Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabrearn (Pagrus major). Mean (+/- S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0 +/- 5.4, 92.8 +/- 1.9, and 91.8 +/- 5.2%, respectively; for fresh sperm, they were 87.5 +/- 7.7, 95.8 +/- 2.4, and 93.8 +/- 4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P < 0.05), there was no effect (P > 0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8 +/- 5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0 +/- 7.2% had normal morphology, 20.6 +/- 3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4 +/- 4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased. (c) 2007 Elsevier Inc. All rights reserved.

Inbreeding depression for various traits in two cultured populations of the American bay scallop, Argopecten irradians irradians Lamarck (1819) introduced into China

This paper examines the effect of inbreeding level of population on the magnitude of inbreeding depression expressed by comparing them between two cultured populations (A and B) in the hermaphroditic animal of the bay scallop Argopecten irradians irradians. Population A is expected to have less genetic variations and higher inbreeding level due to longer cultured history (20 generations) and less "ancestral" individuals (26 individuals) than population B due to shorter cultured history (4 generations) and more "ancestral" individuals (406 individuals). Two groups within each population were produced, one using self-fertilization and one using mass-mating within the same population. Selfed offspring (AS and BS) from two populations both had lower fitness components than their mass-mated counterparts (AM and BM) and exhibited inbreeding depression for all examined traits, e.g. lower hatching, less viability and slower growth, indicating that inbreeding depression is a common feature in this animal. Fitness components in all traits of offspring from population A significantly differed those from population B and the magnitude of inbreeding depression for all traits in population A with higher inbreeding level was significantly smaller than that in population B with lower inbreeding level, indicating that both fitness components and magnitude of inbreeding depression were significantly affected by inbreeding level of populations and genetic load harbored in population A may be partially purged through inbreeding. Moreover, the magnitude of inbreeding depression in the two populations both varied among traits and life history stages. The present results support the partial-dominance hypothesis of inbreeding depression. (C) 2008 Elsevier B.V. All rights reserved.

Effect of long-term cryopreservation on physiological characteristics, antioxidant activities and lipid peroxidation of red seabream (Pagrus major) sperm

The aim of this study was to determine the effect of long-term cryopreservation on physiological characteristics, the antioxidant activities and lipid peroxidation of red seabream sperm which were respectively cryopreserved with 15% dimethylsulfoxide (Me2SO) for 1 month, 13 months, 26 months, 48 months and 73 months. The motility and fertility of post-thaw sperm decreased with the storage time going on. The highest motility (87.67 +/- 2.52%) was obtained in sperm cryopreserved for 1 month and the lowest (50.67 +/- 5.31%) was in sperm for 73 months. There were no significant differences (p < 0.05) in fertilization rates of sperm cryopreserved for 1 month (71.33 +/- 8.84%), 13 months (69.22 +/- 1.02%) and 26 months (60.33 +/- 2.33%); however, the sperm fertility decreased significantly for 48 months (47.22 +/- 3.89%) and 73 months (39.56 +/- 0.69%) storage. In addition, superoxide dismutase (SOD) activities of sperm were at a stable level for less than 26 months storage, then, decreased significantly after 48 months storage. Catalase (CAT) activities of sperm cryopreserved for 13 months, 26 months, 48 months and 73 months were significantly lower than that for 1 month. There were no significant differences in the malondialdehyde (MDA) level of sperm for less than 13 months storage. After 26 months storage, the concentration of MDA increased significantly, and the highest concentration (3.22 +/- 0.05 nmol/mgprot) was obtained in 73 months storage sperm. (C) 2010 Elsevier Inc. All rights reserved.

An efficient methodology for cryopreservation of spermatozoa of red seabream, Pagrus major, with 2-mL cryovials

The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2-mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 +/- 4.7% to 88.6 +/- 8.0%), fertilization rates (89.6 +/- 2.9 to 95.6 +/- 1.9%), and hatching rates (85.3 +/- 5.1% to 91.4 +/- 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.

