Zoological Science

The mouse pink-eyed dilution (p) locus is known to control the melanin content, melanosome morphology, and tyrosinase activity in melanocytes. However, it is not well known whether the p allele is involved in regulating melanocyte proliferation, differentiation, and death. The aim of this study is to investigate in detail the role of the p allele in melanocyte proliferation, differentiation, and death using a cell culture system. The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mice at the p locus (black, C57BL/10JHir-P/P) and their congenic mutant (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with serum-free melanoblast-proliferation medium (MDMDF) and melanocyte-proliferation medium (MDMD). The proliferation and differentiation of p/p melanoblasts in MDMDF or MDMD were greatly inhibited compared with those of P/P melanoblasts and melanocytes. It is possible that apoptosis is related to the reduced proliferative and differentiative activity of p/p melanoblasts/melanocytes. The addition of apoptosis-inhibitors, such as caspase-9 inhibitor (C9I) and Bax-inhibiting peptide (BIP) into MDMDF or MDMD stimulated the proliferation and differentiation of p/p melanoblasts. In contrast, in P/P melanoblasts and melanocytes, C9I and BIP failed to stimulate their proliferation and differentiation. The number of apoptotic keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. Moreover, expression of C9 and Bax in keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. These results suggest that the increased apoptosis in keratinocytes and melanoblasts/melanocytes is related to the reduced proliferative and differentiative activity of p/p melanoblasts.

In most animal species, centrosomes, the main microtubule-organizing centers, usually disintegrate in oocytes during meiosis and are reconstructed from sperm-provided centrioles before the first cleavage division. In parthenogenetic oocytes, however, no sperm-derived centrosome-dependent microtubule nucleation is expected, as fertilization does not occur. The water flea Daphnia pulex undergoes parthenogenesis and sexual reproduction differentially in response to environmental cues. We used immunofluorescence microscopy with anti-α-tubulin and anti-γ-tubulin antibodies to examine spindle formation and the occurrence of centrosomes during parthenogenetic oogenesis and the subsequent cleavage division in D. pulex. The spindle formed in abortive meiosis in parthenogenesis is barrel-shaped and lacks centrosomes, whereas the spindle in the subsequent cleavage division is typically spindle-shaped, with centrosomes. During abortive meiosis, γ-tubulin is localized along the spindle, while in the first cleavage division it is localized only at the spindle poles. Thus, D. pulex should provide a useful comparative model system for elucidating mechanisms of spindle formation and improving our understanding of how evolutionary modification of these mechanisms is involved in the switch from sexual to parthenogenetic reproduction.

In most animal species, centrosomes, the main microtubule-organizing centers, usually disintegrate in oocytes during meiosis and are reconstructed from sperm-provided centrioles before the first cleavage division. In parthenogenetic oocytes, however, no sperm-derived centrosome-dependent microtubule nucleation is expected, as fertilization does not occur. The water flea Daphnia pulex undergoes parthenogenesis and sexual reproduction differentially in response to environmental cues. We used immunofluorescence microscopy with anti-α-tubulin and anti-γ-tubulin antibodies to examine spindle formation and the occurrence of centrosomes during parthenogenetic oogenesis and the subsequent cleavage division in D. pulex. The spindle formed in abortive meiosis in parthenogenesis is barrel-shaped and lacks centrosomes, whereas the spindle in the subsequent cleavage division is typically spindle-shaped, with centrosomes. During abortive meiosis, γ-tubulin is localized along the spindle, while in the first cleavage division it is localized only at the spindle poles. Thus, D. pulex should provide a useful comparative model system for elucidating mechanisms of spindle formation and improving our understanding of how evolutionary modification of these mechanisms is involved in the switch from sexual to parthenogenetic reproduction.

The present study investigated the caste differentiation system of Armitermes holmgreni in natural colonies from the Atlantic Forest in northeastern Brazil. Thirteen measurements were made of the termites encountered in three colonies. The morphometric differences among the instars were analyzed using principal component analysis (PCA). The differentiation patterns of the nymphal lineages of A. holmgreni agreed with earlier descriptions of species of the Termitidae family, with one larval instar followed by five nymphal instars and alates. Nymphoid neotenics became differentiated starting at the fourth or fifth nymphal instar. Two larval instars were observed in the apterous lineage before the appearance of workers, presoldiers, and soldiers. Both workers and soldiers had only one instar each. The A. holmgreni workers consisted of male and female individuals, without sexual dimorphism, a situation that had not been previously recognized among the Syntermitinae. The A. holmgreni soldiers consisted only of female individuals, a pattern observed in most species of Termitinae and Macrotermitinae. The differentiation of nymphoid neotenics starting at the fourth or fifth nymphal instar has also been reported for Armitermes euamignathus. The general patterns of differentiation of A. holmgreni were similar to those previously observed in other species of Syntermitinae.

