Concentrations of nucleophilic sulfur species in small Indian mongoose (Herpestes auropunctatus) in Okinawa, Japan.

Reactive sulfur species (RSS), such as hydrogen per (poly)sulfide, cysteine per (poly)sulfide, glutathione per (poly)sulfide, and protein-bound per (poly)sulfides, can easily react with environmental electrophiles such as methylmercury (MeHg), because of their high nucleophilicity. These RSS are produced by enzymes such as cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) and are found in mammalian organs. Organs of wildlife have not been analyzed for hydrogen sulfide, cysteine, glutathione, and RSS. In this study, low molecular weight nucleophilic sulfur substances, including RSS, were quantified by stable isotope dilution assay-based liquid chromatography-mass spectrometry using ß-(4-hydroxyphenyl)ethyl iodoacetamide to capture the target chemicals in the small Indian mongoose which species possesses high mercury content as same as some marine mammals. Western blotting revealed that the mongoose organs (liver, kidney, cerebrum, and cerebellum) contained proteins that cross-reacted with anti-CBS and CSE antibodies. The expression patterns of these enzymes were similar to those in mice, indicating that mongoose organs contain CBS and CSE. Moreover, bis-methylmercury sulfide (MeHg)2S, which is a low toxic compound in comparison to MeHg, was found in the liver of this species. These results suggest that the small Indian mongoose produces RSS and monothiols associated with detoxification of electrophilic organomercury. The animals which have high mercury content in their bodies may have function of mercury detoxification involved not only Se but also RSS interactions.

Identification of 2-phenylethanol with a rose-like odor from anal sac secretions of the small Indian mongoose (Herpestes auropunctatus).

The small Indian mongoose (Herpestes auropunctatus) is an invasive species in Okinawa and Amami-Oshima, Japan. Major strategies for their eradication have been the use of baited traps, which suffer from decreasing efficiency with declining populations and the bycatch of native animals. To address these concerns, mongoose-specific lures are required. In this study, we aimed to identify species- and/or sex-specific compounds from anal sac secretions of small Indian mongooses. Volatile compounds emitted from male and female mongoose anal sac secretions were analyzed by thermal desorption-gas chromatography-mass spectrometry. In addition to several fatty acids, 2-phenylethanol was identified as a minor compound, which is uncommon in mammalian secretions but a dominant odorant in roses. Female samples emitted higher levels of 2-phenylethanol than male samples did. These findings indicate that 2-phenylethanol is a female-specific volatile compound of anal sac secretions in small Indian mongooses, and it may be useful as an ingredient of mongoose-specific scent lures.

Potential Causative Mutation for Melanism in Rats Identified in the Agouti Signaling Protein Gene (Asip) of the Rattus rattus Species Complex on Okinawa Island, Japan.

The occurrence of black fur, or melanism, in many mammalian species is known to be linked to DNA sequence variation in the agouti signaling protein (Asip) gene, which is a major determinant of eumelanin and pheomelanin pigments in coat color. We investigated 38 agouti (i.e., banded wildtype) and four melanistic Rattus rattus species complex (RrC) lineage II specimens from Okinawa Island, Ryukyu Islands, Japan, for genetic variation in three exons and associated flanking regions in the Asip gene. On Okinawa, a predicted loss-of-function mutation caused by a cysteine to serine amino acid change at p.124C>S (c.370T>A) in the highly conserved functional domain of Asip was found in melanistic rats, but was absent in agouti specimens, suggesting that the p.124C>S mutation is responsible for the observed melanism. Phylogeographic analysis found that Asip sequences from Okinawan RrC lineage II, including both agouti and melanistic specimens, differed from: 1) both agouti and melanistic RrC lineage I from Otaru, Hokkaido, Japan, and 2) agouti RrC lineages I and II from South Australia. This suggests the possibility of in-situ mutation of the Asip gene, either within the RrC lineage II population on Okinawa or in an unsampled RrC lineage II population with biogeographic links to Okinawa, although incomplete lineage sorting could not be ruled out.

The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches.

