius auratus) and to test whether a laboratory procedure could be adapted to field conditions. Caudal fin explants were cryopreserved in culture medium with 125 mM sucrose and 10% Me₂SO. After 14 days of culture, the frozen–thawed explants showed the same cell growth rate and grew the same somatic cell number as the fresh ones. Cells proliferated inside and around the explants as shown by BrdU labeling. Neither the size of the fin pieces nor the freezer type, −70 °C upright or −20 °C chest, influenced the outcome of cryopreservation. Fin pieces were stored 4 days at 4 °C in dry conditions prior to cryopreservation without alteration of the fin explant culture success. This study demonstrated that field collecting of goldfish fin pieces is possible as whole fin pieces can be stored in standard fridge or be shipped at subzero temperature before they are frozen into a plain −20 °C chest freezer. After incorporation in cryobanks in liquid nitrogen, thawed fin pieces reliably produce somatic cells in cell culture conditions.     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 °C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10⁴ cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 °C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

Developmental morphology of the cottid fish Pseudoblennius marmoratus

Embryonic, larval, and juvenile development of a Japanese cottid fish Pseudoblennius marmoratus is described from laboratory-reared specimens. Eggs of P. marmoratus resembled those of two congeners, Pseudoblennius cottoides and Pseudoblennius percoides, but differed in their smaller diameter and yolk color. Although the larvae and juveniles of P. marmoratus were also similar to them in general morphology, the former differed in the lengths of newly hatched larvae, pigmentation patterns, number of pectoral fin rays, and dorsal fin spine elongation.     

Hemimyzon nanensis, a new balitorid fish from the chao phraya basin, thailand

A new balitorid fish,Hemimyzon nanensis, is described from the Mae Nam Nan River basin, a tributary of the Chao Phraya River, in northern Thailand. This species is distinguishable from its congeners by the following unique combination of characters: pectoral fin with 9–11 simple and 10–12 branched, rays; pelvic fin with 3–4 (usually 3) simple and 8–9 (usually 9) branched rays; 59–63 total lateral line scales; distance between pelvic fin bases 8.1–9,6% SL.H. nanensis is the first species of the genus recorded outside China and Taiwan.     

Lycodes nishimurai, a new deep-sea eelpout (Teleostei: Zoarcidae) from the Sea of Japan

A new zoarcid fish, Lycodes nishimuari, is described on the basis of four specimens from the Yamato Bank, the Sea of Japan, at depths of 642–669 m. Although this species resembles Lycodes diapterus, L. hubbsi, L. pectoralis, L. macrochir, and L. nakamurae in having a deep notch in the pectoral fin and a single lateral line on the body, it is distinguished from them by the following combination of characters: no scales on pectoral fin base; scales on dorsum before dorsal fin origin; suborbital, preopercular, and last postorbital pores with risen rim; whitish lips; no whitish transverse bars on body; pectoral fin membrane and anterior part of dorsal fin membrane without black spots; basal color of vertical fins black. Supplementary material to this paper is available in electronic format at .     

Brief migration of the grass puffer, Takifugu niphobles, to fresh water from salt water

The grass puffer, Takifugu niphobles, is a peripheral freshwater fish often seen in brackish water. In the Sai River, Miyazu, Japan, we found that the grass puffer migrate to fresh water from salt water in June and July. Most of the grass puffer stayed in fresh water for 3.6 h on average and returned to the sea during the day. A low salinity tolerance experiment indicated that the grass puffer can live in fresh water for 2 days but not for longer than 4 days. Based on these findings, we discuss the physiological effects and ecological implications of the migration.     

Analysis of early growth of guppy strains,Poecilia reticulata,with different color patterns

Summary The guppy, Poecilia reticulata, is economically the most important species of freshwater ornamental fish cultured in Singapore. About 30 strains with different color patterns and fin shapes are reared in guppy farms practising monoculture in Singapore. To compare the growth rates of domesticated strains with different color patterns, newborn fry of 11 strains were obtained on the same day from a single farm in Singapore and were reared experimentally in the laboratory for about 100 days. Each strain was distributed randomly into 4 tanks with 30 fish/tank. Weekly weighings of 10 fish/tank were made from 17 to 100 days of age. Three strains were homozygous for the autosomal recessive blond gene which gives rise to a pale yellow background pigmentation (bb). These blond strains had significantly smaller body weights than corresponding ones with the same color pattern but with the wild-type grey-brown background coloration due to the dominant allele (BB). The strains with the red tail pattern due to a dominant X-linked gene (Rdt) had more rapid growth than those with other tail color patterns including the blue, black, green snakeskin and variegated. However, no significant differences were detected among the other color pattern strains. Thus among the strains studied, the blond strains were associated with slower growth while those with the red tail color were associated with faster growth.     

Westernmost record of the diamondback puffer,Lagocephalus guentheri (Tetraodontiformes: Tetraodontidae) in the Mediterranean Sea: First record from Greek waters

On 5th November 2017, a puffer fish specimen was caught in the Aegean Sea. Morphological and molecular analysis identified it as Lagocephalus guentheri. This first record from Greek waters represents the westernmost observation of the species in the Mediterranean Sea. The expansion and establishment of puffer fish species in the basin are discussed, with particular emphasis on the importance of the additional use of molecular analysis for increasing the efficiency in understanding species spread and dynamics.     

