2010 Vol. 31, No. 6
2010, 31(6): 565-569. doi: 10.3724/SP.J.1141.2010.06565
Bm-TFF2, an amphibian trefoil factor, which is isolated from skin secretions of frog Bombina maxima, has much stronger biological activities than human TFFs. In the present study, Bm-TFF2 gene was amplified by polymerase chain reaction (PCR) from its cDNA and cloned into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α-factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 1% methanol for 72 hours, the expression of Bm-TFF2 came up to the best quantity which was about 50 mg in 1L medium, and 80% saturation ammonium sulfate was suitable to collect the Bm-TFF2 protein, as identified by SDS-PAGE and Western blotting assay. The results showed that the plasmid of Bm-TFF2-pPIC9K was constructed successfully and expressed abundantly in eukaryotic expression system, which lies basis for researching further the biological activities and the relationship of structure and functions of Bm-TFF2.
Isolation and characterization of Hainantoxin-II, a new neurotoxic peptide from the Chinese bird spider (Haplopelma hainanum)
2010, 31(6): 570-574. doi: 10.3724/SP.J.1141.2010.06570
Hainantoxin-II (HnTx-II), a novel neurotoxin, was isolated from the venom of the Chinese bird spider (Haplopelma hainanum) by cation exchange chromatography and reverse-phase HPLC. The toxin was a single chain polypeptide with calculated molecular weight of 4 253.135 obtained by mass spectrometry. The complete amino acid sequence of HnTx-II was determined by Edman degradation and found to contain 37 residues with three disulfide bonds. Results showed HnTx-II can reversibly paralyze cockroaches for several hours after intra-abdominal injection with ED50 of 16 μg/g and kill the insects immediately at a dose of 60 μg/g. It was also shown to kill mice at a LD50 value of 1.41μg/g after intracerebroventricular injection. Hainantoxin-II shares 91% sequence homology with Huwentoxin-II (HwTx-II), an insecticidal peptide from another bird spider(Haplopelma schmidti) with a unique scaffold. While HnTx-II and HwTx-II both exhibit toxic activities in insects and mammals, HnTx-II shows higher insecticidal activity and lower lethiferous activity of mammals than HwTx-II. These results help clarify structural-functional relationships ofthe polypeptide toxin.
2010, 31(6): 575-580. doi: 10.3724/SP.J.1141.2010.06575
The prolactin exerts obvious adjustment and control function for mammary gland development, lactation and milk protein gene expression in water buffalo. In this study the sequence features and polymorphisms of the exon 4 in prolactin gene were examined in 385 individuals which came from 12 river and swamp type buffalo populations using DNA direct sequencing and PCR-SSCP methods. The results showed that the sequence of exon 4 in prolactin gene was consists of 180 nucleotides, the fragment had high conservative character in different species. The e4. 109 C＞T substitution was detected in nine swamp buffalo populations, and it was a silent mutation and was not associated with the traits of milk yield in buffalo. The PBA gene was the predominant gene in seven swamp type buffalo populations, while PBB gene was the dominant gene in Dehong and Fuzhong populations. The frequencies of PBA in swamp type buffalo was 0.400 − 0.917 and the average value was 0.629±0.049. The polymorphism wasn’t found in river buffalo, all the samples from river buffalo were holding nucleotides e4.109 C. The results indicate that there is distinct genetic differentiation between swamp and river type buffalo.
Correlation and location of EST markers with cold tolerance trait of common carp (Cyprinus carpio L.)
2010, 31(6): 581-586. doi: 10.3724/SP.J.1141.2010.06581
Expressed sequence tags (ESTs) are parts of complementary DNAs (cDNAs) with certain gene function．It provides us more information than other neutral markers. The association of EST markers with phenotypes can increase our understanding of the biochemical pathways and mechanisms affecting economically important traits. In this study, 12 candidate EST markers isolated from the cold-induced brain cDNA of common carp (Cyprinus carpio L.) were conducted the correlation analysis of marker and cold tolerance trait of common carp using GLM model of SPSS 17.0 software firstly, then tried to locate them in the genetic linkage map using OneMap software. As a result, eight out of 12 candidate EST makers were separately located in six linkage groups, in which marker CC009(P<0.05) and CC115 (P<0.01) were associated with cold tolerance andmapped to LG38 and LG2, respectivey. Homology identity alignments showed that marker CC009 was highly homologous to the known Uridine-cytidine kinase I of Danio rerio with an identity of 94%, and marker CC115 was lowly homologous to the putative glycosyl transferase of Prochlorococcus marinus with an identity of 56%.
