Geographical variation of the skull morphology of the common tree shrew (Tupaia glis).

Geographical variation was examined morphologically in the common tree shrew (Tupaia glis) in some Indochinese and Malayan regions. Osteometrical examination and principal component analysis elucidated the morphological differences among various populations. The populations from southern and western Thailand were distinguished morphologically from the other populations. Variation in males from south Thailand and Kuala Lumpur suggests that the Isthmus of Kra may have an influence on the variation of skull morphology. However, the Isthmus of Kra was not completely considered as a factor of geographical separation in this species, because we could not confirm the separation in skull size and shape between the localities at least in females. While, the Kanchanaburi population in western Thailand was significantly smaller than the other population in skull size, and constituted the morphologically separable group in our study.     

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 (3 beta-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 alpha-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.     

Population genomics provides insights into the evolution and adaptation of tree shrews (Tupaia belangeri) in China

Physiological adaptation of tree shrews (Tupaia belangeri) to changing environmental temperature has been reported in detail. However, the T. belangeri origin (mainland or island), population history, and adaptation to historical climate change remain largely unknown or controversial. Here, for the first time, we sequenced the simplified genome of 134 T. belangeri individuals from 12 populations in China and further resequenced one individual from each population. Using population genomic approaches, we first observed considerable genetic variation in T. belangeri. Moreover, T. belangeri populations formed obvious genetic structure and reflected different demographic histories; they generally exhibited high genetic diversity, although the isolated populations had relatively low genetic diversity. The results presented in this study indicate that T. b. modesta and T. b. tonquinia were separated recently and with a similar population dynamics. Second, physical barriers rather than distance were the driving factors of divergence, and environmental heterogeneity may play an important role in genetic differentiation in T. belangeri. Moreover, our analyses highlight the role of historical global climates in the T. belangeri population dynamics and indicate that the decrease of the T. belangeri population size may be due to the low temperature. Finally, we identified the olfaction-associated adaptive genes between different altitude populations and found that olfactory-related genes of high-altitude populations were selectively eliminated. Our study provides demographic history knowledge of T. belangeri; their adaption history offers new insights into their evolution and adaptation, and provides valuable baseline information for conservation measures.     

Immunohistochemical study on the endocrine cells in gut of the stomachless teleost, Zacco platypus (Cyprinidae)

The regional distribution and relative frequency of neurohormonal peptides-producing cells were demonstrated in the gut of the stomachless teleost, Zacco platypus (Temminck et Schegel), using 10 types of specific antisera raised against mammalian regulatory peptides. The gut of Z. platypus was divided into five portions from proximal to distal (segment I-V). Most of immunoreactive cells in the epithelial lining portion, between epithelial cells, were generally spherical or spindle shape having long cytoplasmic process that was reached via the lumen while cells round in shape were found in the basal portions of epithelial lining occasionally. Serotonin-, somatostatin-, glucagon-, cholecystokinin (CCK)-8 and pancreatic polypeptide (PP)-immunoreactive cells were observed in this study. However, no chromogranin A-, secretin-, vasoactive intestinal peptide (VIP)-, substance P- and bombesin-immunoreactive cells were found. Serotonin-immunoreactive cells were demonstrated throughout the entire gut tract and occurred more frequently than other cells. Somatostatin-immunoreactive cells were restricted to proximal segments of the gut (segment I-III) with rare frequencies, and glucagon-immunoreactive cells were demonstrated in the proximal segments of the gut (segment I, II) with moderate to few occurrences. CCK-8-immunoreactive cells were found throughout the whole intestinal tract except for most proximal segment (segment I) with moderate to few frequencies and PP-immunoreactive cells were demonstrated in the proximal to middle segments, segment I-III, with a few, rare and rare frequencies, respectively.     