In initial studies of the eutherian small Indian mongoose (Herpestes auropunctatus), the Y chromosome could not be identified in somatic cells. The male chromosome number is uniquely odd, 2n = 35, whereas that of females is 2n = 36. Previous reports indicated that this unique karyotype resulted from a translocation of the ancestral Y chromosome to an autosome. However, it has been difficult to identify the chromosomes that harbor the translocated Y chromosomal segment because it is an extremely small euchromatic region. Using a Southern blot analysis, we detected four conserved Y-linked genes, SRY, EIF2S3Y, KDM5D, and ZFY, in the male genome. We cloned homologues of these genes and determined their sequences, which showed high homology to genes in two carnivore species, cat and dog. To unambiguously identify the Y-bearing autosome, we performed immunostaining of pachytene spermatocytes using antibodies against SYCP3, γH2AX, and the centromere. We observed trivalent chromosomes, and the associations between the distal ends of the chromosomes were consistent with those of Y and X1 chromosomes. The centromere of the Y chromosome was located on the ancestral Y chromosomal segment. We mapped the complementary DNA (cDNA) clones of these genes to the male chromosomes using fluorescence in situ hybridization (FISH), and the linear localization of all genes was confirmed by two-colored FISH. These Y-linked genes were localized to the proximal region of the long arm of a single telomeric chromosome, and we successfully identified the chromosome harboring the ancestral Y chromosomal segment.

Genetic diversity and differentiation of the Ryukyu endemic frog Babina holsti as revealed by mitochondrial DNA.

We surveyed the genetic diversity and genetic differentiation of an endangered frog, Babina holsti, endemic to Okinawajima and Tokashikijima Islands of the Ryukyus, to elucidate its divergence history and obtain basic data for its conservation. Genetic differentiation between the two island lineages is moderate (3.1% p-distance in the cyt b gene). This result suggests that the two island lineages have been isolated between the late Pliocene and the middle Pleistocene and have never migrated between the current northern part of Okinawajima and Tokashikijima Islands, which were once connected in the late Pleistocene glacial age. On Okinawajima Island, the southernmost sample was constituted by a unique haplotype, without considerable genetic distance from haplotypes detected from northern samples. This unique haplotype composition in the southernmost sample would have resulted from the restricted gene flow between the southernmost population and the other populations in Okinawajima Island. Furthermore, the absence of genetic diversity within the southernmost sample indicates that this population has recently experienced population size reduction, possibly by predation pressure from an introduced mongoose, which is more abundant in the southern part than in the northern part of the island. Lower genetic diversity in the Tokashikijima sample implies a small effective population size for mitochondrial DNA (mtDNA) in B. holsti on the island. Immediate conservation measures should be taken for the populations from the southernmost range in Okinawajima and Tokashikijima.

Genetic characterization of Okinawan black rats showing coat color polymorphisms of white spotting and melanism.

We examined pelage color variation in wild populations of black rats (the Rattus rattus species complex) in the Yambaru forest area, northern Okinawa Island, Ryukyu Archipelago, Japan. Our field study revealed that 8.7% (38/438) and 0.2% (4/2500) of rats exhibited two types of coat color: white spotting and melanism, respectively. Using 34 representative animals, the phylogeography of the population was inferred using a nuclear gene marker, i.e., sequences (954 bp) of the melanocortin-1 receptor (Mc1r) gene responsible for the melanistic form in black rats. Four sequences from Okinawa were characterized as R. tanezumi, the Asian strain of black rat. Notably, neither of the phenotypic characters of white spotting or melanism was associated with the Mc1r haplotypes. Analysis of mitochondrial cytochrome b (Cytb) sequences (1140 bp) revealed that four haplotypes recovered from Okinawa clustered with the clade of R. tanezumi and differed by one or more bases from haplotypes at other localities in Japan and Asian countries. Thus, both variants may have arisen in the native rat population of Okinawa without interaction with the lineage of R. rattus, which exhibits a worldwide distribution and displays such coat color variants. The Yambaru population of black rats has thus experienced its own evolutionary history in allopatry for a substantial period of time (e.g., 10,000 years), which has preserved valuable genetic polymorphisms and will be useful for assessing the ecological consequences of genetic variation in natural populations.

Proposal for Japanese encephalitis surveillance using captured invasive mongooses under an eradication project on Okinawa Island, Japan.

A project to eradicate invasive small Asian mongooses (Herpestes javanicus) is underway to conserve the unique ecosystem of Okinawa Island, Japan. In the present study, we tried to elucidate whether the mongoose is a host of Japanese encephalitis virus (JEV) and to evaluate the reliability of surveillance of Japanese encephalitis (JE) using this species. Culex tritaeniorhynchus, the main vector mosquito of JEV, feeds on the mongoose. Eighty-five (35.4%) of 240 wild small Asian mongooses captured between 2001 and 2005 had neutralizing antibodies against more than one of four JEV strains. Prevalence rates of JEV antibodies tended to increase with body weight and length of the animals. One of three sentinel mongooses showed a temporal change in antibody titer. These results indicate that the small Asian mongooses on Okinawa Island are sensitive to JEV. From the antibody titers and the locations of capture, the JEV active area was clarified. We propose that surveillance of JE using mongooses captured under the eradication program is reliable.