In light of reproductive isolation being a fundamental aspect of the biological species concept, we performed crossing experiments using six species from three genera (Hoplobatrachus, Euphlyctis and Fejervarya) of family Dicroglossidae to explore postmating isolation in dicroglossid frogs. Our results revealed gametic isolation among these genera, although the intergeneric hybrids between female E. cyanophlyctis and male H. chinensis were not viable at the tadpole stage, while the hybrids between female E. cyanophlyctis and male H. tigerinus were inviable at the hatching stage. These results showed complete hybrid inviability between the two genera. Almost all interspecific hybrids between female H. tigerinus and male H. chinensis died of underdevelopment at the tadpole stage, whereas several hybrids developed normally and survived to maturity. Chromosomal observations and mtDNA and allozyme analyses confirmed that these mature hybrids were allotriploid, with two maternal genomes and one paternal genome. The present results suggest that the allotriploids were produced spontaneously, and histological observations confirmed their sex as sterile males. We also investigated the molecular relationships between H. tigerinus, H. chinensis, and the interspecific allotriploids by mitochondrial Cytb, 12S and 16S rRNA gene analyses. The maternal inheritance mode of mitochondrial genomes was retained in the hybrids. Finally, the present results suggest that the degree of postmating isolation reflects phylogenetic relationship. In addition, we speculate that allotriploids may be produced via hybridization among cryptic species.

The red fox (Vulpes vulpes) is the most widely distributed terrestrial carnivore in the world, occurring throughout most of North America, Europe, Asia, and North Africa. In South Korea, however, this species has been drastically reduced due to habitat loss and poaching. Consequently, it is classified as an endangered species in Korea. As a first step of a planned red fox restoration project, preserved red fox museum specimens were used to determine the genetic status of red foxes that had previously inhabited South Korea against red foxes from neighboring countries. Total eighty three mtDNA cytochrome b sequences, including 22 newly obtained East Asian red fox sequences and worldwide red fox sequences from NCBI, were clustered into three clades (i.e., I, II, and III) based on haplotype network and neighbor-joining trees. The mean genetic distance between clades was 2.0%. Clade III contained South Korean and other East Asian samples in addition to Eurasian and North Pacific individuals. In clade III, South Korean individuals were separated into two lineages of Eurasian and North Pacific groups, showing unclear phylogeographic structuring and admixture. This suggests that South Korean red fox populations may have been composed of individuals from these two different genetic lineages.

Genetic studies on protected species can be difficult, particularly when they form a mixed-species flock with other species. The bean goose (Anser fabalis), which is internationally recognized as a threatened species, was observed to form overwintering foraging flocks with white-fronted goose (Anser albifrons) at agricultural lands to feed on the grains in Korea. Non-invasive samples such as feces and feather that are readily available in their foraging ground can be useful for understanding the structure and composition of populations, but they often require specific experimental conditions due to small amount or low quality of DNA. In this study, we designed sets of primers that would allow efficient molecular identification of species and sex of individuals of bean geese and white-fronted geese. Species-specific primers (WFG-F/BG-F and G-R), developed from ND2 region of mitochondrial DNA, produced PCR products with different sizes which allow easy species identification without further sequencing. Based on published CHD 1 sequences, we designed internal primers (Gsex-F and Gsex-R) for sex determination that can be used in nested PCR after applying P2/P8 primers, and our methods clearly showed high success rate of molecular sexing from non-invasive samples. These molecular tools open the possibilities for genetic studies using non-invasive samples collected from a mixed-species aggregation containing bean geese and white-fronted geese.

Many animal species form communal roosts in which they aggregate and sleep together. Several benefits of communal roost have been suggested, but due to lack of data on relatedness among group members, it is unknown whether these benefits can be amplified by the formation of kin-based communal roosts. We investigate the genetic composition of two winter roosts of Eurasian Magpies (Pica pica), using microsatellite markers on non-invasive samples. Using permutation tests by reshuffling the alleles presented in the roosts, we determined that individuals in the communal roosts of magpies were not more related than expected by chance, suggesting that kinship may not be a driving force for the formation of communal roosts in magpies. However, the pairwise relatedness and estimated relationship based on a maximum likelihood approach revealed that the roosts involve both kin and non-kin. Relatedness coefficients varied widely within a roost, indicating that family subgroups form a small proportion of the total number of birds in a roost. Our results suggest that ecological benefits of communal roost in animals are sufficient for the evolution of communal roosts without any involvement of kinship.

