高温对田间小菜蛾DNA损伤及4种抗药性相关酶系活性的抑制

研究了高温对福州上街菜田小菜蛾成虫4种抗药性相关酶系活性的影响。与饲养在25℃下的小菜蛾相比,33．5~C或40℃处理72h导致小菜蛾基因组DNA出现DNA凋亡特征梯度化条带。33~C饲养小菜蛾4、8、12或24h对小菜蛾成虫乙酰胆碱酯酶（AChE）和羧酸酯酶（CarE）活性无显著影响,但33℃饲养小菜蛾12或24h导致小菜蛾成虫谷胱甘肽s转移酶（GSTs）酶活性和细胞色素P450含量显著下降。36℃、24h可导致AChE活性显著下降,36℃、12h和24h可导致CarE活性显著下降,36℃、4h,8h,12h和24h可导致GST活性和细胞色素P450含量显著下降。总体上,高温对CarE、GSTs和细胞色素P450的抑制作用大于对AChE的影响,此外,3CC对AChE、CarE、GSTs酶活性和细胞色素P450含量的抑制作用大于33℃的抑制作用。     

小菜蛾有机磷护性季节性变化及毒理机制研究

1998～ 2 0 0 3年期间的田间监测结果显示 ,福州地区小菜蛾 Plutella xylostella对甲胺磷和敌敌畏的抗性水平呈季节性变化 ,每年春秋季的抗性水平显著高于夏季 7月期间的抗性水平。与 2 5℃ 2 d的饲养条件相比 ,35℃ 2 d和 5℃ 30 d可使小菜蛾羧酸酯酶 (Car E)和谷胱甘肽 - S-转移酶 (GST)活力显著抑制 ,35℃ 2 d还可使小菜蛾乙酰胆碱酯酶 (ACh E)活力显著抑制 ,但 5℃4 d对小菜蛾 ACh E、Car E和 GST活力影响差异不显著。当反应温度均为 2 5℃时 ,在 35℃ 2 d、2 5℃ 2 d和 5℃ 30 d下饲养的小菜蛾 ACh E对甲胺磷和克百威的敏感性 (K i值 )没有明显变化 ,但酶与杀虫剂在 37℃的反应温度下 ,甲胺磷、敌敌畏和克百威对在 2 5℃下饲养的小菜蛾 ACh E的抑制作用显著增高。上述结果表明环境温度会影响小菜蛾的解毒代谢及杀虫剂对 ACh E的抑制作用。探讨了环境温度的季节性变化对田间小菜蛾有机磷抗性水平影响的毒理学机制。     

小菜蛾有机磷抗性季节性变化及毒理机制研究

1998～ 2 0 0 3年期间的田间监测结果显示 ,福州地区小菜蛾 Plutella xylostella对甲胺磷和敌敌畏的抗性水平呈季节性变化 ,每年春秋季的抗性水平显著高于夏季 7月期间的抗性水平。与 2 5℃ 2 d的饲养条件相比 ,35℃ 2 d和 5℃ 30 d可使小菜蛾羧酸酯酶 (Car E)和谷胱甘肽 - S-转移酶 (GST)活力显著抑制 ,35℃ 2 d还可使小菜蛾乙酰胆碱酯酶 (ACh E)活力显著抑制 ,但 5℃4 d对小菜蛾 ACh E、Car E和 GST活力影响差异不显著。当反应温度均为 2 5℃时 ,在 35℃ 2 d、2 5℃ 2 d和 5℃ 30 d下饲养的小菜蛾 ACh E对甲胺磷和克百威的敏感性 (K i值 )没有明显变化 ,但酶与杀虫剂在 37℃的反应温度下 ,甲胺磷、敌敌畏和克百威对在 2 5℃下饲养的小菜蛾 ACh E的抑制作用显著增高。上述结果表明环境温度会影响小菜蛾的解毒代谢及杀虫剂对 ACh E的抑制作用。探讨了环境温度的季节性变化对田间小菜蛾有机磷抗性水平影响的毒理学机制。     

