Worawut Rerkamnuaychoke

Cytogenetic differentiation of two sympatric tree shrew taxa found in the southern part of the Isthmus of Kra.

Sympatric populations of the genus Tupaia encompassing two cytotypes (cyt60, 2n = 60 and cyt62, 2n = 62) were found in the southern part of the Isthmus of Kra (the middle region of the Malay Peninsula, Thailand). C-bands, location of rDNA, and location of non-essential telomeric repeats (TRs) were investigated in detail for 23 animals captured in the area. Such chromosomal traits definitely reveal that two distinct cytotypes exist in the sympatric population, though the external morphological traits are similar. Hybrid cytotypes were not observed; thus, these two cytotypes appear to be genetically isolated sibling species. Chromosomal results compared with previous data, geographic distribution and morphological data observed with new insight suggest that, in the sympatric population, ‘cyt60’ represents members of Tupaia glis, while ‘cyt62’ identifies individuals of Tupaia belangeri. The cytogenetic information discovered in the present study offers new insight to morphological classification and, further, may provide substantial diagnostic characteristics for the distinction of tree shrew species.

Molecular Diversity and Phylogeography of the Asian Leopard Cat, Felis bengalensis, Inferred from Mitochondrial and Y-Chromosomal DNA Sequences

Abstract To investigate genetic diversity and phylogeography of the Asian leopard cat (Felis bengalensis), mitochondrial DNA (mtDNA) sequences were determined for 39 individuals from various areas. Sequences combining the complete cytochrome b gene (1,140 bp) with the partial control region (646–810 bp) were classified into 24 haplotypes: 21 types from 21 animals, one from eight animals from Tsushima Islands, one from eight animals from Iriomote Island, and one from two animals from Southeast Asia. Phylogenetic trees of the 24 haplotypes clearly showed three clades: a Northern Lineage and Southern Lineages 1 and 2. The Northern Lineage consisted of animals from Tsushima Islands, the Korean Peninsula, the continental Far East, Taiwan, and Iriomote Island. Within the Northern Lineage, genetic contacts could have occurred between geographically neighboring populations before isolation by straits. Southern Lineage 1, comprising Southeast Asian animals, showed higher genetic diversity. Southern Lineage 2 had large genetic distances from other lineages. Within the control region, the Asian leopard cats shared two to four repetitive motifs, and the number of motifs and their constitution were highly variable among individuals. The motifs were polymorphic even within individuals and could be classified into 31 types. Finally, males of mtDNA Southern Lineage 1 had either of two types of the Y-chromosomal gene ZFY, whereas all males of Northern Lineage shared only one type. Our results indicate that the diversity of southern populations is higher and that genetic differentiation among northern local populations reflects past geographical isolation.

Apoptosis and gene expression in the developing mouse brain of fusarenon-X-treated pregnant mice.

Fusarenon-X (FX), a type B trichothecene mycotoxin, is mainly produced by Fusarium crookwellense, which occurs naturally in agricultural commodities, such as wheat and barley. FX has been shown to exert a variety of toxic effects on multiple targets in vitro. However, the embryonic toxicity of FX in vivo remains unclear. In the present study, we investigated FX-induced apoptosis and the relationship between the genetic regulatory mechanisms and FX-induced apoptosis in the developing mouse brain of FX-treated pregnant mice. Pregnant mice were orally administered FX (3.5 mg/kg b.w.) and were assessed at 0, 12, 24 and 48 h after treatment (HAT). Apoptosis in the fetal brain was determined using hematoxylin and eosin staining, the TUNEL method, immunohistochemistry for PCNA and electron microscopy. Gene expressions were evaluated using microarray and real time-reverse transcription polymerase chain reaction (qRT-PCR). Histopathological changes showed that the number of apoptotic cells in the telencephalon of the mouse fetus peaked at 12 HAT and decreased at 24 and 48 HAT. FX induced the up-regulation of Bax, Trp53 and Casp9 and down-regulated Bcl2 but the expression levels of Fas and Casp8 mRNA remained unchanged. These data suggested that FX induces apoptosis in the developing mouse brain in FX-treated dams. Moreover, the genetic regulatory mechanisms of FX-induced apoptosis are regulated by Bax, Bcl2, Trp53 and Casp9 or can be defined via an intrinsic apoptotic pathway.

