2009 Vol. 30, No. 2
2009, 30(2): 113-120. doi: 10.3724/SP.J.1141.2009.02113
The roe deer is an animal of economic importance in China and a primary food source of the Amur tiger. Understanding the morphological and molecular variation of geographically isolated roe deer populations is therefore important in establishing conservation strategies. Thirty two samples from three differences geographic regions were sequenced and the populations analysed. Twenty seven haplotypes were identified from 32 sequences of the 460 bp D-loop of mtDNA genome. Fifty sites were found to be polymorphic. The population genetic diversity of roe deer was of higher level as indicated by the following parameters: mean value of haplotype diversity (h =0.9780) and mean value of nucleotide diversity (π = 0.02260). Neither the estimate of Tajima’s D nor that of Fu & Li's D deviated significantly from the neutral selection hypothesis (P>0.1) for the three populations. The result suggests no evidence of strong selective sweeps or balancing selection. Of the three populations, the Great Khinghan Mountains geographic region was shown to have the most nucleotide diversity. To ensure conservation of genetic diversity within this population, we propose a high priority conservation classification.
Effects of Sample Size and Loci Number on Genetic Diversity in Wild Population of Grass Carp Revealed by SSR
2009, 30(2): 121-130. doi: 10.3724/SP.J.1141.2009.02121
Sample collection and marker selection are of primary importance in population genetic studies, and can greatly influence the analysis and interpretation of the data obtained. Microsatellite DNA sequences are the most revealing DNA markers available so far for inferring population structure and have been widely employed in genetic studies of populations．Nevertheless，few studies have specifically examined the effects of sample size and loci number on various genetic diversity measures in population genetic work using these markers．Here，we examined this issue using experimental data from an analysis of genetic diversity of wild populations of grass carp at 45 polymorphic microsatellite loci. The following genetic diversity measures were studied: number of alleles per locus (Na), effective number of allele per loci (Ne)，observed hererozygosity (Ho) and expected heterozygosity (He)．We also tried to infer the theoretical minimum sample size and loci number needed for population genetic studies with microsatellite loci. The results are as follows: (1) Na and Mean number of allele over loci (MNA) are significantly affected by sample size. It increase according to the raise of the sample size, only when the sample size exceeds 40 it tends to be stable. The significant correlation between sample size and Na was observed and the correlation curve is Y=3.8403+0.6376, r2=0.967．However, there is no significant correlation between the mean value of Ne, Ho, He and the sample size. Mean value of Ho, He fluctuate with the raise of the sample size, only when the sample size exceeds 40 it manifest a stable trend. (2) Evaluating of different genetic parameters with the makers of different polymorphism information content, a significantly different result were acquired. When PIC>0.5, Na, Ne, Ho, He were 3.6, 1.5934, 0.2824, 0.3540, While PIC<0.5, Na, Ne, Ho, He were 9.5, 5.9209, 0.8497, 0.8242 respectively. Accordingly, marker selecting strategy has much greater impact on evaluation of genetic diversity. (3) According to the rise of loci number, the value of genetic parameter could be divided into three stages: ① Descending stage: loci number between 0−5, values of Na, Ne, Ho, He indicated descending trend. ② Loci number between 5−10, values of Na, Ne, Ho, He demonstrate ascending trend. ③ When loci number exceeded 15 values of Na, Ne, He tend to be stable, while only when loci number exceeded 25 the value of Ho tend to be stable. Analysis above proved that not only the sample size and loci number but also the polymorphism information of microsatellite markers have significant effect on the estimation of population genetic diversity. So, we suggested that when microsatellite markers were used to estimate genetic diversity of population the minimum loci number and sample size are needed to exceed 25 and 40, respectively. Moreover, the conclusion above has been used to evaluate the genetic diversity of wild population of grass carp, values of the parameter were Na=7.26、Ne =4.21、Ho=0.73、He=0.68, which demonstrated that has a relatively high diversity, a totally different conclusion from the former studies. Therefore, further researches are necessary for the evaluation of genetic diversity of geographically different population of grass carp.
2009, 30(2): 131-136. doi: 10.3724/SP.J.1141.2009.02131
Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates. Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics. The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research. In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells. Chromosomes of two amphioxus species from Xiamen waters in China are also observed. The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B. japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.
