2010 Vol. 31, No. 3
2010, 31(3): 205-220. doi: 10.3724/SP.J.1141.2010.03205
To understand spatial patterns of genetic differentiation in the Brachionus calyciflorus species complex, the rDNA ITS regions of 124 rotifer clones collected from eight geographic regions in East China were sequenced and analyzed. A total of 73 haplotypes were defined, and were grouped into three clades by the phylogenetic trees. The divergences of ITS sequence among the three clades ranged from 4.2% to 25.3%, indicating the occurrence of three cryptic species (cryptic species I, cryptic species II and cryptic species III). The sympatric co-occurrence of these cryptic species was observed at Guangzhou, Danzhou and Wuhu. The nucleotide diversity in B. calyciflorus complex was higher, and so was haplotype diversity. Within cryptic species III, the higher genetic differentiation might attribute to fragmentation, restricted gene flow, and interfered effective gene flow and accelerated differentiation of geographic populations by the rapid increase of colonizers after an ambiguous glacial bottleneck. The nested clade analysis suggested to some extent the geographic structure, and the non-significant correlation existed between the geographic distance and the average pairwise difference between populations (Da) as well as Fst. The Younger Dryas Event after the last full glacial period might has considerable effect on the patterns of geographic structure and distribution of cryptic B. calyciflorus species. During the YD event, the three cryptic species probably retreated and co-occurred in multiple relict refugia to the south of Nanling area. Long-distance colonization possibly coupled with subsequent fragmentation might be the main reason of the current patterns of geographic structure. The geographic distribution of B. calyciflorus complex on a global scale might attribute to the motion of continental plates.
2010, 31(3): 221-229.
In the present study, we cloned and sequenced 10 new ND4 and ND5 gene sequences of Cobitoidea. These sequences were used to reconstruct phylogenetic relationships together with those of 15 other species downloaded from GenBank. The results showed that the length of ND4 gene sequence was 1 380−1 387 bp with ATG as starting codon and incomplete termination signal as terminated codon; the length of ND5 gene sequence was 1 821−1 839 bp with ATG as starting codon and TAA or TAG as terminated codon; three tRNA genes coding tRNAs that carry hisidine, serine and leucine respectively, were inserted between ND4 and ND5 genes. A, T, C and G accounted for 30.4%, 27.3%, 14.2% and 28.1% in ND4/ND5 gene (including intervening three tRNA genes). The content of A+T (57.7%) is higher than that of G+C (42.3%). The estimated Ti/Tv ratio was 1.586. With Danio rerio and Cyprinus carpio as outgroups, the phylogenetic relationships of Cobitoidea were analyzed using maximum parsimony (MP) method, maximum likelihood (ML) method and Bayesian analyses (BI). Results of all the three methods indicated that Cobitinae, Nemacheilinae, Botiinae, Balitoridae and Vaillantellidae were all monophyletic respectively, and their interrelationships were: (Vaillantellidae + (Botiinae + (Cobitinae + (Nemacheilinae + Balitoridae)))), which was consistent with the previous studies based on the whole mitogenome and some nuclear genes sequences (eg. RAG1 gene). However, the present results differ from those based upon other mtDNA genes sequences (e.g. the cyt b and D-loop showed that Nemacheilinae grouped with Cobitinae forming sister-group, then they clustered with Balitoridae). The difference could be due to the phylogenetic information carried by different length sequence: Longer sequences carry more information and result in more reliable phylogenetic trees.
