Genetic diversity and evolutionary history of the Schizothorax species complex in the Lancang River (upper Mekong)

The genus Schizothorax (Cyprinidae), one of the most diverse genera of ichthyofauna of the Qinghai‐Tibetan Plateau (QTP), is a good candidate for investigating patterns of genetic variation and evolutionary mechanisms. In this study, sequences from the mitochondrial control region, the cytochrome b gene, and two nuclear genes were used to re‐examine the genetic diversity and investigate the evolutionary history of the Schizothorax species complex inhabiting the Lancang River. Three maternal clades were detected in the Schizothorax species complex, but frequent nuclear allele sharing also occurred among the three maternal clades. A discrepancy between topologies of mitochondrial and nuclear loci might result from introgression or/and incomplete lineage sorting. The divergence of the clades of the Schizothorax species complex was closely related to the Late Pliocene and Early Pleistocene orogenesis of the QTP and Southwest Mountains of China. Demographic analyses indicated that the species complex subsequently persisted in situ with stable populations during Pleistocene glacial cycling, which suggested that Pleistocene climate changes did not exert a remarkable influence on the species complex. Our study provides a comprehensive analysis of the genetic diversity and evolutionary history of the Schizothorax species complex in the Lancang River.     

四川裂腹鱼脑型芳香化酶Cyp19b基因组织分布及温度对其表达水平的影响

为研究脑型芳香化酶基因Cyp19b在四川裂腹鱼(Schizothorax kozlovi)早期性别分化中的作用,采用RACE方法从四川裂腹鱼脑中扩增得到该基因c DNA全长序列,并应用荧光定量RT-PCR技术测定该基因m RNA的相对表达量,探讨该基因在不同规格四川裂腹鱼鳃、脑、心、肝、脾、肾、肌肉、精巢、卵巢组织中的表达差异,以及温度对其早期仔鱼阶段该基因表达的影响。四川裂腹鱼Cyp19b基因的c DNA全长序列共3 021 bp,共编码507个氨基酸,属于脑型芳香化酶基因;四川裂腹鱼Cyp19b基因编码的氨基酸序列与其他鱼类脑型芳香化酶基因编码的氨基酸序列同源性可达70%以上,而与性腺型芳香化酶基因编码的氨基酸序列同源性较低,为64%左右;四川裂腹鱼Cyp19b基因仅在脑组织中表达,具有比较高的组织特异性,且随着个体的增长,逐渐呈现显著的雌雄差异;对不同温度处理12日龄仔鱼6 d后,低温(10℃和14℃)能显著促进Cyp19b基因表达量的升高,但高温(26℃)却对其表达量无显著影响。由此推测,脑型芳香化酶基因Cyp19b可能在低温导致四川裂腹鱼雌性化过程中发挥着重要作用。     

Floral dimorphism, pollination, and self-fertilization in gynodioecious GERANIUM RICHARDSONII (Geraniaceae)

The selective maintenance of gynodioecy depends on the relative fitness of the male-sterile (female) and hermaphroditic morphs. Females may compensate for their loss of male fitness by reallocating resources from male function (pollen production and pollinator attraction) to female function (seeds and fruits), thus increasing seed production. Females may also benefit from their inability to self-fertilize if selfing and inbreeding depression reduce seed quality in hermaphrodites. We investigated how differences in floral resource allocation (flower size) between female and hermaphroditic plants affect two measures of female reproductive success, pollinator visitation and pollen receipt, in gynodioecious populations of Geranium richardsonii in Colorado. Using emasculation treatments in natural populations, we further examined whether selfing by autogamy and geitonogamy comprises a significant proportion of pollen receipt by hermaphrodites. Flowers of female plants are significantly smaller than those of hermaphrodites. The reduction in allocation to pollinator-attracting structures (petals) is correlated with a significant reduction in pollinator visitation to female flowers in artificial arrays. The reduction in attractiveness is further manifested in significantly less pollen being deposited on the stigmas of female flowers in natural populations. Autogamy is rare in these protandrous flowers, and geitonogamy accounts for most of the difference in pollen receipt between hermaphrodites and females. Female success at receiving pollen was negatively frequency dependent on the relative frequency of females in populations. Thus, two of the prerequisites for the maintenance of females in gynodioecious populations, differences in resource allocation between floral morphs and high selfing rates in hermaphrodites, occur in G. richardsonii.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-adrenergic signals, leading to a rise in intracellular 3′,5′-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 ± 0.08. A change in assay temperature from a euthermic value (37 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) K_m values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH₄Cl was reduced. However, temperature change had little or no effect on K_m values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 °C and activation energies that were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the rabbit enzyme showed a breakpoint at 17 °C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 ± 0.18 at 37 °C to 2.19 ± 0.07 at 5 °C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with ³²P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 °C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. Accepted: 10 July 1998     