树鼩实验室繁殖的新方法

在实验室笼养条件下将28只健康成年树鼬按1:2或1:3的雄雌比例配对饲养,并每隔3周进行一次检查,通过一年的试验饲养,研究结果表明,这批实验树鼬共受孕45次,分娩37窝,流产2窝,死产3窝,总产仔147只,成活139只,另有3只母树鼦怀孕待产.每只母树鼩产仔2～3窝,每窝2～5仔,幼仔初生体重为(10.4±1.2)g,40日龄断奶体重为(66.2±15.7)g.该方法树鼩繁殖率和成活率极高,成本相对较低且操作简单,是一种值得参考和借鉴的新方法.     

Relationships between body weight,fasting blood glucose concentration,sex and age in tree shrews (Tupaia belangeri chinensis)

The tree shrew (Tupaia belangeri chinensis) is a squirrel‐like lower primate or a close relative of primates, commonly used as an animal model in biomedical research. Despite more than three decades of usage in research, the clear relationships between body weight, fasting blood glucose concentration, sex and age among tree shrews remain unclear. Based on an investigation of 992 tree shrews (454 males and 538 females) aged between 4 months and 4 years old, we found that male tree shrews have significantly higher body weight and fasting blood glucose concentration than female tree shrews (p < 0.001). The concentration of fasting blood glucose slightly increased with body weight in males (r = 0.152, p < 0.001). Meanwhile, in females, the body weight, concentration of fasting blood glucose and waist circumference positively increased with age (p < 0.001). Additionally, 17 tree shrews with Lee index [body weight (g)*0.33*1000/body length (cm)] above 290 had significantly higher body weight, waist circumference and glycated haemoglobin A1c (HbA1c) than non‐obese tree shrews with a Lee index score below 290 (p < 0.001). Interestingly, 6 of 992 tree shrews (three males and three females, 2–4 years old) displayed impaired plasma triglycerides, HbA1c, low‐density lipoprotein and oral glucose tolerance test, suggestive of the early symptoms of metabolic syndrome. This study provides the first clear relationships between body weight, fasting blood glucose concentration, sex and age in tree shrews, further improving our understanding of this relationship in metabolic syndrome (MetS). Given the similarity of tree shrews to humans and non‐human primates, this finding supports their potential use as an animal model in the research of MetS.     

Immunohistochemical staining of chlamydial antigen in emerald tree boas (Corallus caninus).

Of 120 privately owned captive-bred and wild-collected emerald tree boas (ETBs) (Corallus caninus), 97 died or were euthanatized. Eighteen snakes were necropsied, and tissues were collected from all major organs and processed for light microscopy. Histologic examination demonstrated histiocytic granulomas in the small intestine, heart, and esophageal tonsils of one ETB, small intestine of a second ETB, and in an esophageal tonsil of a third ETB. Within the center of these granulomas, small, basophilic, punctate organisms were demonstrated using hematoxylin and eosin staining. Transmission electron microscopic examination of an intestinal granuloma demonstrated developmental stages of organisms consistent with members of the family Chlamydiaceae. An immunoperoxidase staining technique and 2 different commercially available monoclonal antibodies against chlamydial lipopolysaccharide antigen was used to identify chlamydial antigen in these lesions. Liver of a puff adder (Bitis arietans) with previously reported systemic chlamydiosis served as the positive control. Both monoclonal antibodies stained antigen in these granulomas. Additionally, macrophages within aggregates of lymphoplasmacytic cells in the colon, small intestine, and esophageal tonsils of 3 other ETBs contained antigen. Although both antibodies labeled antigen in serial sections of tissue, a difference in staining intensity was noted.     

Suppression of hepatitis B virus replication in Tupaia hepatocytes by tumor necrosis factor alpha of Tupaia belangeri

Recently, Tupaia belangeri was used to study the full replication cycle of hepatitis B virus (HBV) in the primary hepatocyte cultures. Thus, the Tupaia model represents a suitable model to study the effects of cytokines on HBV infection. Here, Tupaia tumor necrosis factor-alpha (TNF-α) was molecularly cloned and expressed in mammalian cells. A test system for the biological activity of Tupaia TNF-α was established on the basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant Tupaia TNF-α was able to suppress HBV replication in primary Tupaia hepatocytes (PTH). However, the formation of HBV covalently closed circular DNA (cccDNA) and viral RNA was not completely prevented. Therefore, Tupaia TNF-α may contribute significantly to the control of HBV infection though it is not able to completely inhibit HBV replication alone. The characterization of this important cytokine allows further studies on its antiviral actions in the Tupaia model.     