Cloning and expression pattern analysis of a lipopolysaccharide -and β-1,3-glucan-binding protein in Portunus trituberculatus
2010, 31(6): 587-594. doi: 10.3724/SP.J.1141.2010.06587
The lipopolysaccharide -and β-1,3-glucan-binding protein (LGBP) is apattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glycohydro16 domain was identified. The expression of P. trituberculatus in varioustissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytesof the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic processagainsting bacterial infection.
Molecular cloning, sequence analysis and expression pattern of hepcidin gene in ayu (Plecoglossus altivelis)
2010, 31(6): 595-600. doi: 10.3724/SP.J.1141.2010.06595
Hepcidin, a member of cysteine-rich antimicrobial peptides,plays an important role in both fish adaptive immunity and the regulation of iron metabolism. In this paper, the nucleotide sequence of a full-length cDNA clone for ayu (Plecoglossus altivelis) hepcidin gene, 763 nucleotides in length, was determined. Ayu hepcidin gene contained a complete open reading frame (ORF) encoding an 85-amino-acid peptide with a molecular weight of 9.7 k. A signal peptide of 24 residues existed in hepcidin N-terminus. The ayu hepcidin mature peptide sequence contained 25 amino acids with eight cysteines that formed four disulfide bonds. Sequence comparison and phylogenetic analysis showed that ayu hepcidin was most similar to Atlantic salmon (Salmo salar), and the relationships of the different hepcidin coincided well with the evolutionary relationships of their organisms. In healthy ayu, hepcidin mRNA was mainly expressed in the liver, spleen, kidney, heart, and muscle. After Listonella anguillarum infection, liver hepcidin mRNA expression change was determined by quantitative real-time PCR (qRT-PCR) method. Hepcidin transcripts of ayu liver were significantly up-regulated and peaked at 12 h. These results suggest that hepcidin may be involved in the immune response of ayu.
cDNA cloning and tissue-differential expression of Na+/K+/2Cl- −cotransporter 1-α isoform in Sarotherodon melanotheron
2010, 31(6): 601-609. doi: 10.3724/SP.J.1141.2010.06601
The gills are the major apparatus for osmoregulation in fish to acclimate the changes of salinities. Na+/K+/2Cl−cotransporter 1-α (NKCC1α) is one of the key ion cotransporter locoalized in gill chloride cells which has been associated with the maintence of osmotic homeostasis. The transport process mediated by NKCC1α is characterized by electroneutrality with a stoichiometry of 1Na:1K:2Cl. Sarotherodon melanotheron is one of the most euryhaline teleosts able to withstand variations in environmental salinity ranging from freshwater to hyper-saline waters. In this study, the reverse transcription-polymerase chain reaction and rapid amplification of 3' and 5'cDNA ends methods were used to identify the full cDNA of the NKCC1α with an Open Reading Frame which contains 1 151aa of S.melanotheron. The amino acid multiple alignment and phylogenetic analysis showed that this isoform is more similar with isoforms in Oreochromis mossambicus, Salmo salar and Anguilla anguilla, and there is the highest homologous of 99% between Sarotherodon and Mossambique. The predicted protein secondly structure of NKCC1α contains 10 transmenbrane domains, which were highly conserved in sequences and locoalization sites relatively to other species. The quantitative real time polymerase chain reaction (qRT-PCR) assay was developed to estimate the mRNA expression levels in gill, liver, intestine and kidney in freshwater, the results showed a tissue-specific model. Furthermore, the sanility significantly affects the relative expression level of NKCC1α mRNA in gill with a 4.9 times higher in 136 salinity water than that in 0 salinity. The results suggest that the NKCC1α is closely related to the salt tolerance in S.melanotheron.
