2007 Vol. 28, No. 1
Phylogenetic Relationships of Butterflies in the Subfamily Limenitinae Based on Mitochondrial Cytochrome b Gene Sequences
2007, 28(1): 1-8.
The present study investigated phylogenetic relationships of the subfalimy Limenitinae based on sequence data of the mitochondrial Cyt b gene of 25 species, covering 10 genera of the subfamily, using Ariadne ariadne as outgroup. Phylogenetic trees were reconstructed with neighbor-joining (NJ), maximum parsimony (MP) and Bayesian inference (BI) methods. It is indicated that the involved species of this subfamily clustered into two clades: the first clade includes those of the genus Neptis, Phaedyma,Pantoporia and Lintinga; the second one includes those of the genus Parthenos,Moduza,Limenitis, Athyma, Lexias and Euthalia. The genus Limenitis is suggested to be monophyletic; and the genera Euthalia and Lexias proved to be sister to each other and form a monophyletic group. The genus Litinga appears to be more closely related to Neptini than to Limenitini. However, phylogenetic positions of some genera, such as Phaedyma, Pantoporia and Lintinga, are not well-resolved in the present study.
Construction of a cDNA Library from the Testis and Sequence Analysis of the Ubiquitin Gene from Rana nigromaculata (in English)
2007, 28(1): 9-16.
A full-length cDNA library from the testis of dark-spotted frogs (Rana nigromaculata) was constructed with the SMART (switching mechanism at 5′ end of RNA transcript) technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR (LD-PCR). After SfiⅠ digestion and fractionation, cDNA (>500 bp) was ligated to λ TriplEx2 vector and packaged with GigapackR Ⅲ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×106 pfu/mL and 2.4×106 pfu/mL and the titers of the amplified libraries were 0.48×109 pfu/mL and 3.0×109 pfu/mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0?kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in GenBank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.
2007, 28(1): 17-23.
In order to better understand the model of the molecular mechanisms governing spatially and temporally programmed transcription, the Fhx/P25 gene promoter of the fibroin protein from Bombyx mori was cloned and sequenced. An expression vector named pSK-P25-DsRed-PolyA was constructed, in which the reporter gene DsRed was driven by Fhx/P25 promoter. The promoter's activity was then characterized by transient expression assays in BmN cells of B. mori. The results of a sequence analysis showed that the Fhx/P25 promoter possesses the characteristics both of a eukaryotic promoter and a silk gland-specific expression promoter. A conserved TATA box sequence was located at position -28－-32 and had the sequence TATAA. There were three CAAT motifs, of which the two CAAT motifs located at positions -110－-117 and -90－-87 may be active. Secondary structure analysis indicated that the sequence of the promoter region forms a complicated stemloop structure. This may relate to the tissue speciality or the timing and activity of the protein expression. The transient expression product of the red fluorescent gene, driven by the promoter Fhx/P25, can be observed in cultured BmN cells of B. mori.
Isolation and Characterization of Microsatellite DNA Markers from Forest Musk Deer (Moschus berezovskii)(in English)
2007, 28(1): 24-27.
In this report, we describe the isolation and characterization of microsatellite loci for forest musk deer (Moschus berezovskii) through an improved enrichment protocol. Five new markers were isolated from the genomic DNA of forest musk deer and showed high polymorphism with 4-13 alleles in 24 sampled individuals from the population of Jinfeng Mountain, Sichuan Province, China. The observed and expected heterozygosities were from 0.429-0.957 and 0.587-0.902, respectively. The average polymorphism information content (PIC) value in these five loci was 0.730. This suggests that the five microsatellite loci are a valuable tool for further studies about forest musk deer.
Comparison of Food Components Between Rana pleuraden and Rana chaochiaoensis from Kunming and Evaluation of Their Benefit and Harm
2007, 28(1): 28-34.
