2006 Vol. 27, No. 1
This paper studied the influences of forest regeneration on the diversity of ground-dwelling beetles (Coleoptera), in taxonomic level of families, in Wolong Natural Reserve (30°45′-31°25′N，102°52′-103°24′E), Southwestern China. Comparisons were conducted among larch plantations of different forest ages, namely, recently planted (5 years old), young (15 years old), mature plantation (45 years old), and natural deciduous broad-leaved forests (ca. 100 years old). Larch plantations were dominated by non-native Larix kaempferi but interspersed with a few native L. mastersian. Pitfall traps were used in the field collections. During the field research, a total of 7 444 beetles were collected, of which, Carabidae accounted for 40.2%, Staphylinidae 38.3%, and Tenebrionidae 6.4%, and these three families were considered as dominant groups. Family richness, diversity and evenness of beetles were significantly higher in three larch plantations than in natural broad-leaved forest; among the three plantations, the highest values were observed in the recently planted whereas the lowest in the young plantation. Contrary to the above three indices, family abundance was higher in natural broad-leaved forest than in three larch plantations; within the three plantations, the lowest value were showed in the recently planted whereas the highest value in the young plantation. Based on the family composition and abundance, ground-dwelling beetles of three larch plantations and the adjacent natural broad-leaved forest could be separated by ordinations of principal coordinate analysis (PCO) and cluster analysis, but beetle assemblages in the mature plantation was similar to both natural forest assemblages and young plantation assembalges. Seasonal dynamics of family abundance were similar among the three plantations, but significantly different from that of the natural broad-leaved forest. However, family richness, diversity and evenness did not show the similar trends. Multiple linear regression analyses showed that the coverage and height (or depth) of canopy layer, herbaceous layer and leaf litter layer were the most important factors in determining the family richness, abundance, diversity; and evenness. Although some similar characteristics in beetle composition were displayed in the mature plantation as well as in the natural broad-leaved forest, our results showed that the composition of ground-dwelling beetles in larch plantations differed obviously from those in the natural deciduous broad-leaved forest at the family level. Finally we concluded that, in an attempt at reforestation by the establishment of plantations, it is necessary to preserve the undisturbed deciduous broad-leaved forest for the purpose of protecting the diversity of ground-dwelling beetles in Wolong Natural Reserve.
Acoustic Characters of Chinese White-bellied Rat's Voice in Different Individual Encountering Settings in Captive
2006, 27(1): 12-17.
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The voices of 11 individuals (3♂, 8♀) of Chinese White-bellied Rat (Niviventer confucianus) from field and their behaviors were recorded and observed in different individual encountering settings in captive. The results showed that Chinese White-bellied Rat made 4 kinds of voices in different individual encountering situations, I.e. defeated male's voice, defeated female's voice, female's voice when male occurred and young's voice when adult occurred, and the significant differences on values of most measured acoustic parameters between voices could be found. The acoustic characters based on our studies suggested that Chinese White-bellied Rat might have two types of voices when individuals encountered each other: One is fearing call with multi-harmonic frequency conversion, including defeated individual's voice and young's voice when adult occurred; another is rejecting call with multi-harmonic constant frequency, such as female's voice when male occurred.
2006, 27(1): 18-22.
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Dens of Tibetan fox in Shiqu County, Sichuan Province were surveyed to understand their characteristics and the mechanism of habitat adaptation of the Tibetan fox. Four fixed line transects with a total length of 40 km, covering 230 km2 research area were set from July to August 2001, 2002, August to November 2003, and March to April 2004. The mean entrance number of Tibetan fox dens was 1.8±1.8 (n=156). Multi-entrance dens (n=47) were more distributed on slope than mon-entrance (n=109)(U=1 417，P＜0.001). The mean of width, height and the first tunnel length of 91 measured dens were (17.0±2.5) cm, (24.9±7.2) cm and (169.9±88.4) cm respectively. Tibetan fox dens were mainly constructed by soil (74.7%). Rock dens were significantly more distributed on slope than the other two kinds of dens (χ2=10.227，P=0.006). Den entrance exposure did not show concentration on mean angle (Rayleigh，Z=0.898, n=201,P＞0.05), but slope direction concentrated significantly on the mean angle (249.9±77.1)°(Rayleigh，Z=7.907,n=201,P＜0.05). No correlation was found between entrance exposure and slope direction (Hotelling，F1,400=5.365,P＜0.05) but the first tunnel obliquity correlated with the gradient (t=0.350,n=116，P＞0.05), four breeding dens were found during our field research. These breeding dens were all made of soil with the slope direction from 220° to 320°. No fox dens were found on the plain area.