2010, 31(3): 230-238. doi: 10.3724/SP.J.1141.2010.03230
The genus Lepturichthys is endemic in China, including two valid species, L. fimbriata and L. dolichopterus. The former is widely distributed in the Upper and Middle Yangtze River, while the latter only in Minjiang River. In the present study, the mitochondrial cytochrome b gene sequences were analyzed for 20 individuals of L. fimbriata collected from the Upper and Middle Yangtze River and eight individuals of L. dolichopterus collected from Minjiang River. This paper aimed to investigate the geographical speciation process of the genus Lepturichthys. The results showed that the genetic variations among different haplotypes of L. fimbriata ranged from 0.1% to 1.8%, with an average of 0.7%, and from 0.6% to 1.3%, with an average of 0.7% among different haplotypes of L. dolichopterus. The variations between L. fimbriata and L. dolichopterus were also very low, ranging from 0.2% to 1.8%, with an average of 0.9%. Two methods, Neighbor Joining (NJ) and Bayesian analysis (BI), were used for constructing the molecular phylogenetic trees. Both NJ and BI trees indicated that all Lepturichthys specimens formed as a monophyletic group; the L. dolichopterus samples also formed as monophyletic group, having a derived position in the phylogenetic trees, while the L. fimbriata samples formed as a paraphyletic group. Both L. fimbriata and L. dolichopterus are valid species in morphology. Based on molecular and morphological data, we presumed that the geographical speciation process of the genus Lepturichthys could be as following: during the Quaternary Glacial period, L. fimbriata in the Middle and Lower Yangtze River dispersed to the drainages of Southeast China along the Continental Shelf of East China Sea, one population spread to the Minjiang River and envolved as a new species (L. dolichopterus) because of considerable environmental difference, while the remaining L. fimbriata populations in the Yangtze River formed as a paraphyly. The other similar geographical speciation patterns are discussed in the present study as well.
In order to study the structural, functional and molecular evolutional relationship of fish liportein lipase (LPL) and hepatic lipase (HL) genes, seven liver LPL and HL cDNA partial sequences were isolated from Acipenser sinensis, Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idellus, Cirrhinus molitorella, Oreochromis niloticus, Channa maculate by RT-PCR. Three full-length cDNA sequences of LPL, HL of Acipenser sinensis and LPL of Hypophthalmichthys molitrix were obtained by RACEs. From the sequence analysis and homologous results, the amino acid sequences of LPL and HL are relatively conserved in mammals, birds and fishes. Taken together with these obtained amino acid sequences and sequences of all known LPL, HL, EL and PL from other vertebrates, a phylogenetic tree was constructed by neighbor-joining method. The result supports that all of them belong to lipase family.
The phagocyte NADPH oxidase plays a crucial role in host defense against invading microorganisms by catalyzing the formation of reactive oxygen species, which is the precursor of a variety of microbicidal oxidants such as hydrogen peroxide (H2O2). In the present study, full-length cDNAs of three regulatory subunits of NADPH oxidase, including p40phox, p47phox, p67phox were cloned from head kidney of mandarin fish utilizing the reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis showed that the full length cDNA of p40phox is 1 406 nt, containing a 1 050 nt open reading frames that encodes a 349 amino acid protein, the full length cDNA of p47phox is 1 686 nt, containing a 1 209 nt open reading frames that encodes a 402 amino acid protein, the full length cDNA of p40phox is 2 185 nt, containing a 1 488 nt open reading frames that encodes a 495 amino acid protein. Semi-quantitative RT-PCR analyses from various tissues indicated that mRNAs of the three subunits can be detected in the blood, brain, heart, spleen, kidney and thymus, but their expression intensity are different in tissues. Stimulating the mandarin fish with formalin killed Flavobacterium columnare G4 significantly up-regulated the expression of p40phox in blood and head kidney; and p47phox in head kidney and spleen; and p67phox in blood, head kidney and spleen. The results suggested that mandarin NADPH oxidase was involved in the immune responses against bacteria.
cDNA Cloning, Characterization and mRNA Expression of a Profilin from the Swimming Crab Portunus trituberculatus
2010, 31(3): 261-267. doi: 10.3724/SP.J.1141.2010.0326
We isolated and characterized the profilin full-length cDNA from hemocytes of swimming crab Portunus trituberculatus. The profilin cDNA consists of 742 bp and encodes a polypeptide of 125 amino acids, having a predicted isoelectric point of 5.87. The deduced amino acid sequence shows 42.9% amino acid sequence identity to the profilin of mosquito Anopheles gambiae. The profilin mRNA was highly expressed in hemocytes and moderately in hepatopancreas of normal crab. The higher expression of profilin mRNA is observed in crab challenged by the pathogenic bacterium Vibrio parahaemolyticus. These results suggest a potential role for profilin in pathogen host defense mechanisms.