Embryology of Swertia cincta (Gentianaceae) and Its Systematic Value

This paper reports the mega-, micro-sporogenesis and female-, male-gametogenesis of Swertia cincta for the first time, with the aim of discussing the systematic position of section Platynema and section Ophelia of Swertia. Anthers are tetrasporangiate. The development of anther walls conforms to the dicotyledonous type. The tapetum cells have dual origin and are similar to the glandular type. There are two middle layers. The endothecium and epidermis persist. Cytokinesis in the microsporrocyte meiosis is simultaneous type and the microscpore teads are tetrahedral. Pollen grains are 3-celled. The ovary is bicarpellum and unilocular. The placentation is of suparietal placentation with 12 series of ovules. The ovules. The ovule is unitegmic, tenuinucellar and ana-campylotropous, The embryo sac orignates from the single-archesporial cell. The one chalazal megaspore in lienar tetrad become the functional megasore. The development of embryo sac is of the polygonum type. Before fertilization, two polar nuclei fuse into one secondary nucleus. Three antipodal cells persisted and divided into 5-8 cells. A comparison between two sections indicates that section Plathnema is better treated as distinct section and is more advanced than section Ophelia according to the evolutionary trends of embryological characters.

Embryology of Swertia (Gentianaceae) relative to taxonomy

The embryological features of three species of Swertia (s.l.) - S. erythrosticta, S. franchetiana, and S. tetraptera were characterized, and the observations were used, together with previously gathered data on other species, to evaluate a recently proposed polyphyly, based on molecular data, of Swertia s.l. Comparisons of species within the genus showed that they have diversified embryologically, and there are significant between-species differences. Notable features that vary between species include the number of cell layers that form the anther locule wall, the construction of the wall of the mature anther, tapetum origin, the cell number in mature pollen grains, the structure of the fused margins of the two carpels, the ovule numbers in placental cross-sections, the shape of the mature embryo sac, the degree of ovule curvature, antipodal variation and the presence of a hypostase, and seed appendages. They share characters that are widely distributed in the tribe Gentianeae, such as a dicotyledonous type of anther wall formation, a glandular tapetum with uninucleate cells, simultaneous cytokinesis following the meiosis of the microsporocytes, tetrahedral microspore tetrads, superior, bicarpellary and unilocular ovaries, unitegmic and tenuinucellar ovules, Polygonum-type megagametophytes, progamous fertilization, nuclear endosperm, and Solanad-type embryogeny. The presence of variation in embryological characters amongst the species of Swertia s.l. strongly supports the view that Swertia s.l. is not a monophyletic group. Frasera is better separated from Swertia s.l. as an independent genus, and is only distantly related to Swertia s. s. judging from the numerous differences in embryology. Swertia tetraptera is very closely related to Halenia, as they show identical embryology. (C) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 383-400.

Defoliation, nitrogen, and competition: effects on plant growth and resource allocation of Cleistogenes squarrosa and Artemisia frigida

Two species, Artemisia frigida Willd. (C-3, semishrub, and dominant on overgrazed sites) and Cleistogenes squarrosa (Trin.) Keng (C-4, perennial bunchgrass, and dominant or codominant on moderately grazed sites) were studied to determine the effects of defoliation, nitrogen (N) availability, competition, and their interactions on growth, biomass, and N allocation in a greenhouse experiment. The main treatments were: two nitrogen levels (NO = 0 mg N pot(-1), N1 = 60 mg N pot(-1)), two defoliation intensities (removing 60% of total aboveground biomass and no defoliation), and three competitive replacement series (monocultures of each species and mixtures at 0.5:0.5). Our results were inconsistent with our hypothesis on the adaptive mechanisms of A. frigida regarding the interactive effects of herbivory, N, and competition in determining its dominant position on overgrazed sites. Cleistogenes squarrosa will be replaced by A. frigida on over-grazed sites, although C. squarrosa had higher tolerance to defoliation than did A. frigida. Total biomass and N yield and N-15 recovery of C. squarrosa in mixed culture were consistently lower than in monocultures, whereas those of A. frigida grown in mixtures were consistently higher than in monocultures, suggesting higher competitive ability of A. frigida. Our results suggest that interspecific competitive ability may be of equal or greater importance than herbivory tolerance in determining herbivore-induced species replacement in semi-arid Inner Mongolian steppe. In addition, the dominance of A. frigida on overgrazed sites has been attributed to its ability to shift plant-plant interactions through (lap colonization, root niche differentiation, and higher resistance to water stress.