2006, 27(1): 23-28.
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Changes of soil fauna communities were studied by Tullgren dry funnel method in the grassland, pine, mixed Schima superba, Acacia mangium and mixed Acacia forests during four seasons of 2003-2004 at the Heshan Research Station. It suggested that there were significant effects of season, plantation and soil depth on soil fauna (P<0.01, three-way-ANOVA). Soil fauna were conspicuously abundant in autumn (P<0.05, DMRT), but conspicuously succinct in summer (P<0.05, DMRT). Soil fauna decreased significantly (P<0.05, DMRT) into deeper soil and most of them concentrated in 0-5 cm soil layer. The order of all plantations was mixed Acacia forest > Acacia mangium forest > mixed Schima superba forest, pine forest and grassland. Significant interaction can also be found between plantation and season, and plantation and soil depth respectively. Soil fauna varied mostly in different plantations in autumn when soil fauna developed to the highest level, and the differences of soil fauna were discovered largely among soil depths with better plantation. It was concluded that man-made reconstruction to ecological system was beneficial to forest restoration, and the differences among various plantations might be correlated closely to the amount and quality of the litter in the forests.
Four pine forests (6-10, 11-15, 16-20 and 31-40 year-old) located in the Cangshan Mountain and Erhai Lake National Reserve and seven pine forests (1-5, 6-10, 11-15, 16-20, 21-30, 31-40 and more than 50 year-old) located in the non-protective area near the national reserve were selected. Three replications of each forest was set and a total of 33 sites were investigated. At each site, we quantified six habitat variables (species richness, abundance and percentage of grasses and shrubs coverage respectively at the bottom layer of forests) within randomly determined 5 m×5 m areas. One hundred cages were set in five lines at each site to trap small mammals, whose species and numbers were recorded. Dominance of Dremomys pernyi and Callosciurus erythraeus in small mammal communities, time niche breadth and time niche overlap between the two small mammals were calculated respectively. Step-wise regression was used to analyze relationship between small mammals and habitat factors. Our results indicated that D. pernyi occurred earlier than C. erythraeus in protective pine forests. D. pernyi was captured in 6-10 year-old forest initially, and C. erythraeus was captured in 16-20 year-old forest initially. D. pernyi and C. erythraeus were captured in the 31-40 and 21-30 year-old forests initially in the non-protective area, respectively. Populations of D. pernyi and C. erythraeus in the 31-40 year-old protective forests were 3 and 3.75 times of those in the same-aged non-protective forests, respectively. Shrubs significantly influenced populations of the two small mammals. Populations of Dremomys pernyi was positively correlated with density of shrubs; populations of C. erythraeus was positively correlated with coverage of shrubs, and negatively correlated with coverage of grasses. D. pernyi and C. erythraeus were important for pine forests to scatter pine seeds. Human activities in the non-protective pine forests decreased the vegetation heterogeneity at the bottom layer of pine forests, postponed the occurrence of D. pernyi and C. erythraeus, and decreased populations of the two small mammals.
2006, 27(1): 34-40.
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Historically the black crested gibbon (Nomascus concolor,N. sp. Cf. nasutus) has ever been distributed widely in southern and southeastern Yunnan. A series of field surveys and interviews were conducted with local people, from July 2003 to December 2004 to assess the conservation status of the gibbons in southeast Yunnan. Results demonstrated that the eastern black crested gibbon (N. sp. Cf. nasutus) and the western black crested gibbon (N. concolor) disappeared in the Niuluohe Nature Reserve of Jiangcheng County, Gulinqing Nature Reserve of Maguan County, Laojunshan Nature Reserve of Malipo County and probably in Daweishan Nature Reserve of Pingbian County. Less than 25 individuals of 4-7 groups were found in isolated habitats in Bajiaohe (2 groups), Xilongshan (1-2 groups) nature reserves located in Jingping County, and Huanglianshan (1-3 groups) nature reserve in Luchun County. Great loss of habitats and heavy hunting caused distributions reduction and population decline.
In Vitro Culture of Bovine Fetal Fibroblast Cells Using Tissue Explant Attachment and Gene Transfection Through Electroporation
2006, 27(1): 41-47.