A Tetrodotoxin-Producing Vibrio Strain, LM-1, from the Puffer Fish Fugu vermicularis radiatus

Identification of tetrodotoxin (TTX) and its derivatives produced from a Vibrio strain in the intestine of the puffer fish Fugu vermicularis radiatus was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that Vibrio strains are responsible for the toxification of the puffer fish.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus</Emphasis> species capable of producing tetrodotoxin from the puffer fish <Emphasis Type="Italic">Fugu obscurus</Emphasis>

We obtained puffer fish Fugu obscurus from Wudi, China and analyzed the level of tetrodotoxin (TTX) toxictiy by mouse bioassay. The ovary showed the highest potency (125 MU/g), followed by the liver, intestine, and skin. A TTX-producing strain, namely, W-3, was isolated from the ovary of puffer fish F. obscurus. After culturing at 28 °C for 48 h, toxins were extracted from the liquid medium and analyzed by mouse bioassay, high-performance liquid chromatography, and gas chromatography-mass spectrometry. The results showed that strain W-3 produced TTX and related compounds. Physiological and biochemical characterization and 16S rRNA analysis indicated that this strain represents a novel species within the Bacillus genus; we named this strain as Bacillus sp. W-3. Our results suggested that marine bacteria play a role in the production of TTX in puffer fish F. obscurus.     

Experiments on the Feeding Behavior of the Hematophagous Candiru, Vandellia cf. Plazaii

Hematophagous candirus (family Trichomycteridae Vandellia cf. plazaii) did not respond to potential chemical attractants (ammonia amino acids fresh fish slime and human urine) added to their aquarium water. They seemed to respond visually to live goldfish Carassius auratus and Amazonian cichlids Cichlasoma amazonarum. One hypothesis tested is that the goldfish (an ostariophysan) might be preferred to the cichlid (a perciform fish). Goldfish were attacked first significantly more often when both prey species were present (one-sided difference in proportions test, N = 15, p = 0.02). However neither the elapsed time to the first attack nor the duration of the attacks differed significantly (Mann-Whitney's U-test N = 32 p = 0.27 and p = 0.92). These two species seemed incapable (goldfish) or weakly capable (cichlid) of avoiding or repelling candiru attacks     

Multiple origins of the green-sensitive opsin genes in fish

Vertebrate opsins are divided into four major groups: RH1 (rhodopsins), RH2 (rhodopsinlike with various absorption sensitivities), SWS (short-wavelength sensitive), and LWS/MWS (long and middle-wavelength sensitive) groups. The green opsin genes (g101 _Af and g101 _Af ) in a Mexican characin Astyanax fasciatus belong to the LWS/MWS group, whereas those in goldfish belong to the RH2 group (Yokoyama 1994, Mol Biol Evol 11:32–39). A newly isolated opsin gene (rh11 _Af ) from A. fasciatus contains five exons and four introns, spanning 4.2 kilobases from start to stop codons. This gene is most closely related to the two green opsin genes of goldfish and belongs to the RH2 group. In the LWS/MWS group, gene duplication of the ancestral red and green opsin genes predates the speciation between A. fasciatus and goldfish, suggesting that goldfish also has an additional gene which is orthologous to g101 _Af and g103 _Af .Correspondence to: S. Yokoyama     

Genes encoding giant danio and golden shiner ependymin

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, trueepn genes have been characterized in one order of teleost fish,Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the completeepn genes,gd (1480 bp), andsh (2071 bp), fromCypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid, genome. Each gene was found to contain six exons and five introns. Genegd encodes a predicted 218-amino acid (aa) protein GD 93% conserved to goldfish EPN, whilesh encodes a predicted 214-aa protein SH 91% homologous to goldfish. Evidence is presented classifying proteins previously termed “EPNs” into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark features of true EPNs.     

Microdontochromis rotundiventralis, a new cichlid fish (perciformes: Cichlidae) from Lake Tanganyika

Microdontochromis rotundiventralis, a new species of cichlid fish, is described on the basis of 13 specimens from Nkumbula Island, Lake Tanganyika (Zambia). It is distinct from its only congener,M. tenuidentatus, in having two (rarely one) rows of teeth on both jaws, a rounded distal margin on the pelvic fin, the outermost pelvic fin soft ray length 1.23–1.43 times the innermost ray, a deeper body (depth 26.8–29.1% standard length) and the anal fin with 9 (rarely 8 or 10) soft rays.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Annual reproductive cycle and sexual dimorphism in the dragonet,repomucenus valenciennei, in tokyo bay, japan

The annual reproductive cycle of the dragonet,Repomucenus valenciennei, from Tokyo Bay, Japan, was studied by analysis of seasonal trends in gonadosomatic indices and histological observations of gonads. Sexual dimorphism in the growth of several body parts relative to standard length (SL) and changes in color pattern of the first dorsal fin with growth were also investigated. The spawning season lasted from spring (April [1991] or February [1992]) to autumn (October) with two spawning peaks, in spring and autumn. In spring, only one-year-old (age 1+) fish spawned, age 0+ females not spawning until autumn, at which time they had reached age 1+. Likewise, histological observations of males indicated that testes had reached full maturity by 80 mm SL (age 1+). The minimum mature size of females was estimated as 60 mm SL. In males 45–80 mm SL, the first spine of the first dorsal fin, last ray of the second dorsal fin, last ray of the anal fin, and caudal fin ray showed strongly positive allometry, indicating rapid growth of these structures relative to SL. Subsequently, their growth returned to an isometric pattern in males>80 mm SL. In females, on the other hand, the same body parts showed slightly positive allometry throughout their growth. The color pattern on the first dorsal fin also changed in males 45–80 mm SL.