Food components of 1 105 individuals of Rana pleuraden and 428 Rana chaochiaoensis collected from Bailongsi, Huahongdong and Xijiekou of Kunming from May 1998 to November 2003 were analyzed by dissecting their stomachs. It was calculated that percentage of number and frequency, α diversity index, index of percent similarity, width of trophic ecological niche, overlap of trophic ecological niche and beneficial coefficient of the two frog species. According to results, major food components of different populations of R. pleuraden collected from different localities as well between R. pleuraden and R. chaochiaoensis were basically identical. However, there were differentiation in food diversity and trophic ecological niche between R. pleuraden and R. chaochiaoensis as well between their males and females. The annual beneficial coefficients of both R. pleuraden and R. chaochiaoensis were positive, which implied that their benefit for mankind was larger than harm. The beneficial level of R. pleuraden was higher than one of R. chaochiaoensis. The study results showed that beneficial coefficient could be changed among populations of a species collected from different regions and/or the same population collected from different seasons or years, which meant that the beneficial coefficient could not be compared. This coefficient only expressed the level of benefit and harm for mankind in the certain period.
2007, 28(1): 35-40.
During the falls of 2002 and 2003, we studied the migration dynamics of birds using mist nets, on Fenghuang Mountain, Yunnan Province, China. A total of 6 677 individuals, which included 176 species belonging to 29 families and 13 orders, were caught and banded. The night bird migration on Fenghuang Mountain started in early August and finished in mid-November approximately. This migration period can be divided into the beginning stage, maximum stage and finishing stage. Among these stages, the maximum stage was from mid-September to early October. The night-time migration began approximately one hour after sunset and finished at dawn. There were two peaks during the night. The first occurred from 20:30 to 24:00, and the second occurred from 4:00 to 5:30. The day-time migration began in mid-October (beginning stage), and rose to the maximum stage from the last ten days in October to the first ten days in November. The finishing stage was from mid-November to the beginning of December. In the daytime, migration began from approximately one hour before sunrise, and then rose to the peak between 8:00 and 9:30. The migrants decreased gradually until 11:00. The migration period continues for about 100 days on Fenghuang Mountain. The migrating occurred during three migration peaks in nights, which are foggy, have southwest winds and no moonlight, but only one migration peak during all other nights.
2007, 28(1): 47-52.
Samples of subtidal macrobenthic fauna were collected and environmental factors were measured in the Yangtze River Estuary in April, 2005. The community structure of macrobenthic fauna and its correlation with environmental factors were analyzed. Thirty-eight species were identified, belonging to five ecological assemblages. The total species numbers were low, but were higher in the outer sampling sites of the estuary. The average abundance was 32.9 ind./m2 and the average biomass was 5.035 g/m2 (fresh weight) at all sampling stations. Compared with historic data from the 1970s and 1980s, the community structure of macrobenthic fauna has changed obviously and the biomass has decreased rapidly. Total abundance and biomass of the species were obvious higher at the outer sampling sites of the estuary. Salinity was the main factor affecting the distribution of the macrobenthic fauna. The distribution of the macrobenthic fauna communities corresponded to three structural orders of the Yangtze River Estuary.
Population Density and Habitat Utilization of Ibex (Capra ibex) in Tomur National Nature Reserve, Xinjiang, China(in English)
2007, 28(1): 53-55.
Surveys was conducted to study the population density and habitat utilization of the ibex (Capra ibex) in Tomur National Nature Reserve during October-December 2005. The results are as follows: ① The mean group size of the ibex in Tomur Nature Reserve is 8.43 and the population density is 269.76／100 km2; ② The ibex prefer areas with steep slope (between 30°-45°), broken substrates and cliffs nearby (<100 m). They avoid grasslands and flat hillsides; ③ The probable reason for this habitat selection is self-protection.
2007, 28(1): 56-62.
