Follistatin is a functional antagonist of several members of the TGF-β family of secreted signaling factors, including myostatin, the most powerful inhibitor of muscle growth characterized to date. Myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. To assess the potential benefits of follistatin in treating muscle degenerative diseases, we examined the expression of myostatin and follistatin in Mdx mice, a model for Duchenne muscular dystrophy, and in B10 mice as a control. Our results demonstrated a temporary and coincident expression of follistatin and myostatin in both mouse strains, but this expression was significantly higher in Mdx mice than in B10 mice. The maximum expression of follistatin and myostatin in the presence of restoring necrotic muscle was detected 4 weeks after birth in Mdx mice. Interestingly, during the stage of complete regeneration, the absence of myostatin and follistatin proteins and a marked decrease in the expression of both genes were observed 9 weeks after birth in both mouse strains. These findings suggest that follistatin not only blocks myostatin but also allows other activators to function in muscle development, emphasizing that follistatin could be a very potent molecule in combating muscle loss during dystrophies and muscle ageing, disuse, or denervation.

Insect eggs store many lipid droplets as an energy source for embryonic development. We previously reported that lipid droplets are incorporated into embryos in three steps in the silkworm, Bombyx mori. The midgut plays important roles in lipid incorporation during the second and third steps, whereas the manner of lipid incorporation during the first step is still unknown. In this study, we focused on how lipids were incorporated into the embryo in the first step, compared with the mechanisms used in the second step, by means of transmission electron microscopy using the high-pressure freezing and freeze substitution method. At the beginning of the first step (blastoderm formation stage), some lipid droplets were observed in each cell of the embryonic tissues. Lipid droplets were seen to be derived from the oocyte peripheral cytoplasm by superficial cleavage. At the end of the first step (late appendage formation stage), some lipid droplets were attached to the elongated rough endoplasmic reticulum (rER). It seemed that formation of the lipid droplets occurred in embryonic cells at the end of the first step, because the rER is the site of biogenesis of lipid droplets. The incorporation of lipid droplets in the first step may be subdivided into two stages: the blastoderm formation stage and the subsequent stage before blastokinesis.

The tenthredinid sawflies Macrophya apicalis and M. infumata, which range throughout Japan and adjacent areas, are very similar in external morphology and share the same host plant, Sambucus sieboldiana. We investigated their genetic distances within the genus and their intraspecific variation, and estimated their time of divergence to clarify the history of their speciation and colonization. We collected 33 specimens of the two species from several locations in the Japanese archipelago (Hokkaido, Honshu, Shikoku, and Kyushu) and Cheju Island (Korea). We extracted total DNA from each specimen, and PCR amplified and sequenced 1300 bp of the cytochrome oxidase subunit I (COI) gene. We constructed phylogenetic trees by using the UPGMA and maximum likelihood methods. We estimated divergence times based on previously published COI substitution rates. The genetic distance between the two species indicated that their ancestors differentiated in the late Miocene, probably on the Asian continent. Based on the calculated time of intraspecific diversification in Japan, we estimated that both species immigrated to Japan in the early Pleistocene and crossed the Tsugaru Strait land bridge between Hokkaido and Honshu after the middle Pleistocene. Genetic analysis of the two sibling species elucidated their relative taxonomical positions within the genus, and their intraspecific variation. Estimated from previously published COI evolutionary rates, the time of arrival of both species in Japan is consistent with the history of colonization of the country by other animals during the last ice age.

The growth hormone secretagogue-receptor (GHS-R) is an endogenous receptor for the gut hormone ghrelin (GRLN). Two isoforms of GHS-R have been identified in several animals: functional GHS-R1a and a splice variant of unknown function, GHS-R1b. Here we report identification of a GHS-R-like receptor (GHSR-LR) in the Mozambique tilapia, Oreochromis mossambicus. The cDNA is 1584 bp in length and encodes a 384-amino acid GHS-R1a ortholog. The amino acid sequence of tilapia GHS-R1a is 54, 60, 80 and 89% identical to that of rat, chicken, pufferfish, and seabream GHS-R1a, respectively. Genomic PCR revealed that the tilapia GHS-R gene is composed of two exons separated by a single intron. In addition, a GHS-R1b ortholog, which is generated by alternative splicing of the GHS-R gene and contains part of the intron, was identified and predicted to be a 298-amino acid protein. Functional analyses of tilapia GHS-R1a were conducted using mammalian HEK 293 and CHO cells, but the expected increase in intracellular calcium ions by tilapia or rat GRLN was not observed. We found that the GHS-R1a ortholog is expressed in greater quantities than the GHS-R1b ortholog in all tissues assayed. Further studies are required to conclude that our identified protein is the GHS-R for tilapia, although the gene structure and amino acid sequence showed high similarities to other GHS-R genes; thus, we designated this protein GHSR-LR.