Summer habitat selection of alpine musk deer in Xinglongshan National Nature Reserve, Northwestern China
2010, 31(6): 610-616. doi: 10.3724/SP.J.1141.2010.06610
During July and August from 2006 to 2008, summer habitat selection was studied in Alpine musk deer (Moschus sifanicus) in Xinglongshan National Natural Reserve in northwestern China. In total, seventy one musk deer utilized habitat plots and 246 random habitat plots were surveyed. Seventeen habitat characteristics were recorded and compared between the two habitat types, using Mann-Whitney U test and chi-square to compare the differences between utilized and random habitat plots, and principal component analysis (PCA) to determine the main factors influencing the habitat selection of musk deer. Mann-Whitney U test and chi-square was conducted to test whether there was significant difference between utilized and random plots. The results showed that musk deer prefers habitat with taller arbor height (7.57 ± 0.83 ) m, higher food-plants abundance (12.97 ± 1.80), increasing foliage cover for concealment, lower water dispersion, and higher anthropogenic disturbance. Furthermore, PCA results suggested that the arbor characteristics (arbor canopy and arbor DBH), altitude characteristic, food characteristics (ground-plant cover and food-plant abundance) and shrub characteristics (shrub canopy, shrub height and related arbor density) influence summer habitat selection patterns of alpine musk deer in Xinglongshan National Nature Reserve. The general pattern of summer habitat utilization and selection of alpine musk deer is an adaptive strategy to the changing food, concealment, water source and the physical condition of summer habitat.
Temporal stability in song dialects of black redstarts (Phoenicurus ochruros rufiventris) in Menyuan, Qinghai Province
2010, 31(6): 617-622. doi: 10.3724/SP.J.1141.2010.06617
Bird song dialects provide a useful model for the study of cultural evolution and its interactions with genetic evolution. By comparing song dialects over an 8-year span in the black redstarts (Phoenicurus ochruros rufiventris) of Haibei station (37°29'N, 101°28'E, 3, 200 m a.s.l.) in Menyuan county, Qinghai Province,This study examined the temporal stability of the birds’ song dialects. Songs of black redstarts were recorded from mid-April to late May in 2001 by Uher-CR 210 tape recorder with Sony directional microphone and in 2009 by Marantz PMD 670 digital recorder with Sennheiser ME 66 directional microphone. Songs were analyzed by using Avisoft-SASLab pro. The similarity of songs between different years were analyzed by investigating the sonagram, measuring temporal and frequency properties and processing cluster analysis by Spss 10.0 on windows. The results showed that species-specific syllable, typical song forms, and the typical strophe keep high temporal stability. Males investigated from 2001 and 2009 shared the entire strophe or some of the syllables. Males sampled from 2009 have a larger strophe repertoire than that from 2001. The study also indicated that the divergence of shared strophe in 2009 is correlated with distance between territories. The degree of strophe type sharing and song similarity are higher between neighbours than that from more distant males.
Shape change in viable eggs of the collembolan Folsomia candida provides insight into the role of Wolbachia endosymbionts
2010, 31(6): 623-626. doi: 10.3724/SP.J.1141.2010.06623
The endosymbiotic bacteria of the genus Wolbachia that infect the collembolan species Folsomia candida are responsible for facilitating parthenogenetic reproduction in their hosts. This study made empirical observations of the development of eggs of F. candida which contained normal populations of Wolbachia and of eggs which were cured of Wolbachia by treatment with the antibiotic rifampicin. A marked increase in egg size accompanied by a significant change in shape from spherical to discoid occurred in viable eggs three to four days after laying. These changes did not occur in the universally inviable eggs which came from the antibiotic treatment or in the 7% of untreated eggs which were naturally inviable. We infer that Wolbachia plays a critical role in zygotic or embryonic development during or before the first three days after laying and we draw on existing knowledge in speculating on the developmental mechanisms that Wolbachia may influence.
Digestive enzyme and alkaline phosphatase activities during the early stages of Silurus soldatovi development
2010, 31(6): 627-632. doi: 10.3724/SP.J.1141.2010.06627
To provide a theoretical basis to improve the survival and growth rate and optimize diet of sheatfish (Silurus soldatovi), the activities of certain digestive enzymes and alkaline phosphatases were investigated during larval development ofone - ten day old individuals. Results indicated that sheatfish larva (~ three days after hatching) had high levels of alkaline protease activity, which peaked at five days old and dipped by eight days old, although the trend was generally upward. Acid protease activity at one - eight days old was low, after which it increased rapidly. Amylase activity reached the highest value at five days old, after which it began to decline. Lipase activity fluctuated markedly and showed two peaks at three - four days old and six - eight days old. Larval digestive enzyme activity and alkaline phosphatase activity were higher when fed live food than when fed an artificial diet. Throughout the early development process, alkaline protease activity was higher than acid protease, alkaline protease and amylase specific activity decreased significantly for eight-day-old transition larvae, while acid protease activity increased rapidly. These results indicate that the changes in digestive enzyme activity were relevant to digestive function conversion during fish larvae development. Alkaline phosphatase activity showed an upward trend over the first ten days of life, which indicated that the gastrointestinal function of sheatfish improved gradually.