Endemic to the mountains of south-west mainland China, the Sichuan hill partridge (Arborophila rufipectus) is a Galliform bird with high conservation concern. Up until now, little is known about the vocal behaviour of the Sichuan hill partridge. We studied the vocal behaviour of the Sichuan hill partridge in Pingshan, Sichuan Laojunshan Nature Reserve with spectrographic analysis from April to December 2005. Based on observation in the wild and playback experiments, the behaviours and characteristics of vocalization of the Sichuan hill partridge were analysed.The results showed that males had three types of one-syllable call, which were crowing call, courtship call and preserving territory call. The syllable duration was significantly different between calls, but the difference of main peak frequency was not significantly different. The males also had five types of two-syllable call, which were crowing call, courtship call, competing call and two types of preserving territory call. The duration of the first syllable, duration of the second syllable, the interval time between the first and the second syllables and the main peak frequency was positively significant between the five types, but the duration of the whole sentence was not significantly different. In addition, they had three types of several-syllable call, which were alarm call, screaming call and preserving territory call. Sub-adult males had two types of several-syllable calls, which included alarm call and escape call. The syllable duration, interval time and main peak frequency of alarm calls was not significantly different between sub-adult males and adult males, but the duration of the whole sentence was significantly different. The syllable duration, interval time, main peak frequency of screaming call and the duration of the whole sentence was not significantly different between sub-adult males and adult males. The findings suggest that vocal behaviour of hill partridge will benefit to preserve mates and avoid the predator pressure so that the population may exist forever.
2007, 28(1): 63-67.
Four species of Pangasiid catfishes were once recorded in China. In recent years, several important revisions have been made to the taxonomy of the family Pangassiidae by some ichthyologists. As a result of these revisions, the original taxonomic system has changed greatly; a total of 22 valid species belonging to three genera is now recognized in the world. Because Pangasiid fishes in China have not been revised according to the new system, it is unavoidable that some confusion is present in species description. Based on specimen examinations and records in China since 1960, the present paper reconfirms that there is a total of three species of pangasiid fishes in China: Pangasius sanitwongsei Smith, Pangasius djambal Bleeker, Pangasius micronema Bleeker. Based on all the data and specimens available, the population status and threatening factors have been analyzed. According to the data, all three species of pangasiid fishes are rare critically endangered in the lower Lancangjiang River. Three specimens of P. sanitwangsei were collected in 1959 and no more have been collected since. Aside from two specimens of P. djambal collected in 1959, one more specimen has been caught from Buyuanjiang at Menglun by an ichthyologist of the Institute of Hydrobiology. Pangasius micronema is slightly more common than the above two species. One specimen was collected from Buyuanjiang River at Menglun in 1959 and five individuals have been collected since from the reach of the Lancangjiang River between Ganlanba and Jinghong in 1966. One specimen of P. micronema was also collected from the reach of Jinghong in 1967 by the Yunnan Fishery School. The most recent three specimens were caught close to the join of Buyuanjiang River to Lancangjiang River in 1978. All specimens were collected around May and June, which is recognized as the breeding season of pangasiid fishes. This suggests that pangasiid fishes migrate to the lower Lancangjiang River mainly for breeding, rather than feeding. The occurrence of pangasiid fishes in Xishuangbanna was analyzed together with the discharge records of the Lancangjiang River in March and April in Jinghong. A high correlation was demonstrated between these two factors, with 81.3% of specimens caught in years when a high discharge appeared in March and April. A high discharge in March and April may be a signal of summer monsoon, prompting the pangasiids to begin their breeding migration towards the upper MekongLancangjiang River. Populations of the three pangasiid species have been declining since 1970. The decline is not correlated to dam construction because so far no dams have been constructed in the stem rivers of the lower Lancangjiang River. Threatening factors may include overfishing and damage from boat turbines. If any dams are planned to be built in the lower Lancangjiang River, the conservation of pangasiid fishes should be carefully considered. We strongly suggest setting up the Buyuanjiang River as a nature reserve for fishes and other aquatic organisms.
2007, 28(1): 68-72.