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The low efficiency and high cost of pronuclear microinjection technology has been the main barrier for transgenic animal production. The production of somatic cell clones by using cultured cells derived from different tissues sheds new lights on transgenic technologies. In order to prepare the nuclear donor cells for bovine transgenic cloning, bovine fetal fibroblast (BFFb) cells were isolated by attaching tissue explants from ear skin of a bovine fetal at 3-4 months gestation stage. The cells grew to confluence 7 days after attachment and were cryopreserved after purification and amplification for 2-3 passages. The cell growth curve was plotted, and the karyotype of the cells within the 10th passages and exceed the 20th passages was analyzed. The plasmid pNEI, which contained the Neor gene, the EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the human pro-insulin gene regulated by bovine α-lactalbamin promoter for expression specifically in mammary gland, were linearized by digestion with XhoⅠ and purified with phenol chloroform extraction. BFFb cells at passage 3 were harvested at 70% confluence and suspended in HeBES buffer to 5×106 cell/ml. Two hundred microliters of the cell suspension were electroporated with 4 μg linearized pNEI vector in a 2mm Gap cuvette for 1, 5, 10, 15 and 20 ms at 800, 900, 1 000 V/cm, respectively. Cells were checked 24-48 hours after electroporation under fluorescence microscopy for GFP expression, and G418 selection (800 μg/mL) was applied since then. After 2 weeks, selected colonies were counted and maintained in culture medium containing 300 μg/mL G418 for 2-3 passages before cryopreservation. A small part of the cells were analyzed by PCR for gene integration. The data showed that the isolated BFFb cells grew actively, and maintained diploid karyotype even after 20th passages. Bright green fluorescence can be detected from 24 to 48 hours after electroporation, and more colonies were selected at the electroporation condition of 900 V/cm, 5 ms. PCR detection demonstrated that the foreign gene was integrated into the genome. The results indicated that the isolated BFFb cells might be competent for transgenic cloning.
Human peripheral blood mononuclear cells (PBMC) were separated from blood by ficoll-hypaque density gradient centrifugation and rhesus monkey spleen cells were isolated from fresh spleens. To isolate inner cell masses (ICMs) by immunosurgery from human or rhesus monkey embryos and establish embryonic stem cell (ES cell) lines, antiserums were prepared by injecting rabbits with human PBMC or rhesus monkey spleen cells, with 4×108 cells every time, once a week and three times in all, respectively. Our results were listed as below: 1) Rabbit anti-human PBMC antiserum has a titer around 1∶320 and rabbit anti-rhesus spleen cell antiserum has a titer around 1∶640; 2) The trophectoderm were lysed by immunosurgery using the rabbit anti-human PBMC or anti-rhesus spleen cell antiserum followed by exposure to guinea pig complement. ICMs were successfully isolated from 15 human blastocysts and 33 monkey blastocysts. These results indicated that our antiserums could be used for collecting human or monkey ICM cells and they would play important roles in isolating new human or rhesus monkey ES cells in the future.
Correlation Between Dopamine D2 Receptor in the Orbital Frontal Cortex in Rat and the Processing of Morphine Dependence
2006, 27(1): 54-62.
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With conditional place preference (CPP) of morphine dependent rats, the study focus on the effects of the DAD2 receptor in the orbital frontal cortex (OFC) in rat associated with the processing of opioid dependence. CPP models of morphine dependent rats were established by intraperintoneal injection and combined with environmental stimulation. Rats were devided into three groups received Quinpirole or Raclopride injection (agonist or antagonist of the DAD2 receptor) in OFC by a miniminjector after the last conditioning session and CPP was assessed by the time spent in the morphine-associated environment after conditioning. All dates were analysed by statistical method. The time in drug place of Raclopride intervene group showed respectively significantly longer than the control group in the 2nd withdrawal day (P<0.05), however the time of Quinpirole intervene group didn't show significant difference compared with control group (P>0.05). The results indicated that: (1) CPP models of morphine dependent rats were established successfully by intraperintoneal injection with the proper dosage with the reference of many related papers; (2) The DA system in OFC plays an important role in the procession of morphine dependence; (3) The damnification in orbital frontal cortex of animals who addicted in drugs will increase their drugs craving behavior. Clinically, it might indicate that we must cautiously operate intervention to the patients of drug addiction.
Assessment of Homozygosity in Gynogenetic Diploid Using Microsatellite Markers in Japanese Flounder (Paralichthys olivaceus)
2006, 27(1): 63-67.