2010, 31(6): 633-644. doi: 10.3724/SP.J.1141.2010.06633
Several studies have characterized the Yungas as a separate biogeographic unit, mainly based in floristic components. However, these characterizations were mainly qualitative and did not include faunal groups. The Yungas have been assumed as a region with rich floral and faunal diversity, but without testing how well they are described by animal distributions. Our study consists of a formal analysis of endemism based on distribution of small mammals in the southernmost portion of the Yungas. This area is biogeographically very interesting because the Yungas are comprised of discontinuous fragments of forests that extend into temperate arid and semiarid habitats. As a first approximation, we contrasted a group of volant species (bats) versus a group of non-volant species (marsupials). Our results show that small mammals are efficient indicators of endemism in Yungas. Eighty percent of the species of small marsupials included in the analysis supported the identified areas as being zones of endemism. Regarding bats, almost 55 percent of the species supported a designation of endemism. The results also show that the areas we considered are congruent with the botanical definition of the Yungas of northwestern Argentina and their subdivisions, an assumption that had not been previously tested with a formal quantitative method. We also found that non-volant species are better indicators of endemism than volant ones at regional scales, but volant species are better indicators than was previously thought.
Found in the Trans-Himalayas of north-west Yunnan and south-east Tibet, the black-and-white snub-nosed monkey (Rhinopithecus bieti) is one of the world’s most endangered primates. A recent survey indicates that only 15 groups with 2500 individualsremain in the wild. However, the Tibetan Xiaochangdu group may be the only equilibrium group in the field since the last investigation in 1988. To evaluate the effects of traditional culture and socioeconomic activity on biodiversity conservation of R. bieti, we conducted a case study in the Honglaxueshan National Nature Reserve in southeast Tibet from June 2003 to May 2005. Interviews, direct observations, and analysis of socioeconomic data indicated major advantages to the conservation of R. bieti, which included that: 1) traditional culture mainly depended on raising livestock and collecting non-timber products rather than forest planting of Tibetan highland barley; 2) religious beliefs, against to kill any wildlife living on the sacred mountain, were mainly influenced by Tibetan Buddhism; and 3) bigger household numbers were induced by the polyandrous marriage system, which resulted in lower per capita resource consumption than smaller ones.
2010, 31(6): 651-656. doi: 10.3724/SP.J.1141.2010. 06651
Multivariate analysis was adopted to analyze 30 morphometrical characteristics of 121 one-year-old juvenile silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) bred during the 1950s (“the former population”) and 2008 (“the current population”) and collected from the middle reach of the Yangtze River. The average discriminant accuracies of the former and current silver and bighead carp population were 94.2% and 98.0%, respectively. Discriminant analysis also revealed that significant differences in morphology occurred between the former and current populations of both carp in overall characteristics. One-way analysis of variance indicated that between former and current populations, silver carp showed highly significant differences (P<0.01) in twelve of their characteristics and significant differences (P<0.05) in eight of their characteristics, while bighead carp showed highly significant differences (P<0.01) in eight of their characteristics and significant differences (P<0.05) in eight of their characteristics. Six head morphology variables of the current silver and bighead carp were significantly or highly significantly larger than the former populations; fourteen characteristics of silver carp and ten characteristics of bighead carp of the current populations, mainly reflecting truck and tail morphology, were significantly or very significantly smaller than the former populations. Our results indicate that silver and bighead carp have developed a larger head and smaller truck and tail during the last 50 years. Due to such morphological changes, it seems apparent that the heads of these fish species need to be considered in regards to human diets, particularly in relation to economic and nutritious value.