Sympatric distribution of the two morphological types of the common tree shrew in Hat-Yai districts (South Thailand)

The two color types (grayish northern and reddish southern types) of the common tree shrew (Tupaia glis and Tupaia belangeri) were co-distributed in Hat-Yai region (South Thailand). Although the Isthmus of Kra in South Thailand has been considered as distribution barrier of the two types, the sympatric distribution of both types was confirmed in southern side of the Isthmus. In the principal component analysis, the skull measurement character from Hat-Yai region could also be separated into the northern and southern groups according to the skin color identification of corresponding individuals. We could generally distinguish the common tree shrew into two types by skull morphology as well as external skin color.     

Osteometrical skull character in the four species of tree shrew

The skull size and shape were osteometrically examined in the four species of the tree shrews (Tupaia tana, T. javanica, T. minor and T. dorsalis). We suggest that the skull characters were affected by the species specific behavior and terrestrial, arbo-terrestrial and arboreal life, among the genus Tupaia. The neurocranium was laterally narrower in the braincase area, and the splanchnocranium was longer only on dorsal side in T. tana, and these characters were opposite to T. minor. The principal component analysis confirmed the obviously separated clusters among T. tana, T. javanica and T. minor, affected by the adaptation for each behavior. T. dorsalis was considered as terrestrial species from the results of proportion analysis and the principal component analysis.     

Multivariate analysis in skull osteometry of the common tree shrew from both sides of the Isthmus of Kra in Southern Thailand

The Isthmus of Kra in Southern Thailand consists of a lowland of about 70 km in length. It has been suggested that the Isthmus may sink beneath the sea surface according to the change of level of the sea, and may function as a zoogeographical barrier in land mammals in this region. So, the geographical variation was osteometrically examined in skull of the common tree shrew (Tupaia glis) from the both sides of Isthmus of Kra. The osteometrical examination demonstrated that the skull is larger in southern population than in northern one. In the charts of the principal component analysis, however, the morphological separation between two populations can not be obviously seen in female. While, the results of the discriminant analysis indicated the morphological separation between the two populations. These findings suggest that the zoogeographical barrier of Isthmus of Kra may have influence on the osteometrical variation in the common tree shrew, when the Isthmus was covered with sea.     

Immunohistochemical study of endocrine cells in the gastrointestinal tract of the barking deer, Muntiacus muntjak

The morphology, distribution and relative frequencies of endocrine cells in the gastrointestinal tract of the barking deer (Muntiacus muntjak) were investigated. The immunohistochemical method employed used seven types of antisera against chromogranin, serotonin, gastrin, cholecystokinin, somatostatin, glucagon and insulin. Chromogranin and serotonin immunoreactive (IR) cells were found throughout the gastrointestinal tract. Conversely, gastrin-IR cells were only found in the oxyntic gland, pyloric gland and duodenum, while cholecystokinin-IR and somatostatin-IR cells were detected in the oxyntic gland, pyloric gland and small intestines. Somatostatin-IR cells were also seen in the caecum. Glucagon-IR cells were found in all parts of the gastrointestinal tract apart from the colon and rectum. No insulin-IR cells were found in the gastrointestinal tract of this species. The cells in the small intestine were generally spindle shaped with long cytoplasmic processes ending in the lumen (open type), while in the stomach and large intestine, they were occasionally round or spherical in shape (closed type). An uncommon distribution pattern of endocrine cells in the gastrointestinal tract of the barking deer was noted for cholecystokinin- and glucagon-IR cells.     