饲料蛋白质和能量水平对齐口裂腹鱼生长、体组成和蛋白质利用的影响

齐口裂腹鱼（Schizothorax prenanti Tchang）隶属鲤科、裂腹鱼亚科,俗称“雅鱼”,为长江上游重要经济鱼类,也是川西高原特有的冷水性鱼类。其肉质细嫩,味道鲜美,是名贵野生经济鱼类。近年来由于过度捕捞、环境污染等因素的影响,齐口裂腹鱼种群数量不断下降,分布区域日趋缩小,而随着人们生活水平的不断提高,对齐口裂腹鱼等名优水产品的需求却在不断增加。为保护和持续利用这一野生动物资源,     

齐口裂腹鱼血细胞的发生

为了探讨齐口裂腹鱼(Schizothorax prenanti)血细胞发生发育的模式,采用Wright’s、碘酸雪夫氏(PAS)和苏丹黑B(SBB)染色方法对齐口裂腹鱼的头肾、中肾、脾和肝组织印片进行染色,了解其发生的具体组织和发育的一般过程。结果表明:齐口裂腹鱼血细胞可分为红细胞系、淋巴细胞系、单核细胞系、粒细胞系和其他细胞,且这些细胞系的发育均经过3个阶段,即原始阶段、幼稚阶段、成熟阶段。通过观察和统计不同阶段各种血细胞的形态、大小、比例及染色特征,发现原始阶段的血细胞体积较大,其中原始单核细胞最大,大小为(18.90±1.59)μm×(16.32±0.70)μm,在4种组织中原始阶段的红细胞和单核细胞在头肾中所占比例最大,分别为0.64%和0.59%,原粒细胞和原淋巴细胞分别在中肾和脾中比例最大,在肝中少量存在,此外在各组织印片中均发现血栓细胞的存在,在肝中发现巨噬细胞。因此头肾、中肾和脾是齐口裂腹鱼的主要造血组织,少量细胞在肝中产生。粒细胞发育过程中,除原始粒细胞PAS染色阴性外,其他阶段均呈阳性,且阳性随不断成熟逐渐增强;单核细胞从原单核细胞到成熟的单核细胞,PAS阳性逐渐增强;而SBB染色发现,粒细胞、淋巴细胞及单核细胞均呈阳性,未成熟的细胞染色程度不一致,成熟的细胞阳性染色最为强烈。在红细胞系发育过程中经历了细胞由大变小再变大的过程,而粒细胞和淋巴细胞发育过程仅出现由大变小的过程,在红细胞的发育过程中还出现了染色质固缩和血红蛋白增加。     