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Gynogenesis was thought to be a useful method to generate inbred lines in fish. In this study, analysis of 5-8 heterozygous loci in Japanese flounder (Paralichthys olivaceus) was used to measure the increment of homozygosity in meiogynogenetic diploid (Meiotic-A & Meiotic-B) and mitogynogenetic diploid (Mitotic-A). The result showed all homozygosity in Mitotic-G1, while a high percentage of heterozygosity in Meiosis-A and Meiosis-B except Poli11TUF. The rate of heterozygosity in meiogynogenetic progenies at the locus of Poli9TUF, Poli9-8TUF, Poli11TUF, Poli13TUF, Poli23TUF, Poli30TUF, Poli123TUF and Poli130TUF were 1.000 0, 1.000 0, 0.194 4, 0.945 9, 0.861 1, 1.000 0, 0.777 8 and 0.800 0, respectively. The average recombination rate of these eight loci is 0.822 4. The high proportion of heterozygotes for seven loci demonstrates that it is not a practical method for producing homozygous inbred lines in the gynogenetic fish produced by retention the second polar body; and treatments of suppressing the first cell division were more promising for this purpose.
2006, 27(1): 68-74.
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Nixi chicken as an indigenous breed in Yunnan plateau displays some particular genetic characteristics, such as disease resistance, fecundity and the ability to adapt to high altitude and the cold weather. In order to elucidate background information on its genetic diversity and population history and to support the decision on its conservation and further utilization, the polymorphisms of 33 microsatellite loci from 24 chromosomes were examined to assess the level of genetic diversity in this breed. The results showed that 122 alleles were identified in 50 individuals and the mean value was 3.7 per locus. The average heterozygosity and polymorphic information content of 33 microsatellite loci were 0.635 0 and 0.551 4, respectively. The genetic polymorphism observed in macrochromosomes was higher than that in microchromosomes. These results indicated that the Nixi chicken breed has high level of genetic diversity.
2006, 27(1): 75-80.
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Using the Bac-to-Bac baculovirus expression system, the acetylcholinesterase 1 gene (ace1) of Aphis gossypii was successfully expressed in Sf9 cells. The enzyme was secreted into the cell culture medium and tested. The affinity (Km) of the expressed AChE1 for the substrate analog ATChI was 144.5±28.6 μM. The sensitivity index (indicated by Ki value) of expressed AChE1 to 2 carbamate insecticides (pirimicarb and triazamate) and 2 organophosphate insecticides (omethoate and demeton-S-methyl) were 513, 1 883, 5 and 23 (mM·min) -1, respectively, which is in agreement with previously reported values obtained with the natural enzyme. The expressed enzyme showed highest activity when harvested at 2-3 d after infection of the insect cells. The expressed AChE1 was very stable without obvious loss in enzyme activity after 90 d when stored at -20 ℃, although 70% of the enzyme activity was lost after 7 d when stored at room temperature.
2006, 27(1): 81-85.
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A new species of cyprinid fish genus Sinocrossocheilus has been recognized on basis of collections from Hongshuihe River, a tributary of Xijiang, Guangxi, China, September, 1993. All type specimens of Sinocrossocheilus megalophthalmus sp. Nov. (Holotype: KIZ 9309011, 49.1 mm SL) were collected from an underground river of Hongshui He in Tian-E County, Guangxi, China. S. megalophthalmus sp. Nov. is closely related to S. guizhouensis, S. microstomatus and S. labiatus by having 2 rows of pharyngeal teeth, while the other 7 species of the genus having 3 rows. It can be distinguished from S. guizhouensis by following characters: no black spot above midpoint of pectoral fin vs. having a prominent black spot; branched dorsal fin ray 7 vs. 8; branched pelvic fin ray 7 vs. 8; predorsal scales 15-16 vs. 12-14; bigger eye, eye diameter 2.5-3.1 in head length vs. 4.0-5.0; eye diameter 44.7%-57.8% head width vs. 16.6%-20.7%; rostral barbel reaching between anterior and posterior nostrils or reaching anterior edge of eye vs. not reaching posterior edge of posterior nostril; maxillary barbel reaching between anterior edge of eye and midpoint of eye or reaching between midpoint of eye and posterior edge of eye vs. reaching anterior edge of eye; caudal peduncle depth 8.9-10.7 times in SL vs. 7.2-8.2; snout length 2.5-3.7 times in head length vs. 1.9-2.2. It can be distinguished from S. labiatus by following characters: predorsal scales 15-16 vs. 22; lateral line scales 39-40 vs. 42-45; scales above lateral line 4.5-5.5 vs. 6.5; branched dorsal fin rays 7 vs. 8; gill rakers 13 vs. 10; pelvic fin reach anus vs. not reach; eye diameter 44.7%-57.8% head width vs. 23.3%-30.0%. S. megalophthalmus is most similar to S. microstomatus in fin ray counts, lateral line scales and color pattern, but it can be distinguished from S. microstomatus by following characters: circumpeduncle scales 14/16 vs. 12; bigger eye, eye diameter 2.5-3.1 times in head length vs. 4.4-4.6; pelvic fin reaching anus vs. not reaching anus; maxillary barbel reaching between anterior edge of eye and midpoint of eye or reaching between midpoint of eye and posterior edge of eye vs. reaching below eye; prefringe of rostral cap split into fimbriations vs. not split or not prominent split; dorsal fin origin closer to snout tip than caudal fin base vs. equal to; dorsal fin longer than head vs. almost equal to; caudal peduncle length 4.8-5.9 times in standard length vs. 6.1-6.4; snout length 2.5-3.7 times in head length vs. 2.1-2.4; caudal peduncle length 1.6-2.1 times its depth vs. 1.4-1.5.