四种农药对小菜蛾体温的影响

【目的】本研究旨在为阐明小菜蛾Plutellaxylostella体温在其防治中的应用价值提供资料。【方法】在不同人工气候箱内温度(环境温度)下,测定小菜蛾2, 3和4龄幼虫的体温,建立各龄幼虫体温(y)与环境温度(x)的关系方程;同时测定了不同环境温度下不同浓度阿维菌素、毒死蜱、氟虫腈和高效氯氰菊酯分别处理后小菜蛾3龄幼虫在不同处理时间的体温。【结果】小菜蛾2, 3和 4龄幼虫体温(y)与环境温度(x)关系方程分别为y＝0.95x+1.19(r=0.9463), y＝0.95x+1.18(r=0.9988),以及y＝0.93x+1.45(r=0.9989),等温点分别为22.16℃,21.40℃和21.41℃。在气候箱温度设定为15℃或40℃时,4种农药都对小菜蛾3龄幼虫体温无影响;而其他温度条件下,农药处理都可能改变小菜蛾3龄幼虫体温。对于阿维菌素,25℃下 2, 4和8 mg/L处理12 h,2和4 mg/L处理24 h, 0.5, 2, 4和8 mg/L处理36 h以及0.5, 1, 2和8 mg/L处理48 h时3龄幼虫体温均显著高于对照, 8 mg/L阿维菌素处理24 h时3龄幼虫体温显著低于对照;30℃下0.5 mg/L处理24 h及1 mg/L处理36 h 3龄幼虫体温显著低于对照,1 mg/L处理48 h和各浓度处理60 h时3龄幼虫体温均显著高于对照;35℃下只有1和8 mg/L处理48 h时3龄幼虫体温显著低于对照。对于毒死蜱,20℃下50, 200和800 mg/L处理24 h, 100, 400和800 mg/L处理36 h时3龄幼虫体温都显著低于对照;25℃下100和200 mg/L处理12h, 800 mg/L处理24 h, 100, 200和800 mg/L处理60 h时3龄幼虫体温均显著低于对照,而50, 100, 200和400 mg/L处理24 h, 100和200 mg/L处理36 h及100和400 mg/L处理48 h时3龄幼虫体温均显著高于对照;30℃下只有800 mg/L处理24 h时3龄幼虫体温显著低于对照,50, 100, 200和800 mg/L处理60 h时3龄幼虫体温显著高于对照。对于氟虫腈,20℃下只有0.5 mg/L处理36 h时3龄幼虫体温显著低于对照;25℃下 4 mg/L处理12 h和各浓度处理60 h时3龄幼虫体温显著低于对照,0.5 mg/L处理24 h以及0.25, 1和2mg/L处理48 h时3龄幼虫体温均显著高于对照;30℃下 0.25和0.5 mg/L处理12 h, 0.25和2 mg/L处理24h, 4 mg/L处理48 h以及2 mg/L处理60 h时3龄幼虫体温显著低于对照;35℃下只有0.25和0.5 mg/L处理60 h时3龄幼虫体温显著高于对照。对于高效氯氰菊酯,20℃下2和8 g/L处理36 h, 4和8 g/L处理48 h时3龄幼虫体温显著高于对照;25℃下 2, 4和8 g/L处理12 h时3龄幼虫体温均显著低于对照, 0.5, 4和8g/L处理24 h, 1, 4和 8 g/L处理36 h以及1, 2和4 g/L处理60 h时3龄幼虫体温均显著高于对照;30℃下 0.5和1 g/L浓度处理12 h, 0.5, 1, 4和8 g/L处理24 h以及1, 2和8 g/L处理60 h时3龄幼虫体温都显著低于对照。【结论】小菜蛾幼虫自律性体温调节能力低;阿维菌素、毒死蜱、氟虫腈或高效氯氰菊酯处理影响小菜蛾3龄幼虫的体温,影响形式随农药种类和浓度,环境温度及处理时间不同而不同。本研究拓宽了农药毒理学及害虫防治研究内容。     

高温对二化螟实验种群生长、存活和繁殖的影响

以2 7℃和30℃恒温为对照,研究了平均温度在2 7～2 9.2℃内的5种变温条件下(每天2 0 h的适温2 7℃和12 :0 0～16 :0 0的高温处理,分别为日最高气温2 7℃、30℃、33℃、36℃、4 0℃)二化螟实验种群的发育历期、存活率和繁殖力的影响,组建了特定年龄生命表和繁殖力生命表。结果表明高温可导致二化螟种群的发育速率延缓、存活率和生殖力下降,主要表现在幼虫历期随日最高温度的升高而延长,36℃时延至12 d以上;在日最高气温高于33℃时,其存活率从2 7℃时的30 .3%降至36℃时的13.1%和4 0℃时的5 .5 % ,繁殖力也从30℃时每雌产卵量14 8.7粒减至36℃时的70 .5粒。因此,夏季异常高温可能是二化螟田间种群数量增长的一个重要抑制因子     

高温对抗性和敏感小菜蛾生命表参数及后代杀虫剂敏感性的影响

为研究高温对抗性和敏感小菜蛾Plutella xylostella生物学适合度及抗药性进化的影响,本研究测定了温度对杀虫剂抗性和敏感小菜蛾实验种群生命表参数及后代对阿维菌素敏感性的影响。 结果表明：与25℃相比,33.5℃条件下抗性和敏感种群的平均世代历期（D）、净生殖率（R₀）、内禀增长率（r_m）、周限增长率（λ）值、总产卵量、雌蛾平均寿命、日产卵量和孵化率均显著下降。 在25℃下,抗性和敏感种群的r_m,λ,D和日产卵量无显著差异,但抗性种群的R₀略低于敏感种群（为敏感种群的87%）。 在33.5℃下,抗性种群的r_m和R₀显著低于敏感种群（分别为敏感种群的75%和64%）,抗性种群雌成虫寿命、总产卵量和孵化率显著也低于敏感种群。 抗性种群在33.5℃下饲养一代后,后代对阿维菌素的抗性水平大幅下降,而相同处理下敏感种群对阿维菌素的抗性水平没有显著下降。 上述结果表明,无论在常温还是高温条件下,抗性种群生物学适合度均低于敏感种群,尤其是高温下抗性种群适合度远低于敏感种群。 抗性种群经高温饲养一代后对阿维菌素的抗性水平大幅下降可能与高温下抗性种群适合度显著较低有关。     