Development and application of an indirect enzyme-linked immunosorbent assay for serological survey of Japanese encephalitis virus infection in dogs.

Japanese encephalitis virus (JEV) causes serious acute encephalitis in humans and horses. Although dogs are good sentinels for assessing the risk of JEV infection to humans, a virus neutralization test has been the only method available for measuring the levels of JEV antibody in dogs. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) using purified viral particles as an antigen, was developed for serological survey of JEV infection in dogs. In dogs inoculated experimentally with JEV, the ELISA detected anti-JEV IgM 3 days after infection, with IgM levels peaking 7 days after infection. Anti-JEV IgG was detected 14 days after infection and peaked on 21-28 days after infection. Virus neutralization titers correlated with anti-JEV immunoglobulins measured by the ELISA. To test the utility of the new assay, the seroprevalence of JEV infection among 102 dogs in Kyushu, Japan, was examined by IgG ELISA and by virus neutralization. The correlation coefficient between the IgG ELISA and virus neutralization was 0.813 (p<0.001); comparison of the IgG ELISA and virus neutralization showed a sensitivity and specificity of 82% and 98%, respectively. The IgG ELISA was used to survey dogs in Bangkok, Thailand and 51% of these dogs were found seropositive for JEV. These data suggest that in the capital city of Thailand, the risk of infection with JEV remains high.

Cardiac-like musculature of the intrapulmonary venous wall of the long-clawed shrew (Sorex unguiculatus), common tree shrew (Tupaia glis) and common marmoset (Callithrix jacchus)

The cardiac‐like musculature is distributed not only in the heart wall but also in the intrapulmonary venous wall in a few species of insectivores. It has been suggested that the evolutionary origin of venous cardiac‐like musculature may be traceable to a basic stock of certain mammalian lines of descent. So, it is important to clarify whether the musculature may be a common structure in lower mammals from insectivores to primates and to examine the functional significance of the structure.

Mitochondria DNA Sequences of Finlayson's Squirrel Found in Hamamatsu, Shizuoka Prefecture, Japan

Alien Callosciurus squirrels cause severe damage to trees in forests, gardens, and commercial plantations (Jouanin 1986; Setoguchi 1990; Torii 1993; Aprile and Chicco 1999; Gurnell and Wauters 1999). Pallas’s squirrel (Callosciurus erythraeus) originally occurs in eastern India, Bhutan, southeast China, Taiwan, Indochina, and Malaya (Corbet and Hill 1992; Wilson and Reeder 2005). This squirrel was introduced into other countries as an exotic pet. At present, C. erythraeus populations are found in Argentina (e.g., Aprile and Chicco 1999), France (Jouanin 1986; Gurnell and Wauters 1999), and Japan (Ishii 2005; Tamura 2002, 2009; Ikeda et al. 2011). Under law number 78 of the Invasive Alien Species Act (Ministry of the Environment, Government of Japan, 2004: http://www.env.go.jp/nature/intro/index. html), import of C. erythraeus to Japan is strictly prohibited. This species is treated as ‘invasive alien species’ in Japan. Finlayson’s squirrel (C. finlaysonii), which originally occurs in the Indochina Peninsula (Corbet and Hill 1992; Wilson and Reeder 2005), was also introduced to Italy (Bertolino et al. 1999; Currado et al. 1999). Similar to C. erythraeus, C. finlaysonii is thought to cause damage to environments (Bertolino et al. 2004). It is difficult to distinguish this species from C. erythraeus based on external characteristics such as pelage patterns (e.g., Lekagul and McNeely 1988). Based on mitochondrial (mt) DNA control region sequences, Oshida et al. (2007) reported that C. finlaysonii was probably introduced to Hamamatsu, Shizuoka Prefecture, Japan (Fig. 1). They captured eight Callosciurus individuals in Hamamatsu. Of them, seven individuals had haplotypes closely related to that of C. finlaysonii from Laos (two C. finlaysonii haplotypes were detected), and one individual had a C. erythraeus haplotype. Therefore, we expect these two Callosciurus species exist sympatrically in Hamamatsu. Callosciurus finlaysonii, however, is not treated as an invasive alien species in Japan (law number 78 of the Invasive Alien Species Act, Ministry of the Environment, Government of Japan, 2004). Currently this species is treated as unidentified alien species. If the population of C. finlaysonii increases, this species should be added to the list of invasive alien species. To estimate whether C. finlaysonii is increasing, we investigated ratios of C. finlaysonii haplotype and C. erythraeus haplotype in the Hamamatsu population by using mtDNA control region sequences. We discuss the present situation of introduced C. finlaysonii in Hamamatsu. In addition, we compare control region sequences of specimens from Hamamatsu, Laos and Thailand, to identify the origin of introduced C. finlaysonii. The exact origin would be difficult to determine because we do not have sequence data from all C. finlaysonii populations occurring in the Indochina Peninsula. We, however, obtained successfully C. finlaysonii sequences from Thailand and Laos. Identifying whether the Hamamatsu population is more closely related to specimens from Thailand or Laos may help to prevent subsequent introductions of this squirrel into Japan with international legal and political action.