Molecular evidence for the sister relationship of the eastern Asia-North American intercontinental species pair in the Podophyllum group (Berberidaceae)

The presumed pair relationships of intercontinental vicariad species in the Podophyllum group (Sinopodophyllum hexandrum vs. Podophyllum pelatum and Diphylleia grayi vs. D. cymosa) were recently, considered to be paraphyletic. In the present paper, the trnL-F and ITS gene sequences of the representatives were used to examine the sister relationships of these two vicariad species. A heuristic parsimony analysis based on the trnLF data identified Diphylleia as the basal clade of the other three genera, but provided poor resolution of their inter-relationships. High sequence divergence was found in the ITS data. ITS1 region, more variable but parsimonyuninformative. has no phylogenetic value, Sequence divergence of the ITS2 region provided abundant, phylogenetically informative variable characters. Analysis of ITS2 sequences confirmeda sister relationship between the presumable vicariad species, in spite of a low bootstrap support for Sinopodophyllum hexandrum vs. Podophyllum pelatum. The combined ITS2 and trnL-F data enforced a sister relationship between Sinopodophyllum hexandrum and Podophyllum pelatum with an elevated bootstrap support of 100%. Based on molecular phylogeny, the morphological evolution of this group was discussed. The self-pollination might have evolved from cross-fertilization two times in this group. The different pollination and seed dispersal systems of Sinopodophyllum hexandrum and Podophlyllum pelatum resulted from their adaptations to different ecological habitats. The divergence time of Sinopodophyllum hexandrum-Podophyllum pelatum is estimated to be 6.52+/-1.89 myr based on the ITS divergence. The divergence of this species pair predated or co-occurred with the recent uplift of the Himalayas 4-3 myr during the late Miocene and the formation of the alpine habitats. Sinopodophyllum hexandrum developed a host of specialized characters in its subsequent adaptation to the arid alpine surroundings. The present study confirmed the different patterns of species relationship between Asian-North American disjuncts. The isolation of plant elements between North America and eastern Asia must have been a gradual process, resulting in the different phylogenetic patterns and divergence times of the disjuncts.

Embryology of Crawfurdia delavayi (Gentianaceae) and its systematic value

The embryological characters of Crawfurdia delavayi Frabnch. are described and the systematic relationships of Crawfurdia discussed. Anthers are tetrasporangiate. The development of anther walls conforms to the Dicotyledonous type. The tapetum is of single origin. The development of the tapetum with uninucleate cells is of the glandular type. The tapetal cells on the connective side show radial elongation or periclinal division and intrude into the anther locule. The epidermis of anther walls persists and its cells become pillar and fibrous, and the endothecium degenerates. The ovary is bicarpellary and unilocular. The placentation is typically parietal with 8 rows of anatropous ovules. The development of embryo sac is of the polygonum type. Before fertilization, two polar nuclei fuse into a secondary nucleus. Three antipodal cells persist. Flowers are protandrous. Fertilization is porogamous. The development of the endosperm is of the nuclear type. The embryogeny corresponds to the solanad type physalis II variation. The embryological data indicate that it is better to separate Crawfurdia from Gentiana as an independent genus.