黄芪多糖对齐口裂腹鱼生长、体组成和免疫指标的影响

试验研究黄芪多糖对齐口裂腹鱼生长性能、体组成及免疫指标的影响。以450尾健康的齐口裂腹鱼[体重(6.98±0.43)g;体长(9.11±0.25)cm]为试验对象,随机分为5组(C1、C2、C3、C4、C5),每组3个重复,每重复30尾试验鱼。C1、C2、C3、C4、C5组分别投喂在等氮等能(蛋白质含量38.29%,能量15.73 mJ/kg)的基础料中分别添加0、0.02、0.04、0.06、0.08%的黄芪多糖制成5种试验饲料,养殖齐口裂腹鱼50d。结果表明:饲料中未添加黄芪多糖组的增重率(WGR)、特定生长率(SGR)、饲料蛋白效率(PER)均显著低于黄芪多糖添加组(P<0.05),而饵料系数(FCR)则显著高于黄芪多糖添加组(P<0.05)。当黄芪多糖添加水平为0.04%时,试验鱼的WGR、SGR、PER均达到最大(分别为110.31%、1.86%/d和182.07%),FCR最低(1.44),与其他各组差异显著(P<0.05);以WGR、SGR、PER、FCR为指标,利用直线和抛物线回归分析表明,齐口裂腹鱼生长性能最佳时黄芪多糖添加水平为0.045%—0.074%;对齐口裂腹鱼机体组成分析表明,黄芪多糖对鱼体粗灰分和水分影响不显著(P>0.05),黄芪多糖添加水平为0.06%时机体粗蛋白最高,但与黄芪多糖添加水平为0.04%时无明显差异(P>0.05);粗脂肪含量在黄芪多糖添加水平为0.04%时最高,与其他各组差异显著(P<0.05);未添加黄芪多糖组血清免疫酶活性显著低于黄芪多糖添加组,齐口裂腹鱼血清免疫酶活性在一定范围内随黄芪多糖的增加而增强。黄芪多糖添加水平为0.04%时,碱性磷酸酶(ALP)活性最高;酸性磷酸酶(ACP)活性在黄芪多糖添加水平0.06%时最大;黄芪多糖添加水平应在0.06%—0.08%时,溶菌酶(LSZ)、超氧化歧化酶(SOD活性趋于稳定。这说明黄芪多对齐口裂腹鱼的生长和免疫力有明显的促进作用。综合考虑齐口裂腹鱼生长性能和免疫能力最佳时黄芪多糖添加水平为0.04%—0.074%。     

基于mtDNA Cyt b序列分析齐口裂腹鱼群体遗传多样性

研究测定了长江上游4个齐口裂腹鱼(Schizothorax prenanti)野生群体(重庆巫溪、重庆城口、四川雅安、四川阿坝)共104个个体的线粒体Cyt b基因部分序列, 以探讨齐口裂腹鱼野生群体的遗传多样性和遗传结构。结果表明: 在104个个体Cyt b序列中共检测到43个多态性位点, 25个单倍型。4个齐口裂腹鱼群体的单倍型多样性介于0.704—0.884, 核苷酸多样性介于0.007—0.012。群体间Kimura双参数遗传距离介于0.008—0.017, 其中四川雅安群体与四川阿坝群体间遗传距离最近, 基因交流频繁。重庆城口群体与四川雅安群体间遗传距离最远, 基因交流受阻。AMOVA分析表明, 齐口裂腹鱼的遗传分化主要来自群体内部, 且组群间、组群内群体间和群体内存在显著的遗传分化。中性检验得到Tajima’s D和 Fu’s F_s的值不显著, 且歧点分布图呈多峰, 表明长江上游4个齐口裂腹鱼野生群体未经历过种群扩张。研究旨为齐口裂腹鱼野生资源保护提供必要参考意见, 同时为齐口裂腹鱼种质资源合理开发和利用提供理论依据。     

齐口裂腹鱼脑内5-羟色胺免疫组织化学的定位观察

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齐口裂腹鱼耳石早期生长发育与日轮特征研究

文章研究了在实验室条件下齐口裂腹鱼仔稚鱼耳石早期形态发育与生长特点、第一轮纹出现时间和轮纹沉积规律。结果表明: 在13.5-17.2℃孵化条件下,微耳石和矢耳石在出膜前形成,而星耳石于出膜后第12天出现。在仔稚鱼生长过程中,微耳石由近圆形发育成贻贝形,矢耳石经历近圆形、锲形后发育为箭矢状,星耳石形状由近圆形发育为星芒状。微耳石的前区、背区和腹区及矢耳石的背区和腹区生长呈幂函数关系,而微耳石的后区、矢耳石前区和后区生长以及两对耳石的前后区半径之和与全长均呈线性相关。在(18.50.5)℃和(15.61.1)℃条件下,50%矢耳石样本第一轮纹均在出膜后第 2 天形成(分别为出膜后18h和19h),以后每天形成一轮。微耳石和矢耳石轮纹数均与日龄呈线性相关,方程斜率均与1差异不显著(P0.05),表明两对耳石的轮纹沉积均为日周期性。这些结果为研究齐口裂腹鱼野生种群繁殖期和早期生活史特征等生态学问题提供了重要依据。