2010, 31(6): 657-662. doi: 10.3724/SP.J.1141.2010.06657
A field fish survey of Dalian Lake, Shanghai was undertaken in Apr. 11-19, Apr. 27-May 8 and May 20-29, in total 24,061 fish individuals were collected, representing 22 species from 17 genera and 11 families. The dominant specie is Carassius auratus, accounting for 76.38% of the total. The eigenvalues of species diversity were showing below : Shannon-Wiener’s index (H¢) being 1.0027, Simpson’s index (l) being 0.5959, Pielous’s index (J¢) being 0.3244, Margalef’s index (D) being 2.0816 and relative rare species (R) being 90.91%. The fish community could be classified into 3 ecological types, which including river-sea migratory fish (3 species), river-lake migratory fish (1 species) and sedentary fish (18 species). Also they can be subcategoried into five types according to feeding habits, i.e., piscivorous fish (9 species), invertebrativorous fish (2 species), omnivores fish (7 species), planktotrophic fish (3 species), herbivorous fish (1 species). The results suggested that the biodiversity index and fish community stability are both at a low level. Compare to the lower reaches of Huangpu River, the proportion of piscivorous fish in Dalian Lake is higher, which suggested the water quality of Dalian Lake, located in the upper reaches of Huangpu River, is better than that in the downstream. It’s required to intensify supervision and strengthen the environment protection of Dalian Lake to guarantee the sustainable development.
2010, 31(6): 663-669. doi: 10.3724/SP.J.1141.2010.06663
We used the pendulum device to study Doppler-shifted compensation ofgreat leaf-nosed bat(Hipposideros armiger). The bats’ echolocation calls were recorded by the Ultrasound Detector both under the rest condition and Doppler shift condition. Then we analyzed the calls with Avisoft software. Our results suggested that when H. armiger was approaching the target, it showed positive Doppler shift compensation: call frequency and the velocity (v) were positive correlated. Call frequency fell to minimum when the bats’ relative velocity reached to maximum; likewise call frequency raised to the resting condition frequency when the relative velocity became zero. Negative Doppler shift compensation occurred when bats were far away from the target. Under negative Doppler shift compensation condition, we found call frequency and velocity were positive correlated as well, and moreover, call frequency raised to maximum again while the bats had their minus direction’s maximal relative velocity. However, under this status, the elevated value was much lower than the depressed value under positive compensation at the same velocity. The frequency of occurrence of negative compensation was obviously less frequent than that under positive compensation condition. Therefore, we inferred that the two characteristics of the negative Doppler shift compensation mentioned above may be the coactions consequence of the bio-structural restriction and natural selection.
Food habits and hunting patterns of Tibetan brown bear during warm seasons in Kekexili region on Qinghai-Tibetan Plateau
2010, 31(6): 670-674. doi: 10.3724/SP.J.1141.2010.06670
Tibetan brown bear (Ursus arctos pruinosus) is an endemic subspecies of brown bear on the Qinghai-Tibetan plateau. We once reported a preliminary study on the summer food habit of Tibetan brown bear from July to August, 2005 inthe Kekexili (Hoh Xil).. However, the hunting modes of the Tibetan brawn bear have not been reported. From July to August, 2009, we collected additional data on food habits and hunting patterns of Tibetan brown bear in the Kekexili region. We found Tibetan brown bears are more raptatorial than vegetarian, and their basalfood was plateau pika (Ochotona curzoniae), relative frequency occurrence of pika in the fecal residual of brawn bear was 37.3%, dry weight of pika residuals was 44.7%, respectively, followed by wild yak (Bos grunniens) , 18.7% and 30.2%, and Tibetan antelope (Pantholops hodgsoni)15.0% and 16.2%. Both prey actively mode and scavenge mode were used by Tibetan brown bears in Kekexili. Tibetan brawn bears actively dug and hunted for pika and scavenged bodies of wild yak, Tibetan antelope and Tibetan gazelle. We observed that Tibetan brawn bears spent about 10% of their time to dig and to hunt for pika but we had never seen Tibetan brawn bears actively hunted large mammals such as wild yak, Tibetan antelope and Tibetan gazelle. The total amount of Tibetan antelope, Tibetan gazelle and wild yak ingested by Tibetan brown bear through scavenge was about the same amount of Plateau pika and Himalayan marmot(Marmota himalayana) eaten by the bear.
2010, 31(6): 675-676. doi: 10.3724/SP.J.1141.2010.06675
One specimen was collected from the main course of Lancangjiang River (upper reach of Mekong) in Guanlei Port, Xishuangbanna, Yunnan, China in April, 2008. It is identified as Dasyatis laosensis, a new record of Dasyatidae species in China. It could be distinguished from other Dasyatis species by a combination of the following characteristics: one enlarged venomous spine on the tail, ventral surface of body with orange marginal coloration, tail length greater than body length.