Ultrastructure of Bat (Pipistrellus kuhlii) Epidermis with Emphasis on Terminal Differentiation of Corneocytes
2006, 27(1): 86-93.
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The ultrastructure of the skin of air-adapted mammals (bats) is not known. The study at the electron microscope of the skin of the back and the flying membrane of Pipistrellus kuhlii showed that the thickness of the epidermis is very low (10-12 μm), and that 1-2 flat spinosus cells are present beneath the stratum corneum which is formed by very thin corneocytes that resemble those of avian apteric epidermis. The stratum granulosum is discontinuous and few small (less than 0.3 μm large) keratohyalin granules are present. The epidermis is reduced to one flat basal layer in contact with the stratum corneum in many areas of the flying membrane. Transitional corneocytes are almost absent suggesting that the process of cornification is very rapid. In the basement membrane numerous hemidesmosomes are present and form attachment points for the dense dermis underneath. Numerous collagen fibrils directly contact with the hemidesmosomes and the dense lamella of the basement membrane. Sparse elastic fibrils allow the stretching of the epidermis during flight and the rapid folding of the epidermis after flying without damaging the epidermis. Like in avian epidermis, the production of lipids is high in bat keratinocytes, and multilamellar bodies discharge lipids extra- and intra-cellularly. This may compensate the lack of a thick fat layer in the dermis of the flying membrane as lipids may help in thermical insulation against the cooling air currents flowing on the bat skin during flight. Fur hairs are very thin (4-7 μm), and they have an elaborated cuticle made of pointed expansions similar in texture with that of the cortex. Cuticle cells form hook-like grasping points that allow to keep hairs stuck together. In this way the pelage remains compact in order to maintain body temperature.
2006, 27(1): 94-97.
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The Lugu Lake, shared by Yunnan and Sichuan Province, is a fault lake in the Northwest of Yunnan Plateau in China. Its original fish fauna was composed of 4 species, including three Schizothorax species and Misgurnus anguillicaudatus. During our surveys to the Lugu Lake in 2001, 2002 and 2004, 518 specimens were collected and the current fish fauna was documented. Besides previous collections and abundant reports, 12 species have been recorded in the Lugu Lake, which belong to 10 genera, Four families and three orders. Change of fish fauna in the Lugu Lake was primarily due to over-fishing and debris flow caused by deforestation.
2006, 27(1): 98-99.
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Amphibian Skin Secretions and Bio-adaptive Significance —Implications from Bombina maxima Skin Secretion Proteome
2006, 27(1): 101-112.
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The studies on Bombina maxima revealed the rich molecular and functional diversity of amphibian skin secretion proteins and peptides. Discovered peptides from B. maxima skin secretions are three classes of antimicrobial peptides, bradykinin analogs and its gene associated peptides with diverse biological functions, proline-rich bombesin and its gene associated peptides, neuromedin U analog, Bv8 peptides, trefoil factors and protease inhibitors. The molecular and functional diversity, gene formation mechanisms and expression patterns of the peptides in B. maxima skin well reflect the molecular basis of bio-adaptation of the frog in certain living environments. In addition, B. maxima albumin with a heme b cofactor is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, indicating its important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc. The extraordinary complexity of peptides found in amphibian skin, coupled with the high probability of their novel molecular structures and possible counterparts in mammals, make amphibians an important target group in biomedical research and new drug development. Meanwhile, amphibian skin functional genome should be a nice model to study molecular biology of bio-adaptation, new gene formation and evolution.