Mouse lung rudiments express eight members of fibroblast growth factor (FGF) family genes from embryonic day 10 (E10) to E13. Some of these are expressed in either the epithelium or mesenchyme, while others are expressed in both. Incorporating the results of our previous study, we characterized the branch-inducing activities of all of FGFs expressed in the early lung rudiment. Of these, FGF1, FGF2, FGF7, FGF9 and FGF10 induced branching morphogenesis in Matrigel-embedded E11 epithelium, and their effective concentrations varied (10 nM, 10 nM, 3 nM, 1 nM, and 100 nM, respectively). Whereas shaking culture dishes containing medium supplemented with FGF7 or FGF10 showed reduced branching morphogenesis, those supplemented with FGF1, FGF2, or FGF9 did not, suggesting the involvement of autocrine growth factor(s) in branching morphogenesis induced by FGF7 or FGF10. In the presence of heparin, a well-known activator of FGF signaling, cystic morphology with lumen expansion was observed in cultures containing FGF1, FGF7, or FGF10, but growth arrest was observed in cultures containing FGF2 or FGF9. These results indicate that several paracrine and autocrine FGFs function during branching morphogenesis of lung epithelium.

Chinese sturgeon, Acipenser sinensis, is a critically endangered anadromous fish species spawning in the Yangtze River of China during October and November. In this study, we analyzed the effects of hydrological factors, such as water temperature and discharge, on the natural reproduction time of the Chinese sturgeon and evaluated the impact of the impoundment of the Three Gorges Reservoir (TGR) based on our survey data from 1998 to 2011. The results showed that the first spawning dates were significantly (P < 0.05) correlated with the date of water temperature reaching 20°C (20°C WT dates), October mean discharge (Oct. discharge), and the discharge change from October to November (Oct—Nov Δdischarge). Regression analysis suggested that oneday delay of 20°C WT dates could postpone the first spawning date by 0.673 days. A discharge increase of 1000 m³s^-1 in October could bring forward the first spawning date by two days. Our results also indicated that the impoundment of the TGR had delayed the first spawning time due to water temperature lag and flow regulation. We suggest the following ecological regulations in order to facilitate conservation of the Chinese sturgeon: to eliminate water temperature lag by regulating water temperature downstream of the Three Gorges Dam (TGD), to enhance water discharge downstream in October, and to complete impoundment before October.

We carried out a primary survey by quadrat sampling to quantify breeding habitat characteristics of blackthroat (Luscnia obscura), a poorly documented and vulnerable bird species, in Foping nature reserve on the central southern Qinling Mountains. Tree layer information was collected in 10 m × 10 m plots, bamboo and shrub layer information was collected in 2 m × 2 m plots, and grass and ground layer information was collected in 1 m × 1 m plots. Our observations showed that blackthroat lives in coniferous forest and coniferous broadleaved forest with dense bamboos at the elevation ranging from 2130 m to 2600 m. Shrub cover, density and height, and ground cover in sites with blackthroat were significantly higher than those in random sites, while tree density, grass height and cover in habitat sites were significantly lower than those in random sites. These results suggest that shrub cover, shrub density, and canopy cover may be relevant to breeding habitat selection by these birds. Our study suggests that Qinling Mountains may be an important blackthroat breeding site.

The morphology of the nephrons of the coelacanth Latimeria chalumnae was investigated by electron microscopy. Each nephron is composed of a large renal corpuscle with well vascularized glomerulus, ciliated neck segment, proximal tubule divided into first and second proximal segments, ciliated intermediate segment, distal tubule, collecting tubule, and duct. The podocytes of visceral epithelium contain large bi-lobed nuclei and their surface membranes pinch off vesicles into the cytoplasm. The processes of the podocytes give rise to pedicels that enclose narrow filtration slits. The endothelium of glomerular capillaries is attenuated and fenestrated. The short cytoplasmic processes of mesangial cells do not penetrate deeply into the sub-endothelial lamina. The glomerular basement membrane is about 286 nm in thickness. The pedicels also arise from podocyte cell bodies, and are connected by diaphragms and enclose slits, which open into narrow urinary spaces between podocytes. The cuboidal cells of the short neck segment display cilia with a characteristic pattern of 9 2 microtubules. The first proximal tubule segment differs from the second proximal segment in having densely packed microvilli, prominent endocytotic-lysosomal apparatus, and numerous basal membrane infoldings associated with mitochondria. The lateral cell membranes like those of other segments are straight and joined by desmosomes and apical adhering and tight junctions. The distal tubules display few short luminal microvilli and numerous basal mitochondria. The distal tubule, collecting tubule and duct are devoid of intercalated cells. The ultrastructure of the L chalumnae nephrons correlates well with their osmoregulatory function and resembles that of freshwater rainbow trout.

We observed the morphology of the lingual papillae (filiform, conical, fungiform, and vallate papillae, and lateral organ) and their connective tissue cores (CTCs) in the American mink (Neovison vison) using light and scanning electron microscopy. Filiform papillae were distributed on the apex linguae and rostral regions of the corpus linguae. Conical papillae were distributed over the caudal region and absent in the radix linguae. Numerous ridges were present in the radix linguae. Four to six vallate papillae were situated at the border between the corpus and radix linguae. Instead of foliate papillae, a pair of lateral organs was situated on the caudal edge of the corpus. The epithelial surface of each filiform papilla consisted of a single main process and 10–12 accessory processes. Notably, filiform papillae in the apex linguae exhibited morphological variation, and some were dome-like and lacked processes. In contrast, filiform papillae on the rostral part were not variable, were extended to a sharp tip, were associated with an eosinophilic stratum corneum, and lacked nuclei. The CTCs of the filiform papillae consisted of a main core and slender accessory cores surrounding a concavity. Those in the apex linguae were similar in appearance and consisted of main and adjacent accessory cores. The fungiform papillae had a dome-like epithelial surface and their CTCs were columnar, with upper concavities and flanges. The simplified lingual morphology of the American mink, particularly in the filiform papillae in the apex linguae, may be influenced by its diet and semiaquatic lifestyle.