Role of Fas/FasL on Apoptosis of Porcine Follicular Granulose Cells Derived from Isolated Follicles During Culture in vitro
2010, 31(3): 268-274. doi: 10.3724/SP.J.1141.2010.03268
The whole antral follicles were isolated from porcine ovaries and classified as follows: healthy follicles (HF), early atretic follicles (EF) and progressed atretic follicles (PF). The isolated porcine follicles were used for routine histological section and HE staining after examination by eyesight. Morphological research shows that the accuracy rate of eyesight examination for HF is 92%. Healthy follicles were chosen for further experiment and divided into 3 groups: large follicle (Æ＞5 mm), medium follicles (3−5 mm) and small follicles (≤3 mm). All follicles were cultured for 8, 16 and 24 h, respectively and the apoptosis of of their granulose cells were examined by Annexin V-FITC/PI double-labeling. It showed that the total apoptotic rate of granulose cells derived from cultured follicles could reach over 70% at 8 h after culture and be 81.1%-94.6% at 24 h after culture. Granulosa cells from groups were collected at 0, 8, 16, 24, 48 and 72 h after culture without serum and used for the examination of expression of FasL and Fas mRNA with real time PCR SYBRgreen method. The expression level of FasL mRNA of granulose cells from different size of follicles increased with culture time and reached the highest level at 24 h after culture (P＜0.05). Expression level of FasL mRNA of granulose cells from small follicles was higher than those from large and medium follicles. There exists no difference for expression level of Fas mRNA of granulose cells among groups before culture but significantly increased at 8 h after culture and reached the highest level at 48 h after culture. It showed in the present experiment that the follicular culture system without serum used could effectively induce the apoptosis of follicular granulose cells. Cell apoptosis is the main cause of follicular atresia, the degree of which varied with the size of follicles. Small follicles seemed to be easier atretic than medium and large follicles.
2010, 31(3): 275-281. doi: 10.3724/SP.J.1141.2010.03275
200 tadpoles Rana chensinensis at stage 26-27 were exposed to 0.05, 0.1, 0.2 or 0.4 mg/L Cd2+ in tap water respectively. The heteromorphic young frogs in different treatments were anatomized, females and males were identified through gonad observation, and the female rate was calculated. Localization of estrogen receptors (ER) in liver cells was investigated in different treatments by using immunocytochemistry. The results showed that Cd2+ might induce limb abnormality. There was little correlation between abnormality rate and cadmium concentration in lower Cd2+ levels; however, limb abnormality ratio was higher in the 0.4 mg/L group. On the other hand, Cd2+ could affect gonad differentiation. Compared to the control group, the proportion of female population increased in the 0.05 mg/L group and decreased in the 0.1, 0.2 and 0.4 mg/L ones. The sex rate in the 0.2 mg/L group is significantly different from that in the control group. Hermaphrodite gonads were observed in the two treatments with 0.2 mg/L and 0.4 mg/L of Cd2+. Additional, ER expression was positive in both cytoplasm and nucleolus of liver cells in Cd2+ treated groups. But, there was no linear relationship between ER expressions levels and the concentration of Cd2+. These results suggested that cadmium could influence tadpole metamorphosis and gonad development by affecting the secretion of sex hormone.
2010, 31(3): 282-286. doi: 10.3724/SP.J.1141.2010.03283
The pupil size of both human and other animals can be affected by light Many kinds of psychiatrical and psychological disorders associate with abnormal properties of pupil light reflex Thus, the properties of pupil light reflex function can reflect these kinds of mental disorders However, the effect of drug of abuse on pupil light reflex function is till unclear To quantificationally assess the effects of addictive drugs on pupil light reflex function, in the present study, we examined the effects of morphine on pupil diameter and pupil light reflex function in rhesus monkeys We measured the pupil diameter at different timing points after during morphine administration We found that morphine administration reduced the diameter of pupil and decreased the constriction rate Our present results provide an experimental support for applying pupil light reflex property as a reference in addicts’ detection.