Developmental Expression of an Amphioxus (Branchiostoma belcheri) Gene Encoding a GATA Transcription Factor
2009, 30(2): 137-143. doi: 10.3724/SP.J.1141.2009.02137
GATA factors are evolutionarily conserved and play crucial roles during embryonic development in both vertebrates and invertebrates. Vertebrate GATAs can be divided into two subgroups, the GATA1/2/3 and the GATA4/5/6 classes. Through genomic analysis, we have identified three GATA factors, representing the GATA1/2/3 and GATA4/5/6 subfamilies respectively, and one GATA like protein in the genome of the basal chordate amphioxus (Branchiostoma floridae, cephalochordata). Partial sequence of GATA123 in the amphioxus Branchiostoma belcheri (BbGATA123) was cloned and its expression pattern during early embryonic development was studied. Expression of BbGATA123 is first detected in the mesendoderm during gastrulation. Interestingly, in the late neurula and early larva stages, it is expressed strongly in the cerebral vesicle and the mid gut region. Its expression is compared to Otx, a gene known crucial for the development of anterior structures. Our observations suggest that GATA123, together with Otx, might play an important role in the development of amphioxus cerebral vesicle, the counterpart of the vertebrate brain.
2009, 30(2): 144-150. doi: 10.3724/SP.J.1141.2009.02144
According to the conserved sequence of the ferritin gene, a homologous sequence was obtained from the EST database through a BLAST search against the GenBank database. This sequence was amplified with the method of RT-PCR, false sequencing was corrected, and full length cDNA of the ferritin subunit from the Chinese sturgeon was obtained. After being submitted to the GenBank database, the sequence accession number EU348782 was assigned. With the length of 896 bp, this cDNA includes entire coding regions of 53 1bp, which encodes 176 amino acids (aa). The molecular weight was predicted to be 20339.9Mr and the theoretical isoelectric point 5.66. It shares 82.9% protein sequence homology with the ferritin of the Atlantic salmon. This gene is expressed in many organs of the Chinese sturgeon, for example, the liver, pancreas, muscle, brain, heart and gastric mucosa. The highest expression level was found in the pancreas and the heart, while the muscular tissue showed the lowest. Homology modeling was used to predict the 3-D structure of the protein, which included 5 alpha helices and 10 turns. The ferritin protein structure could be overlapped and showed high similarity with that of human, frog and bacteria. It was revealed that this kind of ferritin was highly conserved in structure and function.
2009, 30(2): 151-157. doi: 10.3724/SP.J.1141.2009.02151
In this paper, DMSO was used as cryoprotectant for cryopreservation of Sparus macrocephalus spermatozoa in 0.5 mL straws. Detection of DNA damage in response to a cryopreservation process in Sparus macrocephalus spermatozoa was also carried out. The results demonstrated that there were no significant differences between frozen-thawed sperm conserved by Cortland solution diluted with 5%, 10%, 15%, 20% DMSO and fresh sperm in motility. The best motility of frozen-thawed sperm were obtained when DMSO concentration was 10%, and the activation rate, moving time, living time and fertilization rate of frozen-thawed sperm were 92.91±1.25%, 39.90±2.70 min, 53.82±2.84 min and 89.35±1.99% respectively. However, a significant drop in sperm motility and fertilization rate was observed in sperm cryopreserved with 25% and 30% DMSO. The comet rate and damage coefficient of frozen-thawed sperm conserved with 5%, 10%, 15%, 20% DMSO was similar to fresh sperm, but at 25% and 30% were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of frozen-thawed sperm and concentration of DMSO in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 25% and 30% DMSO. Our analysis suggests that high concentration of DMSO is the main factor that causes the DNA damage in frozen-thawed sperm nucleus.
2009, 30(2): 158-164. doi: 10.3724/SP.J.1141.2009.02158
The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses (ICMs) and fetal neural stem (FNS) cells. In result, three rat chimeras were produced by day 5 (D5) Sprague-Dawley (SD) blastocysts injected with ICMs derived from day 6 (D6) and D5 Dark Agouti (DA) blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs are able to contribute to chimeras, but their potential decreases significantly after culture in vitro (P<0.05), and rFNS cells only have the potential to contribute to early fetal development.