It is increasingly accepted that conservation work should consider the evolutionary history of target species. Fishes in the subfamily Acheilognathinae, family Cyprinidae, are, with the exception of three species exclusively distributed in Europe, restricted to Asia and show a distinct spawning behavior in laying their eggs in gill chambers of freshwater mussels. At present, many of the 70 species recognized in this group are facing with serious population decline in China and Japan, and their phylogenetic relationships are not well resolved. In the present study, based on mtDNA cyt b and 12S rRNA gene sequences, we reconstructed a more detailed species-level phylogenetic tree of this group, and assessed species conservation priorities based on their evolutionary distinctiveness. Molecular phylogenetic analyses showed that the Acheilognathinae contains two major clades: Acheilognathus clade and Tanakia-Rhodeus clade. Based on this phylogenetic result, conservation priority analyses were conducted using ED (evolutionary distinctiveness)/HED (heightened evolutionary distinctiveness), and EDGE (evolutionary distinctiveness and global endangeredness)/HEDGE (heightened evolutionary distinctiveness and global endangeredness) methods. The results suggested that T. himantegus, T. lanceolata, A. gracilis, A. imberbis, T. tanago, and A. longipinnis should be ranked as the top-priority species for conservation. According to our results, we also discussed the current conservation efforts of the bitterling fishes and gave suggestions for future work.

The Kuroiwa's eyelid gecko Goniurosaurus kuroiwae is an endangered species in a state of relict endemism in the Central Ryukyus, Japan, and is divided into five subspecies. We analyzed variations in sequence data for approximately 1900 base positions of mitochondrial 12S and 16S rRNA, and cytochrome b genes from samples representing all recognized subspecies of G. kuroiwae together with those from congeneric species in order to test the relevant previous phylogenetic hypotheses and discuss biogeographical implications in the degree and pattern of genetic divergence within G. kuroiwae. Our results, while confirming a previous molecular phylogenetic hypothesis proposed on the basis of much smaller data set, negate the relationships hypothesized on morphological grounds by explicitly supporting: 1) the primary dichotomy, with substantial genetic divergence, between G. k. splendens from the Amami Island Group and the remaining subspecies all from the Okinawa Island Group; and 2) the presence of at least six independent lineages within the latter, indicating non-monophyly for two of the subspecies, G. k. kuroiwae and G. k. orientalis, in the current taxonomic definitions. The marked genetic divergence between populations of the two island groups seems to have initiated in the middle Miocene, i.e., prior to formation of straits that have consistently been separating these two island groups since the early Pleistocene. All populations of G. kuroiwae are regarded as endangered from the viewpoint of conservation genetics.

Bitterling (Teleostei: Acheilognathinae) are small cyprinid fishes with a discrete distribution in East Asia and Europe. We used a complete mitochondrial cytochrome b sequence (1141 bp) from 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus), sampled across the major part of their distribution, to elucidate their phylogeny and biogeography, focusing particularly on their origin and dispersal. Based on high support value, the monophyletic Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In the latter clade, the monophyly of Rhodeus was poorly supported, though it was topologically nested in Tanakia. On the basis of molecular-clock calibration, both clades diverged in the middle Miocene, with Tanakia-Rhodeus diverging slightly earlier than Acheilognathus. The Tanakia-Rhodeus clade expanded its distribution westward from the Far East, eventually reaching Europe, while Acheilognathus dispersed in the temperate regions of East Asia. A feature common to both clades is that most extant species, including Japanese endemics, appeared by the end of the Pliocene, corresponding with the present delineation of the Japanese archipelago. Autumn-spawning species with an embryonic diapause, unique to bitterling among cyprinid fishes, formed two distinct lineages (barbatulusrhombeus and longipinnis-typus) within Acheilognathus. The estimated time of divergence of the two lineages was approximately from the late Pliocene, a period characterized by glaciations. The timing of divergence suggests that the shift of spawning from spring to autumn, coupled with embryonic diapause, convergently emerged twice in the evolution of bitterling, possibly as an adaptation to the climate of the late Pliocene.

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10^-5 and 10^-4 M) and MeHg (10^-6 to 10^-4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10^-5 and 10^-4 M) and MeHg (10^-6 to 10^-4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone.

In order to establish protocols for gamete recovery from accidentally killed wild animals, or to take advantage of those slaughtered by captive breeders, we assess the influence of two methods on the recovery of epididymal sperm from collared peccaries, and verify the effect of centrifugation on such gametes. Genitalia from nine animals were used. For each animal, one epididymis was processed by flotation and the other was processed by retrograde flushing, both using a buffered media based on Tris. Following recovery, sperm were evaluated for motility, vigor, viability, functional membrane integrity, and morphology. A 1-mL aliquot of each sample was centrifuged, the supernatant removed, and the pellet suspended and evaluated as fresh samples. The sperm characteristics did not differ between the samples collected by flotation or retrograde flushing (P < 0.05). Centrifugation promoted an increase in head and tail defects, thus reducing the percentage of viable sperm (P < 0.05). No other parameter assessed for both methods was affected by centrifugation. In conclusion, epididymal sperm from collared peccaries can be efficiently collected through flotation or retrograde flushing, but not when either is followed by centrifugation.