A New Method to Explore the Modulation from the Saccadic System on the Pupillary Light Reflex System
2010, 31(3): 287-291. doi: 10.3724/SP.J.1141.2010.03288
The saccadic system has anatomical and functional connections with the pupillary light reflex (PLR) system. But it is not known whether the saccadic system modulates the PLR system. To investigate this issue, it is necessary to know whether the uneven light stimulus to retina and the near responses influence the change of pupil diameter. We designed a new behavioral method to investigate the issue on human subjects. Methods: one eye of the subject was stimulated by pulse light stimulus from a horizontal linear array of light emitting diodes (LEDs) presented across visual field in an ocular mask. The changes of the eye position and pupil diameter of another eye were recorded by an infrared eye tracking system. Results: The relative constriction ratios of PLRs when the fixation points of subjects were in the nasal visual field were not significantly different from them when the fixation points of subjects were in the temporal visual field（P=0.148, Non-paired t test）.Conclusion: The influences from uneven light stimulus to retina and the near responses were eliminated by this method. The method can be used to study the modulation from the saccadic system on the pupillary light reflex system.
2010, 31(3): 292-310. doi: 10.3724/SP.J.1141.2010.03318
2010, 31(3): 303-309. doi: 10.3724/SP.J.1141.2010.03311
Combining line-transect method and sampling method, the geographic coordinates of footprint links for Williamson’s Mouse-deer (Tragulus williamsoni) were collected in Mengman and Longmen regions of Mengla area, southern Yunnan, from June to December 2007. A distribution pattern map of the mouse-deer was produced by GIS software. Eleven ecological factors were measured in collected and available samples. The results indicated that the mouse-deer preferred valleys along banks of rivers or streams in low altitude, and were mainly distributed in Sanchahe of Mengman region and Darongshu of Longmen region. Comparing nominal factors between collected and available samples, the results showed that the mouse-deer tended to inhabit middle and lower slopes and favored the plant species of Ficus hirtal, Baccaurea samiflora, Alpinia kwangsiensis and Phrynium capitatum. These plants occur along banks of rivers or streams, one of the important reasons affecting distribution of the mouse-deer populations. Comparing quantitative factors between collected and available samples, the results showed that four factors including tall tree coverage, shrub coverage and herb coverage, and distance to water, were significantly different. These four factors were key in discriminating collected and available samples and main factors affecting the habitat use of the mouse-deer. The dense shrub with taller trees could form good shady condition for the mouse-deer, and greatly strengthen their security. Due to their preference for areas near water, there were more flourishing tall trees and dense shrubs at valleys in low altitude, which was one of important reasons affecting the distribution of the mouse-deer populations massing in valleys in low altitude.
The Understory Bird Communities in Monsoon Evergreen Broadleaved Forest of Mengyang, Xishuangbanna, Yunnan During the Dry Season
2010, 31(3): 311-318. doi: 10.3724/SP.J.1141.2010.0331
Understory birds were sampled by mist nets in monsoon evergreen broadleaved forest of Mengyang, Xishuangbanna, Yunnan from October 2008 to March 2009. A total of 1423 individuals of 90 species representing 28 families and 7 orders were captured. The dominant birds were Sliver-breasted Broadbill (Serilophus lunatus), Brown-cheeked Fulvatta (Alcippe poioicephala), White-throated Bulbul (Alophoixus pallidus), Golden-spectacled Warbler (Seicercus burkii), White-tailed Robin (Cinclidium leucurum), Black-breasted Thrush (Turdus dissimilis), Streak-Breasted Jungle Babbler (Pellorneum ruficeps), and Buff-breasted Jungle Babbler (Trichastoma tickelli). Resident birds, the major species of the understory, accounted for 89.3 percent of total captures. The mean capture rate was 9.0 individuals/(100 net-hours). The capture rate differed significantly between months, highest in December 2008 (12.5±1.3）individuals/(100 net·hours), lowest in February 2009 (5.2±0.6 )individuals/(100 net·hours). One important characteristic is more species richness and lower density of birds in Xishuangbanna compared to understory bird composition of South China. Another characteristic is lower babblers' abundance. Sliver-breasted Broadbill, National Key Protection Bird, is one of dominant birds. Thus its habitate conservation needs further concern in Xishuangbanna.
