2011 Vol. 32, No. 4
2011, 32(4): 353-362. doi: 10.3724/SP.J.1141.2011.04353
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Microsatellite analysis of genetic variation of the oriental river prawn Macrobrachium nipponense in Qiantang River
2011, 32(4): 363-370. doi: 10.3724/SP.J.1141.2011.04363
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Genetic diversity and genetic structure of 7 wild stocks of oriental river prawn Macrobrachium nipponense in Qiantang River, i.e. Wen-yan, Fu-yang, Chang-kou, Tong-lu, Xin-an-jiang, She-xian and Xiu-ning, were investigated using 10 microsatellite DNA markers. The result showed that all the 10 loci were highly polymorphic. There was a trend that the level of genetic diversity of wild stocks in downstream and midstream were higher than the upstream ones’. Sign test and Wilcoxon sign rank test results showed that the stocks in Qiantang River had no bottleneck effect, and the number of stocks had not declined recently. FST among stocks ranged from 0.0201 to 0.1069. Analysis of molecular variance (AMOVA) revealed that a higher portion (93.48%) of variations existed within individuals, while lower portion (6.52%) existed among stocks. FST and AMOVA analysis across all stocks and loci indicated the medium level of divergence among the stocks. The NJ clustering tree based on DA genetic distance demonstrated that the stocks of adjacent geographical position clustered together. 413 individuals obtained from six wild stocks could be divided into two potential populations based on the genetic structure. This study demonstrated that genetic diversity and genetic structure of M. nipponense stocks were relevant to geographical position where they survived.
Cloning and expression analysis of a long type peptidoglycan recognition protein (PGRP-L) from Xenopus tropicalis
2011, 32(4): 371-378. doi: 10.3724/SP.J.1141.2011.04371
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Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) of the immune system, which bind and hydrolyze bacterial peptidoglycan. Here, a long type PGRP (PGRP-L) was first cloned in the lower vertebrate species Xenopus tropicalis (Xt). The XtPGRP-L possessed a conserved genomic structure with five exons and four introns. The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP. The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb. During embryonic development, XtPGRP-L was not expressed until the 72 h tadpole stage. In adult tissues, it was strongly expressed in the liver, lung, intestine, and stomach. Furthermore, after LPS stimulation, the expression of XtPGRP-L was up-regulated significantly in the liver, intestine and spleen, indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.
2011, 32(4): 379-385. doi: 10.3724/SP.J.1141.2011.04379
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Bm-TFF2, a trefoil factor from the large-webbed bell toad (Bombina maxima), can stimulate cell migration and inhibit cell apoptosis. To study the structure-function relationship of Bm-TFF2, we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E. coli. The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR, while the N-terminal, C-terminal and two arginine mutated Bm-TFF2 clones were constructed, and ligated into pET-32a(+) expression vectors. The fusion proteins were induced by IPTG at 37°C. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni2+-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant. The fusion proteins were analyzed by SDS-PAGE and Western blotting. The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L. All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.
2011, 32(4): 386-390. doi: 10.3724/SP.J.1141.2011.04386
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Zebrafish (Danio rerio) Z-OTU, containing OTU and TUDOR domains, was predicted to be a member of OTU-related protease, a family of the deubiquitylating enzymes (DUBs). A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA. Here, we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis. After prokaryotic expression, the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU, which was used for immunohistochemistry in zebrafish ovaries and embryos. Interestingly, obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis. In stage I oocytes, z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle. In addition, its protein was distributed during entire oogenesis, while mRNA was not detected in oocytes at stage IV or mature oocytes. The z-otu mRNA disappeared after midblastula transition (MBT) and its protein gradually decreased after this stage. We inferred that Z-OTU protein, like other OTU-related protease with DUB activity, was required for germinal vesicle breakdown of oocytes during meiosis, germinal vesicle migration, and embryo cleavage maintenance.
Notch signaling dependent differentiation of cholangiocyte-likecells from rhesus monkey embryonic stem cells
2011, 32(4): 391-395. doi: 10.3724/SP.J.1141.2011.04391
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Rhesus monkey embryonic stem (rES) cells have similar characteristics to human ES cells, and might be useful as a substitute model for preclinical research. Notch signaling is involved in the formation of bile ducts, which are composed of cholangiocytes. However, little is known about the role of Notch signaling in cholangiocytic commitment of ES cells. We analyzed the effect of Notch signaling on the induction of cholangiocyte-like cells from rES cells. About 80% of definitive endoderm (DE) cells were generated from rES cells after treatment with activin A. After treatment with BMP4 and FGF1 on matrigel coated wells in serum-free medium, rES-derived DE gave rise to cholangiocyte-like cells by expression of cholangiocytic specific proteins (CK7, CK18, CK19, CK20, and OV-6) and genes (GSTPi, IB4, and HNF1β). At the same time, expression of Notch 1 and Notch 2 mRNA were detected during cell differentiation, as well as their downstream target genes such as Hes 1 and Hes 5. Inhibition of the Notch signal pathway by L-685458 resulted in decreased expression of Notch and their downstream genes. In addition, the proportion of cholangiocyte-like cells declined from ~90% to ~20%. These results suggest that Notch signaling may play a critical role in cholangiocytic development from ES cells.