To investigate the bioactive component from the skin secretion of Amolops loloensis , isolated peaks with insulin-releasing activity were purified and structurally determined. Skin secretions were isolated by Gel filtration and Reversed phase high-performance liquid chromatography, and tested with insulin-releasing assay. The amino acid sequence of bioactive peak was determined by Edman degradation and identified by gene clone method. A 16-amino peptide with obvious insulin-releasing activity was obtained and sequenced as FMPIVGKSMSGLSGKL-NH2 , which was designated amolopin-1. The precursor peptide composed of 64 amino acid residues deduced from cloned skin cDNA (open reading frame 192 bp) exhibited a highly-conserved signal peptide (22aa), an acidic amino acid residue-rich domain and an amolopin-1 encoding domain. Structural alignment with database records revealed that a novel insulinotropic peptide was obtained from skin secretion of Amolops loloensis therefore further study of its acting mechanism and pharmacokinetics may lead to the discovery of a new treatment for diabetes.
2009, 30(2): 171-177. doi: 10.3724/SP.J.1141.2009.02171
OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli (ATCC 25922) is observed by scanning electron microscopy (SEM) and transmitted electron microscopy (TEM). The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died.
2009, 30(2): 178-184. doi: 10.3724/SP.J.1141.2009.02178
SgI-29 is a newly characterized antibacterial peptide derived from human semenogelin I. Using SgI-29 as model, 4 peptides with different length were synthesized. Physico-chemical characteristics and structure prediction of SgI-29 and its derived peptides were analyzed by software packages and helix-wheel plot. The antibacterial activities of SgI-29 and its derived peptides against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were determined. The structure-function relationship of SgI-29 and its derivatives was analyzed. Our results indicated that SgI-22 has the strongest antibacterial activities against the tested bacteria among the synthetic peptides and might be used as a good model for the structure optimization.
Investigation of Fish Resources in Hechi Prefecture of Guangxi Province,with Comparison of Fish Biodiversity Between Two Tributaries
2009, 30(2): 185-194. doi: 10.3724/SP.J.1141.2009.02185
Field surveys were conducted in the freshwater ichthyfauna of the Liu River and the Hongshui River in the Hechi Prefecture, Guangxi Province between 1994 to 2008. According to the field surveys and reference records, the two rivers harbor a total of 185 species belonging to 9 orders, 21 families, and 107 genera. Compared with previous investigations, 21 species were newly discovered in this area, including 1 new genus and 19 new species. There is a total of 134 fish species in the Liu River, under 8 orders, 19 families, and 88 genera. The species of Cypinifomes has the highest number in popoulation. The Perciformes came in second, and the Siluriformes is the third. The numbers of species for other orders are comparatively smaller than the three orders, only 1-3 species. The total number of species in the Hongshui River is 157, belonging to 8 orders, 20 families, and 101 genera. The number of species for Cypinifomes is the most. The Siluriformes takes second place, and the Perciformes is the third. The numbers of species of other orders are comparatively smaller than the three orders. The fish diversity of Hongshui River is richer than Liu River and the reasons are that Hongshui River flows through such regions that karst and underground rivers are developed and drainage areas are broad, with plenty of water.
A New Nemacheiline Loach of Genus Yunnanilus (Balitoridae) from Guangxi, China—Yunnanilus jinxiensis
2009, 30(2): 195-198. doi: 10.3724/SP.J.1141.2009.02195
A nemacheiline loach Yunnanilus jinxiensis sp. nov. has been discovered in collections from a Pearl River Drainage in Ludong village, Jingxi county, and Guangxi of China in May 2008. Yunnanilus jinxiensis is a new member of the Yunnanilus pleurotaenis group. It can be distinguished from all other congeners in this group by the following characters: body entirely covered by scales, except between pectoral fins and pelvic fins; lateral line present, with 15–20 pores; dorsal fin with 8 branched rays; pectoral fin with 13–14 branched rays; the air bladder has two chambers, the anterior ones into bony capsules and a large free posterior chamber, filling body cavity; caudal fin slightly emarginated; body depth 3.9–4.6 times the standard length (SL); head length 4.1–4.4 times SL; caudal peduncle depth 7.5–9.0 times SL; eye diameter 5.5–6.7 times the head length (HL); interorbital width 2.2–2.4 times HL.
