Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Comparison between visual and passive acoustic detection of finless porpoises in the Yangtze river, China

Recently, sonar signals and other sounds produced by cetaceans have been used for acoustic detection of individuals and groups in the wild. However, the detection probability ascertained by concomitant visual survey has not been demonstrated extensively. The finless porpoises (Neophocaena phocaenoides) have narrow band and high-frequency sonar signals, which are distinctive from background noises. Underwater sound monitoring with hydrophones (B&K8103) placed along the sides of a research vessel, concurrent with visual observations was conducted in the Yangtze River from Wuhan to Poyang Lake in 1998 in China. The peak to peak detection threshold was set at 133 dB re 1 mu Pa. With this threshold level, porpoises could be detected reliably within 300 m of the hydrophone. In a total of 774-km cruise, 588 finless porpoises were sighted by visual observation and 44 864 ultrasonic pulses were recorded by the acoustical observation system. The acoustic monitoring system could detect the presence of the finless porpoises 82% of the time. A false alarm in the system occurred with a frequency of 0.9%. The high-frequency acoustical observation is suggested as an effective method for field surveys of small cetaceans, which produce high-frequency sonar signals. (C) 2001 Acoustical Society of America.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wildtype rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wildtype. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc largurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions. (C) 2003 Elsevier Science B.V. All rights reserved.

Characterization of the sleep architecture in two species of fruit bat

Bats (Chiroptera) are the second-most abundant mammalian order in the world, occupying a diverse range of habitats and exhibiting many different life history traits. In order to contribute to this highly underrepresented group we describe the sleep architecture of two species of frugivorous bat, the greater short-nosed fruit bat (Cynopterus sphinx) and the lesser dawn fruit bat (Eonycteris spelaea). Electroencephalogram (EEG) and electromyogram (EMG) data were recorded from multiple individuals (>= 5) by telemetry over a 72-h period in a laboratory setting with light/dark cycles equivalent to those found in the wild. Our results show that over a 24-h period both species spent more time asleep than awake (mean 15 h), less than previous reported for Chiroptera (20 h). C sphinx spent significantly more of its non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) quotas during the light phase, while E. spelaea divided its sleep-wake architecture equally between both light and dark phases. Comparing the sleep patterns of the two species found that C. sphinx had significantly fewer NREM and REM episodes than E. spelaea but each episode lasted for a significantly longer period of time. Potential hypotheses to explain the differences in the sleep architecture of C. sphinx with E. spelaea, including risk of predation and social interaction are discussed. (C) 2010 Published by Elsevier B.V.

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp PCC 7002

Phyrobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phyrobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities. (C) 2009 Elsevier B.V. All rights reserved.

alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5 degrees C) in the dark but rapidly losses viability when exposed to chill in the light (100 mu mol photons m(-2) s(-1)). Preconditioning at a low temperature (15 degrees C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of alpha-tocopherol after exposure to chill-light stress. Mutants unable to synthesize alpha-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P-petE controlled the level of et-tocopherol and ACLT. We conclude that alpha-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of a-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Cultivation of the brown alga Sargassum horneri: sexual reproduction and seedling production in tank culture under reduced solar irradiance in ambient temperature

As a large conspicuous intertidal brown alga, individuals of Sargassum horneri can reach a length of more than 7 m with a fresh weight of 3 kg along the coasts of the Eastern China Sea. The biomass of this alga as a vital component in coastal water ecology has been well documented. In recent years, a steady disappearance of the algal biomass along the once densely populated coastal areas of the Eastern China Sea has drawn attention in China. Efforts have been made to reconstruct the subtidal algal flora or even to grow the alga by use of long-lines. As part of the efforts to establish an efficient technique for producing seedlings of S. horneri, in this investigation a series of culture experiments were carried out in indoor raceway and rectangular tanks under reduced solar irradiance at ambient temperature in 2007-2008. The investigation demonstrated that: (1) sexual reproduction of S. horneri could be accelerated in elevated temperature and light climates, at least 3 months earlier than in the wild; (2) eggs of S. horneri had the potential to be fertilized up to 48 h, much longer than that of known related species; (3) suspension and fixed culture methods were both effective in growing the seedlings to the long-line cultivation stage; and (4) the life cycle of S. horneri in culture could be shortened to 4.5 months, thus establishing this alga as an appropriate model for investigating sexual reproduction in dieocious species of this genus.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

Loss of allele diversity in introduced populations of the hermaphroditic bay scallop Argopecten irradians

The bay scallop Argopecten irradians is a hermaphroditic bivalve native to the Atlantic coast of the United States that was introduced to China for aquaculture production in 1982. It now supports a major aquaculture industry in China. Introduced species often start with limited genetic variability, which is problematic for the further selective breeding. Bay scallop aquaculture is exclusively hatchery based and as the initial introduction consisted of only 26 scallops, there have been concerns about inbreeding and inbreeding depression in cultured populations in China. In this study, eleven simple sequence repeat (SSR) markers were used to compare genetic variation in cultured populations from China with that in a natural population from the east coast of America. Although the difference in heterozygosity was small, the Chinese populations lost 9 of the 45 alleles (20%) found in the wild population. The reduced allele diversity suggests that the Chinese bay scallop populations experienced a bottleneck in genetic diversity that remains significant despite several recent introductions of new stocks aimed at expanding the gene pool. The loss of allele diversity may affect future efforts in selective breeding and domestication, and results of this study highlight the need for additional introductions, advanced breeding programs that minimize inbreeding and continued genetic monitoring. (c) 2007 Elsevier B.V. All rights reserved.

Effect of N fertilizer on the productivity and nutritive values of perennial grass mixtures in the alpine region of Qinghai-Tibetan Plateau, China

A study was conducted on grass mixtures that included smooth bromegrass (SB) + drooping wild ryegrass (DW), smooth bromegrass + Siberian wild ryegrass (SW) + crested wheatgrass (CW) and smooth bromegrass + Siberian wild ryegrass + drooping wild ryegrass + crested wheatgrass in the alpine region of Qinghai-Tibetan Plateau. The study was conducted from 1998 to 2000 to investigate the effects of N application rates and growing year on herbage dry matter (DM) yield and nutritive values. Herbage DM production increased linearly with N application rates. The effect of N application on DM yields was greater (P < 0.05) in the 2nd and 3rd production years than in the establishment year. Dry matter yields of SB + SW + CW and SB + SW + DW + CW can reach as high as 15 000 kg ha(-1) at 345 kg ha(-1) N rate in the 3rd growing year. With increased N application rates, crude protein (CP) contents and 48 h in sacco DM degradability of grasses increased (P < 0.05). No effect (P > 0.05) of N application was detected on organic matter (OM) and acid detergent fibre (ADF) concentration. It can be concluded that for increased biomass production in the alpine region of the Qinghai-Tibetan Plateau, a minimum of 345 kg N ha(-1) should be applied to grass stands in three split application of 115 kg N ha(-1), in early June, early July and late July