The relationship of three Brachionus calyciflorus morphotypes (two short-spined, single-spined and spineless ones) was studied in the laboratory. Newly hatched females of each morphotype were cultured individually in an inorganic or Asplanchna-conditioned mediums, and the morphotype of their offspring were checked at 200× magnification. Females of each morphotype, cultured in the inorganic medium, could produce offspring of these three morphotypes in a single generation. Females of all three morphotyes could respond to specific Asplanchna-substances when they were cultured in Asplanchna-conditioned medium. Moreover, we observed that these three morphotype females could coexist in the medium and had three types of egg-bearing females. Based on the above observations we suggest that B. calyciflorus was a morphotype complex and has three basic morphotypes.
2007, 28(1): 73-80.
The migration of the primordial germ cells (PGCs), formation of the genital ridge, gonad sex differentiation, and their variations under different water temperatures were investigated using histological methods in the teleost, Pseudobagrus vachelli. The results showed that on day 1 the PGCs were at the splanchnic mesoderm above the gut. On day 5, PGCs migrated to the peritoneal epithelium. The genital ridge was formed on day 8 and lacked PGCs. The primordial gonads were formed on day 14. On day 23, the primordial gonads differentiated into ovaries or testes. From day 10 on, the larvae were cultivated in different water temperatures (20±0.5℃, 24±1.0℃,30±0.5℃, 34±0.5℃) for 25 days. The control group was cultivated at natural water temperature (24±1.0℃). The sex ratio and percentage of male larvae were calculated after the experiment. At natural water temperature (24±1.0℃) and lower water temperature (20±0.5℃), the sex ratio was approximately 1∶1 (1∶1.09 and 1.22∶1 respectively). However in the group cultivated at 30±0.5℃, the sex ratio was 1∶4.89 and the percentage of males was 83.3±0.7%. The sex ratio was 2.85∶1 and the percentage of males was only 26.4±0.4% in the group cultivated at 34±0.5℃. These results suggest that water at 30±0.5℃ influenced the larval gonad towards differentiating into a testis, and water at 34±0.5℃ gave the opposite result, i.e. the gonad differentiated into an ovary. This experiment showed that the sex determination of P. vachelli is temperature-dependent sex determination.
2007, 28(1): 81-87.
An effective freezing-thawing technique is crucial for the clinical application of newly derived rabbit embryonic stem (ES) cells. The aim of this study was try to find an optimal cryopreservation protocol for rabbit embryonic stem cells using slow freezing-rapid thawing without a programmable freezer. We tested the effects of the following cryoprotective agents (CPAs) on the post-thaw survival, proliferation and differentiation capacity of rabbit embryonic stem cells: ethylene glycol (EG), dimethyl sulphoxide (Me2SO, DMSO), trehalose and glutamine. Trypan blue exclusion tests showed that, among the CPA treatments in this study, EG was more toxic to rabbit embryonic stem cells than DMSO. The highest survival rate (83.7%) was obtained when the rabbit embryonic stem cells was cryopreserved with 10%DMSO and 20 mmol/L glutamine in the ES cell culture media. Thawed ES cells kept their pluripotency and differentiation potential.
2007, 28(1): 88-94.
To study the effects of oestrogen on ischemiainduced neurogenesis in the hippocampal dentate gyrus, thirty-two adult male rats were randomly divided into four groups：the control surgery group with oestrogen administration (SE), the control surgery group with normal saline administration (SN), the middle cerebral artery occlusion (MCAO) group with oestrogen administration (ME) and the MCAO group with normal saline administration (MN). The MCAO rats were occluded for 90 min by an intraluminal filament and then recirculated. After 1, 3, 12, 24 and 28 h of MCAO, the rats of the four groups were killed to investigate the infarct volume, apoptosis and neurogenesis. The cerebral infarct volume in the ME group was significantly smaller than that of the MN group (P<0.05). No significant cell loss was seen in the dentate gyrus. Cerebral ischemia led to increased neurogenesis, which is independent of cell death in the ipsilateral dentate gyrus (P<0.05). BrdU-positive cells in the ipsilateral dentate gyrus of the ME group were significantly increased when compared with those of the MN group(P<0.05). In the SE group, BrdU-positive cells in both the ipsilateral and contralateral dentate gyrus, were increased when compared with those of the SN group (P<0.05). We concluded that oestrogen plays an important role in neurogenesis, which is independent of ischemia-induced by MCAO in the hippocampal dentate gyrus of rats.