Immunohistochemical and ultrastructural study of endocrine cells from the pyloric region of the European mole (Talpa europaea)]

The distribution and the morphology of some endocrine cells (gastrin, somatostatin and 5-HT immunoreactive) in the pyloric region were studied in the Talpa europaea, an insectivore representing one of the most primitive living Eutherians. The immunohistochemical studies enabled us to identify and calculate the percentage of each cell type: the most numerous endocrine cells were gastrin immunoreactive; fairly numerous appeared somatostatin immunopositive; less numerous were 5-HT immunoreactive cells. While the ultrastructural observations let us describe four endocrine cell types: G cells producing gastrin, D cells containing somatostatin, EC cells of the gastric type producing 5-HT and D1 cells whose content is still unknown.     

Immunohistochemical study on the distribution of endocrine cells in the gastrointestinal tract of the babirusa, Babyrousa babyrussa (Suidae)

The distribution and relative frequency of endocrine cells in the gastrointestinal tract of the babirusa were studied immunohistochemically using the avidin-biotin-peroxidase complex method. Thirteen types of gut endocrine cells were detected; they were immunoreactive for chromogranin, serotonin, somatostatin, gastrin, bovine pancreatic polypeptide (BPP), glucagon, secretin, cholecystokinin (CCK), methionine-enkephalin-Arg6-Gly7-Leu8 (MENK8), motilin, gastric inhibitory polypeptide (GIP) and peptide tyrosine tyrosine (PYY). Cells that were immunoreactive for chromogranin, serotonin, somatostatin and glucagon were found in all portions of the gastrointestinal tract. MENK8-immunoreactive cells were observed in the stomach and small intestine. Gastrin-immunoreactive cells were detected in the pyloric region and duodenum. PYY-immunoreactive cells were found in the small and large intestine. Cells immunoreactive for motilin, CCK, GIP, and secretin were observed in the proximal small intestine and those immunoreactive for neurotensin were found only in the ileum. Although the distribution pattern of endocrine cells in the gastrointestinal tract of babirusa was similar to those reported for pig, restricted distribution of several endocrine cells, gastrin, BPP, MENK8, motilin, CCK, GIP, secretin and neurotensin and wider distribution of glucagon and PYY were observed in the babirusa. The unexpected presence of MENK8 in all glandular regions of the stomach and PYY in the small intestine was also noted. The distribution of gut endocrine cells might be related to the regulatory characteristics of the babirusa digestive tract.     

Immunohistochemical studies on the development of endocrine cells in the thyroid and parathyroid glands in the golden hamster

The development of endocrine cells in the thyroid and parathyroid glands in the golden hamster was studied immunohistochemically in relation to the formation of these glands. The thyroid was formed on day 9 of gestation by the ventral outpocketing of the foregut between the first and second branchial pouches. The thyroid epithelial cells were faintly thyroglobulin-immunoreactive on day 10.5 of gestation. This immunoreaction became intense thereafter, but was almost confined to the cytoplasm of epithelial cells until birth. It appeared in the follicular lumen in newborn animals. The ultimobranchial body was derived from the fifth pouch and fused with the thyroid on day 12 of gestation. Calcitonin-immunoreactive cells first appeared on day 14 of gestation in the dorsomedial part of the thyroid derived from the ultimobranchial body and increased in number and intensity thereafter. Somatostatin-immunoreactive cells also appeared in the dorsomedial part of the thyroid derived from the ultimobranchial body on day 13 of gestation, and increased in number in newborn animals, but decreased thereafter. The parathyroid was derived from the third pouch, situated on day 13 of gestation on the dorsolateral side of the thyroid, and surrounded by a common capsule with the thyroid. Parathormone-immunoreactive cells first appeared on day 15 of gestation in the parathyroid and increased in number and intensity after birth.     

Immunohistochemical study of gastroenteropancreatic endocrine cells of the herbivorous Japanese field vole, Microtus montebelli

The gastroenteropancreatic (GEP) endocrine cells of the Japanese field vole were studied immunohistochemically. Somatostatin-, 5-hydroxytryptamine-, glicentin-, glucagon-, bovine pancreatic polypeptide-, gastrin-, gastric inhibitory polypeptide-, cholecystokinin-, substance P-, secretin-, neurotensin- and insulin-immunoreactive cells were revealed. The characteristic findings of the regional distribution and relative frequency of these immunoreactive cells in the GEP system of the vole were as follows. Somatostatin-immunoreactive cells were more numerous in the oxyntic glands than in the pyloric glands. Some somatostatin-immunoreactive cells were found in small clusters in the oxyntic glands. Gastrin-immunoreactive cells were detected not only in the pyloric glands and small intestine but also in the caecum and spiral colon. Gastric inhibitory polypeptide-immunoreactive cells were also detected in the pyloric glands and no motilin-immunoreactive cell was found in the gastroenteropancreatic system.     