Metabolism and thermoregulation between Mrs Hume’s Pheasant (Syrmaticus humiae) and Elliot’s Pheasant (S. ellioti)
2011, 32(4): 396-402. doi: 10.3724/SP.J.1141.2011.04396
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To understand metabolic adaptations, the basal metabolic rate (BMR) of Mrs Hume’s Pheasant (Syrmaticus humiae) and Elliot’s Pheasant (Syrmaticus ellioti) were investigated. Metabolic rate (MR), body temperature (Tb ) and thermal conductance (C) were determined in both species at a temperatrue range of 5 − 35 ℃, respectively. Oxygen consumption was measured with a closed circuit respirometer. The thermal neutral zones (TNZ) were 24.5 − 31.6 ℃, and 23.0 −29.2 ℃, respectively. With a temperature range of 5 − 35 ℃, Mrs Hume’s Pheasant and Elliot’s Pheasant could maintained stable Tb at a mean of (40.47±0.64) and (40.36±0.10) ℃, respectively. Mean BMRs within TNZs were (1.36±0.84) mLO2/(g·h) for Mrs Hume’s Pheasant and (2.03±0.12) mLO2/(g·h) for Elliot’s Pheasant, which were 77% and 86% of the expected value based on their body mass, respectively. Thermal conductance of Mrs Hume’s Pheasant and Elliot’s Pheasant were (0.12±0.01) and (0.17±0.01) mLO2/(g·h·℃), below the lower critical temperature, respectively, which were 119% and 124% of the expected value based on their body mass, respectively. The ecophysiological characteristics of these species were low metabolic rate, high body temperature, and high thermal conductance, which allow both species to better adapt to the warmer climate environment in south China.
2011, 32(4): 403-420. doi: 10.3724/SP.J.1141.2011.04403
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Afferent and efferent neural elements of the retina and central ganglia in the freshwater snail Planorbarius corneus were labelled using retrograde transport of neurobiotin through the optic nerve. Axons of at least some photoreceptor cells become direct contributors to the optic nerve as no synaptic junctions could be detected. The processes enter the cerebral ganglion and form a dense bundle of thin afferent fibres, the so-called optical neuropil. Efferent neurons were revealed in all ganglia, except the buccal ones. Some of the ascending axons branch in the cerebral ganglia, cross the cerebro-cerebral commissure, reach the contralateral eye and form arborizations in the eye cup. Some efferent neurons send axons to different peripheral nerves as well: n.n. intestinalis, pallialis dexter, pallialis sinister internus et externus. Serotonin- and FMRF-amide-ergic fibres were revealed in the optic nerve. These fibres belong to those central neurons which send their axons to the ipsilateral eye only. They form abundant varicoses in the eye cup and nuclear layer of the retina, and possibly help to regulate retinal sensitivity to light.
A traumatic brain injury model for distinguishing between transplanted neural cells and host cells in vivo
2011, 32(4): 421-427. doi: 10.3724/SP.J.1141.2011.04421
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To perform electrophysiological recording and other investigations on transplanted neural cells in vivo, we used mechanical damage to establish a special traumatic brain injury model that could distinguish transplanted cells from host cells. The morphology of the trauma-induced holes in the cortex of the rat brain was regular. The model was stable and repeatable. Neural stem cells were transplanted into the trauma-induced hole, and were able to survive for a long time. Most of the transplanted cells differentiated into neurons, and only a small amount turned into glia cells. There was a clear boundary between the host cells and the transplanted cells. Single cell electrophysiological recording on transplanted neural cells were detected in vivo. This study established a stable and repeatable traumatic brain injury model, which could be used to conduct in vivo electrophysiological recording research on transplanted neural cells.
2011, 32(4): 428-434. doi: 10.3724/SP.J.1141.2011.04428
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Present work investigated the effects of tail suspension stress (TSS) on spatial memory acquisition, consolidation, and its reversal learning in mice. Eighty-one adult male KM mice were divided into four groups (each group including a TSS subgroup and its control subgroup): absolute spatial memory acquisition and consolidation groups (group AA and CA); relative spatial memory acquisition and consolidation groups (group AR and CR). TSS (20 min) was performed immediately before (acquisition) or after (consolidation) a daily training. Results showed that there was no significant difference between control animals and TSS animals in each group in early spatial memory training days (5-8 d of training). Along with training, the performance of control animals improved significantly, but the performance of TSS animals improved slightly (group AA, CA and AR) or even did not change (group CR) (P < 0.01). Reversal learning was also impaired in TSS animals (P < 0.01). The results indicated that TSS could impair spatial memory acquisition, consolidation and reversal learning (especially the relative spatial memory consolidation and its reversal learning) in mice.