2007, 28(1): 95-100.
Perceptual learning of orientation discrimination was investigated using cats. Two adult cats (Cat 1 and 2) were trained to monocularly discriminate between two static striped sinusoidal grates with 30° orientation difference. After greater than 80% correct performance was reached, cats were then required to monocularly perform a discrimination between two grates with consecutively shifting orientation difference(2°, 4°, 6°, 8°, 10°, 12°, 16°, 20°, 24°, 30°) . The staircase method (two correct-down and one error-up) was applied throughout the training to track the threshold of orientation difference that cats could detect. The performance of detecting grates with varied orientation difference was measured respectively for both trained and untrained eyes before and after training. Our results showed that the learning effect of discrimination for grates with a fixed orientation difference transferred completely from the trained eye to the untrained eye, whereas the inter-eye transfer for detecting grates with gradually reducing orientation difference was almost nonegrates. The two opposite learning effects in the same subject strongly suggest that different information processing mechanisms might mediate the learning processes.
2007, 28(1): 101-103.
To study the correlation between weight and length of captive bottlenose dolphins (Tursiops gilli) (n=20), data collected from two aquariums were analyzed using linear correlation and regression. The results show a significant correlation between weight and length (r=0.960,P<0.01). The regression equation, which expresses the relationship between weight and length, is Ｙ=2.19X-368.65. There is a strong tendency to increase body weight with the growth of body length. Based on the relationship between the two parameters, we can control the weight of dolphins and enhance the level of husbandry and management.
We captured two Megaderma lyra (Megadermatidae, Chiroptera) males in Jinlun Cave, Mashan County, Guangxi Province on December 10, 2004. The two males were killed for another experiment, however we collected their stomachs for examination. The stomach contents of these two bats were examined and the remains of other bats species were found, including dentes, hindfoot, skeleton and hair. By careful examination of the dentes we identified that the upper-dental formula was 126.96.36.199. This formula coincided with Myotis, confirming that the Indian false vampire bat feeds on Myotis. We did not find any remains of insects in these two samples.
2007, 28(1): 106-112.
Spatial attributes are an important characteristic of animal behavior. They are also a difficult subject for researchers, as the study methods, techniques and equipment required to study them have been limited in past years. With the rapid development of their technique and function in the last twenty years, Geographic Information Systems (GIS) have been more often applied in the study of animal behavioral ecology, such as habitat selection, territory, migration route and so on. GIS is a computerbased system, capable of capturing, storing, analyzing, and displaying geographically referenced data with its powerful spatial analysis abilities. In this paper, we discuss the principles and concepts of GIS in studies of animal behavioral ecology. Approaches and development of recent animal behavioral ecology studies using GIS are also summarized.
2007, 28(1): 41-46.
We examined the competitive ability of larval toads (Bufo gargarizans) and frogs (Rana kukunoris) which co-occur in natural pools in the Jiuzhaigou Nature Reserve. We measured the activity level, growth rate, mass at metamorphosis and larval period in a laboratory experiment. Tadpoles of B. gargarizans were significantly more active when food was abundant than scarce, while there was no significant difference in the activity of R. kukunoris tadpoles at different food levels. At low food availability, mass at metamorphosis and growth rate of R. kukunoris were significantly increased in the presence of B. gargarizans, whereas the presence of R. kukunoris had no significant effect on the mass and growth rate of B. gargarizans. In all treatments, the larval period of B. gargarizans at low food availability was the shortest. These results suggest that B. gargarizans can adapt to different food levels by changing their activity rate. At high food level, B. gargarizans increased activity to gain more food. At low food level, B. gargarizans decreased activity and achieved early metamorphosis. However, when food resources were limited, R. kukunoris could gain more food than B. gargarizans.