Production and Immunohistochemical Characterization of Monoclonal Antibodies Against Ovarian Antigen of the Common Tree Shrew (Tupaia glis)

To study the ovarian function of the common tree shrew (Tupaia glis), monoclonal antibodies (MAb) against its ovarian antigen were produced. Several positive hybridomas were cloned and MAb-T2C9 (IgG) was examined for reactivity. Strong immunoreactivity of MAb-T2C9 was localized within the cytoplasm of luteal cells and in the theca interna cells of Graafian follicles. MAb-T2C9 reacted also with testicular interstitial cells and adrenocortical cells, except those of the zona glomerulosa. Similar reactions were seen in the goat and sheep. Western blotting analysis of the ovine corpus luteum after reaction with MAb-T2C9 revealed a single positive band of ?60kDa. These findings suggest that MAb-T2C9 recognized protein molecules related to steroid synthesis.     

Bioactive peptides and serotonin in the gut endocrine cells of the crocodile, Crocodylus niloticus (Laurenti 1768): an immunocytochemical study.

Little is known about peptide-storing endocrine cells in the gut of the Nile crocodile. As in the case of other reptiles, particularly the alligator, a limited range of peptide-storing cells was found in the gut of the crocodile. They were somatostatin, glucagon, gastrin, neurotensin and pancreatic polypeptide. The topographical distribution of cells immunoreactive to somatostatin and gastrin in the gut of the crocodile is comparable to the situation in the alligator. Glucagon and neurotensin immunoreactive cells have a much wider distribution in the gastro-intestinal tract of the crocodile compared to the alligator. Cholecystokinin and bombesin cells previously reported in the small intestine of the alligator were not detected in this study. This is the first report to demonstrate pancreatic polypeptide and serotonin immunoreactivity in the gut of a crocodilian specie.     

Immunohistochemical study on the distribution and relative frequency of endocrine cells in the stomach of the Malayan Pangolin, Manis javanica

The distribution and relative frequency of six kinds of endocrine cells in the stomach of the Malayan pangolin, Manis javanica were studied immunohistochemically using the avidin-biotin-peroxidase complex method. The stomach of the pangolin has three regions of mucous gland, one oxyntic gland and one pyloric gland. Cells immunoreactive for chromogranin, serotonin, somatostatin, BPP and glucagon were detected in all of the gastric glands, while gastrin-immunoreactive cells were found in the entire gastric gland except for the oxyntic gland. The distribution pattern of endocrine cells in the mucous gland and pyloric gland was mainly from the middle to apical portions of the glands. The endocrine cells were rare or not detected in the basal portion of all of the mucous glands and pyloric gland, but they were also found in the basal portion of the oxyntic gland. The distribution pattern of the endocrine cells in the mucous and pyloric glands suggested that this position facilitates a quick response to the luminal ingesta. The wide distribution of gastrin-immunoreactive cells in all of the mucous glands and pyloric gland was the most remarkable finding. This distribution suggests a major function of gastrin-immunoreactive cells for the digestive process in the Malayan pangolin stomach.     

Strongyloidiasis in a Cope's grey tree frog (Hyla chrysoscelis).

A male Cope's grey tree frog (Hyla chrysoscelis) died spontaneously with ventral subcutaneous edema and was necropsied. Thickening of the intestinal mucosa was observed histopathologically, with villous atrophy and intraepithelial nematodes present. Adult female Strongyloides sp. nematodes were isolated from the fixed intestinal tract, the first time this nematode genus has been recovered from this frog genus. Intestinal strongyloidiasis should be considered as a cause for protein-losing enteropathy and death in frogs.