新疆东昆仑蛇绿岩岩石学、地球化学特征及其大地构造意义

The East Kunlun area of Xinjiang (briefly EKAX) is the western part of broadly speaking East Kunlun orogenic zone. The absence of geological data (especially ophiolites) on this area has constrained our recognition to its geology since many years. Fund by National 305 Item (96-915-06-03), this paper, by choosing the two ophiolite zones (Muztag and Southwestern Margin of Aqikekule Lake ophiolite zones) exposed at EKAX as the studied objects and by the analysis of thin section, electron probe, XRF, ICP-MS, SEM and Sm-Nd isotope, totally and sys ematically dealt with the field geological, petrological, minerological, petrochemical and geochemical characteristics (including trace, rare earth element and Sm-Nd isotope) and the tectonic setting indicated by them for each ophilite zone. Especially, this paper discussed the trace and rare earth element patterns for metamorphic peridotites, their implications and related them to the other components of ophiolite in order to totally disclose ophiolite origins. Besides, this paper also studied the petrological, geochemical and paleobiological characteristics for the cherts coexsisted with the Muztag ophiolite and the tectonic setting indicated by them. Based on these, the author discussed the tectonic evolution from Proterozoic to Permian for this area. For Muztag ophiolite, their field geological, petrological, minerological, petrochemical and geochemical characteristics show that: ① outcropped along the Muztag-Jingyuhu fault with west-to-east strike, the ophiolite is composed of such three components as metamorphic peridotites, cumulates and volcanic rocks; ② metamophic peridotites consist of such types as lherzolites, serpentinized lherzolites and serpentinites, only pyroxenites is seen of cumulates and volcanic rocks include basalts, basaltic andesites and andesites; ③ mineralogical data on this ophiolite suggest it formed in supra-subduction zone (SSZ)environment, and its mantle wedge is heterogeneous; ④ whole-rock TiO_2 and Al_2O_3 of metamorphic peridotites indicate their original environment with the MORB and SSZ characteristics; ⑤ metamorphic peridotites have depleted LREE and flat REE patterns and volcanic rocks have enriched LREE patterns; ⑥ trace element characteristics of metamorphic peridotites imply that they had undergone Nb and Ta enrichment event after partial melting; ⑦ trace element characteristics of volcanic rocks and their tectonic diagrams show they are formed in the spreading and developed island arc environment with back-arc basin, such as rifted island arc, which is supported by the ε_(Nd)(t) -2.11～+3.44. In summary, the above evidence implies that Muztag ophiolite is formed in SSZ environment, where heterogeneous mantle wedge was metasomatised by the silica-enriched melt from subducted sediments and/or oceanic crust, which makes the mantle wedge enriched again, and this enriched mantle wedge later partially melted to form the volcanic rocks. For Southwestern Margin of Aqikekule Lake ophiolite, their field geological, petrological, minerological, petrochemical and geochemical characteristics show that: ① it outcropped as tectonic slices along the near west-to-east strike Kunzhong fault and is composed of metamorphic perodotties, cumulates and volcanic rocks, in which, chromites are distributed in the upper part of metamorphic peridotites as pods, or in the lower part of cumulates as near-strata; ② metamorphic peridotites include serpentinites, chromite-bearing serpentinites, thlorite-epidote schists and chromitites, of which, chromitites have nodular and orbicular structure, and cumulates include pyroxenits, serpentinites, chromite-bearing serpentinites, chromites and metamorphically mafic rocks and only basalts are seen in volcanic rocks; ③ Cr# of chromites suggest that they formed in the SSZ and Al_2O_3 and TiO_2 of metamorphic peridotites also suggest SSZ environment; ④metamorphic peridotites have V type and enriched LREE patterns, cumulates have from strongly depleted LREE, flat REE to enriched LREE patterns with universally striking positive Eu anomalies and basalts show flat REE or slight enriched LREE patterns with no Eu anomalies; ⑤ trace element and Sm-Nd isotope characteristics of metamorphic peridotites imply their strikingly heterogeneous mantle character(ε_(Nd)(t)+4.39～+26.20) and later Nb, Ta fertilization; ⑥ trace element characteristics of basalts and their tectonic diagrams show they probably formed in the rifted island arc or back-arc basin enviromnent. In summary, the above evidence shows that this ophiolite formed in the SSZ environment and melts from subudcted plate are joined during its formation. Rare earth element, whole-rock and sedimentary characteristics of cherts with the Muztag ophiolite show that they formed in the continental margin environment with developed back-arc basin, and radiolarias in the cherts indicate that the upper age of Muztag ophiolite is early carboniferous. Based on the accreted wedge models of Professor Li Jiliang for Kunlunshan Mountain and combined with study on the two typical ophiolite profiles of EKAX, the author discussed the tectonic evolution of EKAX from Proterzoic to Permian.

贵阳雨水无机氮沉降的氮、氧同位素特征

雨水中氮沉降主要以铵盐(NH+4)和硝酸盐(NO-3)形式存在,这与地表生态氮循环和酸雨等环境问题直接相连.我们测定了贵阳地区雨水中的NH+4和NO-3的氮氧同位素值,讨论了氮素形态分布及其同位素组成特征,探讨了雨水中溶解无机氮的成因.雨水中的NH+4和NO-3平均值分别为0.81和o.51mg N/L;铵盐的δ15N平均值为-4.7‰,较硝酸盐的δ15N平均值负,雨水中硝酸盐δ18O值为25.2‰～40.1‰,平均值为34.2±4.3‰,季节性差别不显著.