Effects of Withdrawal on Y-maze Spatial Recognition Memory in Mice after Long-term Morphine Injection
2009, 30(2): 199-203. doi: 10.3724/SP.J.1141.2009.02199
Acute drug use modifies brain function in critical ways, and prolonged drug use causes pervasive changes in brain function that persist long after withdrawal. Our previous studies proved that acute or four-day morphine injection impaired mice spatial recognition memory in the Y-maze, but the impairment was short and reversible. In the present study, morphine or saline (40 mg/ kg·day, ip) was injected into mice for 21 days. Spatial recognition memory was tested in the Y-maze on the 2nd, 9th and 19th withdrawal day. Our results showed that spatial recognition memory of morphine-injected mice was impaired on each of the three test days. Our study clearly suggests that long-term morphine use leads to more persistent impairments of spatial recognition memory.
2009, 30(2): 204-208.
A species of bats Hipposideros cineraceus (21 individuals captured, two specimens made) was collected in Fangkong Cave (N: 22°07', E: 107°07', H: 120 m a.s.l.), Zhidong Village, Mingjiang Town, Ningming County, Guangxi Province, South China, in 7 September, 2006. This bat species is the smallest hipposideros in China so far with 32.4 – 36.0 mm forearm and 3.7 – 4.5 body mass. The noseleaf is relatively simple that its anterior leaf is without a median emargination or supplementary lateral leaflets. The intermediate leaf has four glands with long vibrissae. The posterior leaf possesses three poorly developed septa, dividing it into four cells. The internarial septum is inflated and bulbous. The ear is relatively large, slight beyond the end of muzzle when laid forward. This bat species is the first record from this country. The specimens were preserved in Guangdong Entomological Institute.
2009, 30(2): 209-214. doi: 10.3724/SP.J.1141.2009.02209
A wild population of the globally restricted and highly threatened freshwater fish Tanichthys albonubes Lin was recently discovered on Hainan Island, China. Prior to the present study, native populations were only known from isolated pockets in Guangdong Province of China and Quang Ninh Province of Vietnam; the Hainan record thus represents the first insular and southernmost distribution of this little-known species. Thus far it is known only from a single site in Hainan, despite repeated surveys of similar habitats throughout the island. The Hainan population occurs in a clean, sluggish coastal stream with abundant hydrophytes, and is sympatric with 20 fish species. Due to the unique geographic distribution of the Hainan population, and the conservation significance of the species, studies of its phylogenetic relationships with conspecifics populations elsewhere are underway. The exact locality of the newly discovered Hainan population cannot be revealed, but relevant government authorities have been notified and we are working to protect the site.
2009, 30(2): 215-220. doi: 10.3724/SP.J.1141.2009.02215
This paper calculated the Bonferroni interval and Bailey’s interval of four sets published data in order to show comparative dominance of Bailey’s method in the study of “use-available” model on habitat selection of animals. Bailey’s method deduced differently on two sets aforesaid data compared to Bonferroni method, one dataset of which couldn’t show whether the animals made habitat selection or not in Bonferroni method’s judgment. Nevertheless Bailey’s method detected the variance. Cherry (1996) pointed out that the Bonferroni method of interval construction is based on a large sample approximation for a confidence interval for a single binomial proportion with the overall confidence level controlled with the Bonferroni inequality. This method will lead to a logically wrong deduction because of lacking coherence and consonance for small samples. As an alternative, Bailey's method calculates correctly with the use of a continuous correcting factor to overcome the above-mentioned limitation, furthermore a Chi-square goodness-of-fit test is undesired. We recommended Bailey’s method in analyzing data of habitat selection. We also suggested that researchers should properly increase the sample size meanwhile decrease the number of type of habitats to control Type I and Type II errors.
Among the most fascinating mysteries of life is the interaction between biological systems and the earth’s magnetic field. Although the earth’s magnetism may have an under appreciated role in biological interpretations, it has been most extensively studied in the processes of avian orientation and migration. Many species of bird are known to have behavioral responses to the earth’s and artificial magnetic fields. These responses may be mediated by a number of potential magneto-biochemical processes. The two most commonly investigated include a magnetosensitive magnetite rich region in the upper beak area and a photo/magnetoreception process in the eyes of various bird species. In addition to external magnetic stimuli, recent findings in visually restricted birds have described a hemispherically lateralized interpretation of this information within the brain. Even with these findings, a considerable amount of work is needed to clarify what information is processed and how it is used to create the bird’s magnetic compass. This review focuses these recently published findings as a means to assess this intriguing phenomenon.