Karyological study on Kloss's mole Euroscaptor klossi (Insectivora, Talpidae) collected in Chiang Rai Province, Thailand

ABSTRACT We present the first karyological study on Klosss mole Euroscaptor klossi from Chiang Rai Province of Thailand. We used differential chromosome staining methods and determined the diploid and fundamental autosomal numbers to be 36 and 54, respectively. Compared with the G- and C-banded karyotypes of the Japanese mountain mole E. mizura, the karyotype of E. klossi involved a reciprocal translocation followed by a pericentric inversion of chromosomes 1 and 16 of the ancestral karyotype of E. mizura. Some minor C-band duplications supplemented the karyological differences between the two species. The karyotype of E. klossi is distinct from that of the Malaysian mole E. micrura malayana examined previously and these species have distinct scenarios in diverging from the ancestral karyotype of the genus Euroscaptor.

Immunohistochemical localization of inhibin and steroidogenic enzymes in the ovary of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina).

To study the ovarian function of the Order Scandentia, the localization of inhibin and steroidogenic enzymes (3β‐hydroxysteroid dehydrogenase/isomerase and aromatase) in the ovaries of common tree shrew (Tupaia glis) and northern smooth‐tailed tree shrew (Dendrogale murina) was immunohistochemically analysed. As in the results reported for other mammals, inhibin α‐chain was localized in the follicular epithelium of secondary or Graafian follicles in the two species. The localization of aromatase in the ovary of these two species, however, was different. In the common tree shrew, the aromatase was localized in the thecal cells, whilst in other mammals it is localized in the granulosa cells. These results indicate that in the ovary of the common tree shrew, the oestradiol may be synthesized in the thecal cells.

Immunohistochemical Localization of the Cytoskeletal Proteins in the Testes of the Lesser Mouse Deer (Tragulus javanicus)

Abstract. This study demonstrated the immunohistochemical localization of cytoskeletal proteins, cytokeratin, desmin, vimentin, &agr;-tubulin and &agr;-smooth muscle actin (&agr;-SMA) in the testes of adult and immature lesser mouse deer (Tragulus javanicus). Desmin was found in the sub-epithelial myoid cells of epididymal ducts. The immunoreactivity for vimentin was shown in the peritubular myoid cells and around the nuclei of the Sertoli cells of which the positions indicated various arrangements. In the Sertoli cells, vimentin filaments between the basement membrane and the nucleus were elongated with the migration of the nucleus toward the upper part. Furthermore, vimentin was detected in the Leydig cells and the sub-epithelial myoid cells of epididymal ducts. Alpha-tubulin was apparently extended to the tip of each Sertoli cell cytoplasm, and &agr;-SMA was found in the peritubular myoid cells and the sub-epithelial myoid cells of epididymal ducts. On the other hand, cytokeratin and desmin were not detected in the Sertoli cells, Leydig cells, spermatogenic cells and peritubular myoid cells. In the mouse deer testes, vimentin filaments of the Sertoli cells may not be involved in the migration of the elongated spermatids and spermiation like as rat Sertoli cells because the supranuclear extension of vimentin was scanty.

Getah virus epizootic among wild boars in Japan around 2012

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014–2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.