碳氮稳定同位素指示苔藓生境特征以及树冠对大气氮沉降的吸收

对贵阳地区一生境内不同生长条件（石生苔藓、无树冠、不同树冠类型以及同种树冠不同树冠厚度）下的细叶小羽藓（Haplocladium microphyllum（Hedw．）Broth）碳氮同位素进行了研究。不同生境的苔藓铲δ^13C和δ^15N值具有相似的变化趋势和明显的相关关系。石生苔藓δ^13C和δ^15N值比土生苔藓偏高指示了其相对干燥和受到干沉降控制的生境；树冠下方光照条件差、湿度高、缺少干沉降氮的输入是苔藓δ^13C和δ^15N值偏低的主要原因；而树冠类型、叶片形态、叶面性质造成树冠对大气沉降氮吸收能力的不同是三种树冠下方苔藓氮同位素组成差异的潜在因素。此外，同一树冠（桂花树Osmanthus fiagrans Lour．）下方苔藓的氮含量和碳氮同位素平均组成与树冠厚度呈线性关系。尤其是δ^15N的响应很好地反映了吸收过程中树冠厚度对大气沉降氮源同位素组成的控制作用。结果表明。苔藓δ^13C值能很好地指示苔藓的生境差异和生理响应（如光合作用能力和氮需求等）；而δ^15N值在示踪氮源和反映不同树冠吸收过程的同位素分馏效应方面更具有优势，二者的相关关系是环境因素相互作用的重要线索。此外，细叶小羽藓Haplocladium microphyllum（Hedw．）Broth具有高效利用大气沉降氮和分布广泛等特点，可进一步应用于该地区大气氮沉降的指示或监测研究.

贵阳地区主要大气氮源的沉降机制与分布：基于石生苔藓氮含量和氮同位素的证据

对贵阳市区到农村地区4个方向的175个石生苔藓样品氮含量和氮同位素（δ^15N）进行了分析。苔藓δ^15N表明贵阳地区大气氮沉降以铵沉降为主，主要来源为城市废水氨释放以及农村地区的农业氨挥发。苔藓氮含量从市区往外随距离呈指数降低，而δ^15N随距离对数升高，揭示了城市来源的铵沉降随距离指数降低，该变化模式与其他点源氨的沉降分布以及点源氨附近苔藓氮含量的变化规律相同。因此，贵阳市区可以看成整个研究区的一个点状氨源。不同方向苔藓氮含量和δ^15N随距离的变化梯度存在差异，表明城市铵向周边不同方向的扩散迁移具有非均一性，其原因可能与地形条件、植被状况、风向、城市化程度等有关。苔藓δ^15N平均值在15km以外没有明显差异，反映了农业面源氨的影响。此外，运用极限法计算，苔藓氮含量随距离的变化关系（y=1．5e^-0.13x＋1．26）表明城市铵的影响范围总体小于41km，而苔藓δ^15N的变化（y=2．541nz-12．23）反映了在17．2km以内的地区，大气氮沉降以城市铵为主，17．2km以外的地区主要受农业铵沉降的控制。本研究为城市氮沉降的变化提供了新的证据，促进了苔藓氮含量和δ^15N方法研究大气氮沉降从定性向定量的发展。

现代湖泊沉积有机氮同位素与废水氮输入演化

有机氮含量和C:N比的变化可能是有机质降解和富营养化水体升高的共同结果。红枫湖接受较多的工业和农业废水，其NO3-的δ15N平均值分别为+15.68‰和+8.11‰。废水氮的输入改变了湖泊DIN的氮同位素组成。由于水生生物在同化吸收水体DIN的时候倾向于优先吸收14N，因此产生的生物体δ15N值低于湖水DIN的δ15N值，但这种变化较工业废水输入引起的δ15N值升高意义可能较小。故该湖泊现代沉积有机物δ15N值的变化可以认为主要是工业和农业废水排放量变化的结果。从上述沉积有机物δ15N值的变化可知，红枫湖

水样硝酸盐氮同位素分析预处理方法探讨

本文采用离子交换色层法和扩散法进行水样硝酸盐δ15N分析预处理。采用的Dowex® 1-X8树脂对50mL浓度为500、1000、2000mg/L的硝酸盐溶液的吸附率均>99.9%，30mL 2mol/L KCl溶液对树脂柱长5cm的色层柱洗脱率>95%。该方法适用于在野外及时对样品进行初步处理，并可保存较长的时间而不影响分析结果。50℃对硝酸盐扩散10d，回收率>95%，且不会引起氮同位素分馏。