The morphology of many organisms seems to be related to the environments in which they live. Many snakes are so similar in their morphological patterns that it becomes quite difficult to distinguish any adaptive divergence that may have occurred. Many authors have suggested that the microstructure of the reptile's scales has important functional value. Herein, we investigate variations on the micromorphology of the external surface of dorsal scales on the head, the mid-body region (trunk), and the tail of Rhomphotyphlops braminus (Typhlopidae), Eryx jaculus (Boidae), Psammophis sibilans (Colubridae), Naja haje (Elapidae) and Echis carinatus (Viperidae). The specimens were metallized and analyzed by scanning electron microscopy. All species displayed unique dorsal scale surface microstructures of the investigated regions. The microstructural pattern of the scales of head, trunk, and tail differs in different species of these snakes. In conclusion, we detected ecomorphologic relationships between extant dorsal scale microstructures and snake microhabitat, enabling us to hypothesize that environmental pressures have significant influences not only on these animals' macrostructure, but also on its microstructure as well.

We examined the phylogenetic status and history of the mountain hare Lepus timidus in and around Hokkaido using mitochondrial cytochrome b (cyt b) sequences from 158 samples from Hokkaido and 14 from Sakhalin, as well as four samples from the Korean hare, L. coreanus. The phylogenetic analysis of the cyt b sequences generated in this study and obtained from DNA databases showed the clear genetic specificity of the Hokkaido lineage as a clade. The Hokkaido lineage was estimated to have diverged from the other conspecific and L. coreanus lineages 0.46 and 0.30 million years ago (Mya), respectively. These results suggest that the common ancestor of the mitochondrial lineage in Hokkaido and Korea inhabited Far East Asia before colonization by the present continental lineages of L. timidus, including the Sakhalin population. We estimated the time of the most recent common ancestor of the Hokkaido population to be 0.17 Mya, and found two distinct haplogroups within the island. One group had greater genetic diversity (mean number of pairwise differences: π = 0.0188 ± 0.0108) and appears to have expanded from the west to the entire island of Hokkaido. The other had lower genetic diversity (π = 0.0038 ± 0.0037) and its distribution was concentrated in the east. These contrasting west/east trends indicate that the Hokkaido population was fragmented in the past, and then subsequently expanded. Our study suggests that Hokkaido was an important refugium for boreal species in the far eastern region, and allowed the formation of various population genetic structures within the island.

We examined the phylogenetic status and history of the mountain hare Lepus timidus in and around Hokkaido using mitochondrial cytochrome b (cyt b) sequences from 158 samples from Hokkaido and 14 from Sakhalin, as well as four samples from the Korean hare, L. coreanus. The phylogenetic analysis of the cyt b sequences generated in this study and obtained from DNA databases showed the clear genetic specificity of the Hokkaido lineage as a clade. The Hokkaido lineage was estimated to have diverged from the other conspecific and L. coreanus lineages 0.46 and 0.30 million years ago (Mya), respectively. These results suggest that the common ancestor of the mitochondrial lineage in Hokkaido and Korea inhabited Far East Asia before colonization by the present continental lineages of L. timidus, including the Sakhalin population. We estimated the time of the most recent common ancestor of the Hokkaido population to be 0.17 Mya, and found two distinct haplogroups within the island. One group had greater genetic diversity (mean number of pairwise differences: π = 0.0188 ± 0.0108) and appears to have expanded from the west to the entire island of Hokkaido. The other had lower genetic diversity (π = 0.0038 ± 0.0037) and its distribution was concentrated in the east. These contrasting west/east trends indicate that the Hokkaido population was fragmented in the past, and then subsequently expanded. Our study suggests that Hokkaido was an important refugium for boreal species in the far eastern region, and allowed the formation of various population genetic structures within the island.

Ceacam6-L (carcinoembryonic antigen-related cell adhesion molecule 6-long) has recently been isolated from rat testis by differential display technique followed by RT-PCR and DNA sequence analysis. CEACAM6-L is a member of the immunoglobulin (Ig) superfamily and composed of an Ig-like domain, three IgCAM domains, a transmembrane region, and a short intracellular domain. We previously reported that CEACAM6-L was localized at the interface between Sertoli cells and elongating/elongated spermatids and might be an adhesion molecule contributing to apical ectoplasmic specialization in testis. In this study, we investigate the adhesive capacities and the complex structures of CEACAM6-L, using COS-7 cells as a study model. Transfection and immunoprecipitation experiments showed that CEACAM6-L expressed in the cells was distributed to the plasma membrane and interacted homophilically between transfected COS-7 cells. A chemical cross-linking experiment and a binding assay with recombinant CEACAM6-L proteins suggested that CEACAM6-L could form trans-tetra complexes, constructed by cis-homodimers at the adhesion site between COS-7 cells expressing CEACAM6-L. We hypothesized that CEACAM6-L expressed in both germ cells and Sertoli cells forms homophilic trans-tetra complexes between these cells in the seminiferous epithelium.