In-vitro effects of insulin-like growth factor-I (IGF-I) on superoxide production in phagocytic head kidney leucocytes (HKL) and in-vivo effects on plasma lysozyme levels were examined in the rainbow trout (Oncorhynchus mykiss). In-vitro administration of salmon IGF-I (sIGF-I), human IGF-I, and human IGF-II increased superoxide production in zymosan-stimulated HKL. A significant effect of sIGF-I on superoxide production in HKL was observed from 0.1 to 100 nM, and was equipotent to that of salmon growth hormone. IGF receptor (IGFR) type Ib was expressed in trout HKL, and also in the brain, pituitary, liver, and gills; however, the expression of another IGFR (type Ia) was not detected in HKL. In-vivo intraperitoneal injection of sIGF-I increased plasma levels of lysozyme, whereas ceruloplasmin or immunoglobulin M did not change. These results indicate that IGF-I stimulates non-specific immune functions in fish.

Chromosomes stained with fluorochromes, including quinacrine mustard (QM), emit the brightest fluorescence immediately after exposure to excitation light, and the fluorescence gradually fades with an increase in exposure time. However, in the QM-stained chromosomes of the small Japanese field mouse Apodemus argenteus, most C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light, and they become brightly fluorescent by prolonged exposure (delayed QM fluorescence). We proposed recently that the delayed QM fluorescence is somehow related to nicks produced in C-heterochromatic DNA by blue light irradiation. To test this possibility, we examined the chromosomal distribution of nicks by in-situ nick translation and changes, if any, in the QM fluorescence pattern after methylene blue (MB) -mediated photooxidation, which is considered to induce nicks in chromosomal DNA. It was found that C-heterochromatic regions fluoresced brightly without any delay after exposure to blue light, and that nicks increased considerably in the same regions after the MB-mediated photooxidation. It seems, therefore, that photooxidation and strand breaks in DNA (including nicks) are responsible for the induction of delayed QM fluorescence. Trypsin digestion, on the other hand, abolished delayed QM fluorescence. Thus, not only DNA but also chromosomal protein(s) are involved in the unusual sequence of QM fluorescence patterns in A. argenteus.

The milkfish (Chanos chanos Forsskål, 1775) is a euryhaline fish widely distributed in tropical and subtropical Indo-Pacific waters. It is unique in having in the cephalic region adipose eyelid tissue that begins to develop in the larval stage and is completely formed by the juvenile stage. The formation of the adipose eyelids coincides with the onset of active swimming ability. Larval and juvenile milkfish have different dietary modes and habitats. This study was aimed to investigate ontogenetic changes in color perception ability with the use of microspectrophotometry (MSP). Larval milkfish had rod cells and red, green, blue, and violet cone cells, while Juvenile milkfish lost the violet cone cells, and the blue cones shifted to shorter wavelengths. Histological sections showed the presence of cone cells of the single type (but no double or twin types) in the retina, which implies that the milkfish may not have polarized vision.

A non-invasive technique to monitor cardiac activity in small fish, such as goldfish, zebrafish, and medaka, is needed. In the present study, we developed photocardiography (PCG), a non-invasive optical method, to record cardiac activity in small fish. The method monitors changes in near-infrared light transmission through the heart using a phototransistor located outside the body. With this technique, heartbeats in fish of various sizes (14–218 mm) were stably recorded. PCG was applied to monitor the heartbeat during fear-related classical heart rate conditioning in goldfish wherein an electrical shock was used as an unconditioned stimulus. The heartbeats were continuously monitored, even when the beat coincided with the electrical shock, showing that PCG is robust even in an electrically noisy environment. This technique is particularly useful when monitoring the heartbeats of fish of small size or in the presence of ambient electrical noise, conditions in which the use of conventional electrocardiography (ECG) is difficult.

Two new species of Diplosoma are described from coral reefs in the Ryukyu Archipelago, Japan: D. watanabei sp. nov. and D. gumavirens sp. nov. Colonies of both species are green due to algal symbionts of Prochloron, which are distributed in the common cloacal cavity. Both species are characterized by the point of emergence of the retractor muscle and a unique combination of stigma numbers among the four rows of the branchial sac. There are three adhesive papillae in the embryos of D. watanabei, as in most ascidian embryos and larvae, whereas the pre-hatching embryos of D. gumavirens sp. nov. posses 12–16 adhesive papillae. Partial sequences of the cytochrome c oxidase subunit I (COI) gene discriminated the new species from each other and from congeners.

A total of 11 specimens of a nereidid polychaete, which had been collected from the inner part (“Bay of Miya” is the old place name) of Ise Bay in central Japan and preserved in the Natural History Museum Vienna, were taxonomically examined. They were all identified as the brackishwater nereidid, Hediste japonica (Izuka, 1908), which was previously known only from the Ariake Sea and Seto Inland Sea in western Japan and the western coast of Korea. The new information indicates that this species formerly had a wider distribution extending to at least Ise Bay in central Japan, though all Japanese populations of this species seem to have been extirpated except for a population remaining in the Ariake Sea.