Comparison of Thermogenic Character of Liver and Muscle in Chinese Bulbul Pycnonotus sinensis Between Summer and Winter
2010, 31(3): 319-327. doi: 10.3724/SP.J.1141.2010.03319
Acclimatization to winter conditions is an essential prerequisite for survival of small passerines. Seasonal changes in a bird's physiology and behavior are considered to be part of an adaptive strategy for survival and reproductive success. Changes in photoperiod, ambient temperature and food availability trigger seasonal acclimatization in physiology and behavior of many birds. In the present study, seasonal adjustments in several physiological, hormonal, and biochemical markers were examined in wild-captured Chinese bulbuls (Pycnonotus sinensis) from the Zhejiang Province in China. Oxygen consumption was measured using the closed-circuit respirometer containing 3.6 L animal chambers. State-4 respiration in liver and muscle mitochondria was measured at 30℃ with a Clark electrode. The activities of cytochrome C oxidase (COX) in liver and muscle were measured polarographically at 30℃ using a Clark electrode. The protein content of mitochondria was determined by the Folin phenol method, with bovine serum albumin as standard. In winter sparrows had higher body mass and basal metabolic rate (BMR). The contents of mitochondrial protein in liver, and state-4 respiration and COX activity in liver and muscle increased significantly in winter. Circulating level of serum triiodothyronine (T3) was significantly higher in winter than in summer. Together, these data suggest that Chinese bulbuls mainly coped with cold by enhancing thermogenic capacities through increased activity of respiratory enzymes activities. The results support the view that prominent winter increases in BMR are manifestations of winter acclimatization in Chinese bulbuls and that seasonal variation in metabolism in bulbuls is similar to that in other small wintering birds.
2010, 31(3): 328-332. doi: 10.3724/SP.J.1141.2010.03336
One male bat was collected in a cave in Cuibi Hill (N: 21°53', E: 101°18', H: 683 m a.s.l.), Menglun Town, Mengla County, Yunnan Province, in 26 November, 2006. This bat is a middle species with 60.7 mm forearm and 21.4 g body mass. The ears are large oval and joined medially to the forehead at about 15% of the height of inner margin. The tragus of each ear is well-developed and distinctly bifid. The noseleaf is simple. Posterior noseleaf is oval with obtuse tip and convex sides, a significant longitudinal ridge laying middle, which connects to intermediate noseleaf at the base. Intermediate noseleaf presents triangle with W-shape at the tip. Frontal noseleaf is horseshoe shape and attached directly to the muzzle. The tail is absent. The second finger of each wing has only one phalanx. There are no upper incisors, Pm3 and Pm3. The upper canine has an anterior and a large posterior basal cusps. This bat is identified as lesser false vampire, Megaderma spasma, which is the new record of China. Its external and craniodental measurements were presented and compared with those of M. lyra. The specimen was preserved in Guangdong Entomological Institute.
2010, 31(3): 293-302. doi: 10.3724/SP.J.1141.2010.03293
We studied the sonogram and timing of duets of three groups of Central Yunnan western black crested gibbons (Nomascus concolor jingdongensis) in Dazhaizi, Mt. Wuliang. The study was based on a 13-month field observation period, from March 2003 to March 2004. A Sony TC-D5 Pro2 recorder, Sony C-76 directed microphone, and Sony tape were used to record the duet bouts in March 2007 and March 2008. Signal/RTS 4.0 was used to analyze the sonogram of the duet. We first presented sonograms of all kinds of sound units in the duets made by western black crested gibbons. The adult male could produce boom, aa notes and modulated figures. Adult females produced great call or abortive great call. The two adult females in each group usually produced great calls synchronously. After the great call, males always immediately produced a modulated figure to coordinate with the female. The interval between the modulated figure and great call was 2.7 s. On average, groups sang on 53% of days monitored, and sang 1.09 duet bouts per singing day. 91.5% of the duets were produced between half an hour before and three hours after the sunrise. The average duration of the duets was 12.9 min, and females produced 4.6 great calls during one duet bout. The intervals between two successive great calls were generally 115 s. There was no significant difference in the duration and frequency of the duet bouts, but there was significant difference in the number of the great calls and intervals between great calls among groups.