2011, 32(4): 435-441. doi: 10.3724/SP.J.1141.2011.04435
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Pinus armandii, a native pine species, has large (about 300 mg), wingless seeds, and distributes from central to western China at an altitude of 1 000 − 3 300 m. To determine how the seed size affects rodents caching behavior, tagged seed releasing and tracking experiments were conducted at 3 sites in Northwest Yunnan province in 2006 and 2007. Our data indicated that for all sites and both years, compared with the smaller seeds, the proportions of cached large seeds were significantly higher, whereas the consumed ones were significantly lower. Meanwhile, the mean and maximum values of caching distances were also significantly increased in large seeds. Seed fate was different between the two years and within the three sites as there have different rodent community compositions.
Behavioral migration diversity of the Yangtze River Japanese Eel, Anguilla japonica, based on otolith Sr/Ca ratios
2011, 32(4): 442-450. doi: 10.3724/SP.J.1141.2011.04442
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The Yangtze River estuary is the main production area of Anguilla japonica in China, as well as the only existing fishery area for adult eels. Japanese eels are distributed in the main rivers and many tributaries from the Yangtze River estuary to the upper Jinsha River, which extend to nearly 3 000 km. However, their migration behaviors remain relatively unknown. We analyzed the biological characteristics of 153 specimens of silver eels collected from the Jingjiang section of Yangtze River (31º30′N, 120º42′E) between September and November, 2008, and tested the sagittal Sr/Ca ratios of 27 specimens. Among the 153 specimens examined, 85 were female and 68 were male, which translated to a female-male ratio of 1 : 0.8. The ages of the female specimens ranged from 3 to 7 a (average 5.52) with an average total length (TL) of (669±80) mm, average body weight (BW) of (555±229)g, average condition factor of 1.77±0.22, and average gonad somatic index (GSI) of 1.32±0.31. The ages of the males ranged from 3 to 5 a (average:4.38) with an average TL of (518±51) mm, average BW of (234±76) g, average condition factor of 1.62±0.18, and average GSI of 0.21±0.11. All biological parameters of females were significantly larger than those of the male specimens (P＜0.05). According to the average Sr/Ca ratio (7.99±1.05) ×10-3 of the elver mark of sagitta, 17 individuals (62.96%) were river eels and 10 individuals (37.04%) were estuarine eels. Of 16 females, 13 individuals (81.25%) were river eels and 3 were estuarine eels, while of 11 males, 36.36% were river eels and 63.64% were estuarine eels. The analysis on Sr/Ca ratios for every growth layer group (GLG) indicated there were no significant differences between second-age males and females. However, significant differences were observed between the third-age, fourth-age, and migration-age male and female specimens. This was likely related to the fact that second-age eels of both sexes stay in the same inhabitation waters; however, as they grow older, they move to different areas.
Brood parasitism and egg mimicry of Himalayan Cuckoo (Cuculus saturatus) on its host Blyth’s Leaf Warbler (Phylloscopus reguloides) were studied in south-western China from April to July 2009. The cuckoo laid a white egg with fine brown markings on the blunt end. The eggs were conspicuously bigger than the host’s own, with 2.06 g in mass and 1.91 cm3 in volume. Visual modeling showed that the cuckoo eggs, which from the human eye appeared to mimic the host eggs to a great extent, were completely different from the host eggs in both hue and chroma. The characters of the Himalayan Cuckoo nestling, reported for the first time, included two triangular and black patches on its gape, which appeared from four days old and became darker with age and growth. While this character also exists in nestlings of Oriental Cuckoo (C. optatus), it has not been found for other Cuculus species. Our results reveal cryptic aspects in the cuckoo-host egg color matching, which are not visible to the naked human eye, and indicate that high mimetic cuckoo eggs rejected by hosts, as determined by human observers in previous studies, might not be mimetic as birds see them.
2011, 32(4): 456-460. doi: 10.3724/SP.J.1141.2011.04456
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We recorded and described the calls and acoustic characteristics of the male tiger frog, Hoplobatrachus rugulosus, in an artificial pond during mating season. Spectral and temporal call parameters, along with call intensity were analyzed. Three harmonics were distinguishable from the spectrogram. Four patterns of dominant frequency were found in calls produced late at night, i.e, 3 patterns in the first harmonic (located in 500 Hz section, 700 Hz section, and 800 Hz section respectively) and 1 in the second harmonic (located in 1 800 Hz section). Call duration, call duty cycle, call intensity, and pulse rate were highly variable among different patterns of dominant frequency. These call properties could provide valuable evidence for further ecological study of this species.
Localization of stationary pronuclei during conjugation of Paramecium as indicated by immunofluorescence staining
2011, 32(4): 461-464. doi: 10.3724/SP.J.1141.2011.04461
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After the third prezygotic division during conjugation of Paramecium caudatum, migratory and stationary pronuclei are produced. The migratory pronuclei remain in the paroral region tightly against the conjugating boundaries; while the stationary pronuclei are located beside the migratory pronuclei. To date, however, it is not clear what causes this close side-by-side localization between migratory and stationary pronuclei. In the current study, immunofluorescence staining with monoclonal antibody of anti-α tubulin indicated that “U” or “V” shaped spindles connected the migratory and stationary pronuclei during the third prezygotic division. This observation accounts for the close localization between these two types of pronuclei.