黄喉拟水龟对冬眠期间急性高温胁迫的氧化应激响应

全球气候变暖加剧,暖冬现象出现的频率及强度不断增加,外温动物冬眠期间将面临更加频繁的高温胁迫。但目前,高温胁迫对爬行动物冬眠期间氧化应激以及抗氧化防御系统影响的研究还较为缺乏。本研究以黄喉拟水龟(Mauremys mutica)南北两个种群的一龄幼龟为研究对象,探究了冬眠期间(6℃)幼龟受高温胁迫(25℃)后的脂质过氧化损伤标志物丙二醛(MDA)含量,以及抗氧化防御和修复系统的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)的活性变化。结果显示,南种群在经历高温胁迫后肝脏MDA含量高于北种群,且放回6℃冬眠条件恢复24 h后仍在升高;北种群肌肉MDA含量受高温胁迫后上升,但在冬眠温度下恢复24 h后即降至对照水平。两种群SOD活力受高温刺激影响不显著。同时,黄喉拟水龟南种群肝脏中CAT的活力在高温刺激2 h后达到最高,并且各处理组间南种群CAT活力均高于北种群;北种群幼龟肝脏GSH-Px活力在高温刺激12 h达到最高。综上,本研究表明,高温胁迫对冬眠期间黄喉拟水龟机体造成一定的氧化损伤,南方种群可能更易受到冬眠期间温度波动的影响,但南方种群机体具有更强的抗氧化酶活力以应对高温胁迫的威胁。     

高温胁迫对葡萄幼树叶绿素荧光特性和抗氧化酶活性的影响

为了探讨短期和长期高温处理后葡萄生理应答反应,本研究在人工气候室环境中模拟夏季高温发生时间段(10:00~16:00)对一年生盆栽葡萄‘夏黑’进行25°C、35°C、45°C温度处理,测定处理6 h时(短期)和150 h时(长期)功能叶片的叶绿素荧光动力学及抗氧化酶活性。结果显示:在35°C处理6 h时‘夏黑’叶片ΨEo、ΦEo、ETo/RC显著上升;45°C处理6 h时‘夏黑’各荧光参数表现出显著性差异,而在150 h时主要荧光参数恢复至原初水平;高温处理下‘夏黑’的叶绿素含量和SOD酶活增长趋势较不明显;35°C/45°C处理均导致抗氧化酶活性POD和CAT以及过氧化物产物MDA(丙二醇)表现出较强的增长趋势。综上可知,短期高温处理(6 h)‘夏黑’叶片PSII活性遭到破坏,但是长期高温处理(150 h)‘夏黑’的PSII活性得到恢复,推测‘夏黑’有较强高温逆境适应能力;高温逆境打破‘夏黑’氧化还原平衡,MDA含量增加。这些结果对于了解葡萄在高温胁迫下的耐受能力具有参考价值。     

不同地理种群小菜蛾对甲胺磷的抗药性

采用浸叶法研究了福州建新镇、福建农林大学校园内、闽侯县上街镇和闽侯县甘蔗镇等地田间小菜蛾种群对甲胺磷的抗性水平及抗性机制。结果表明,与敏感小菜蛾相比,上述4个田间小菜蛾种群已对甲胺磷产生了34-82倍的抗性水平。敏感小菜蛾乙酰胆碱酯酶(AChE)的Ki值是田间抗性小菜蛾AChE的Ki值的17.3-39.1倍。不同小菜蛾种群对甲胺磷的抗性水平也存在着差异,小菜蛾种群AChE对甲胺磷的敏感性大小与其对甲胺磷的抗性水平高低显著相关。     

滇中菜区小菜蛾种群消长动态及其影响因子

【目的】掌握区域小菜蛾Plutella xylostella(L.)田间种群发生消长规律及温度、降雨和种植制度模式对种群的影响。【方法】2009—2014年,采用性诱剂监测和田间系统调查方法连续6年对滇中菜区——通海县小菜蛾田间种群数量开展成虫和幼虫田间种群数量的研究,分析当地月平均气温、月降雨量和种植模式因素对小菜蛾种群数量的相关性。【结果】(1)通海小菜蛾成虫种群消长年度间波动差异明显,全年呈现1个大的虫峰期为2—11月份,年度间出现小的2~3个虫峰。2012年种群数量较高,高峰期提前到4月份,与2013年高峰期的7—8月份差异明显,2010年和2011年田间种群数量是历年较少年份,种群高峰期的虫量少于百头/盆(卡)/月;(2)小菜蛾幼虫种群全年有2~3个危害峰期,3—6月是发生主峰期,9—11月是弱峰期;与成虫高峰期比较,幼虫田间种群,高峰期推迟20~30 d左右,全年的波动消长情况基本一致;(3)小菜蛾成虫与温度显著相关r=0.442,与降雨量成正相关r=0.283,但相关程度弱;幼虫种群与温度显著相关r=0.530,相关程度显著,与降雨没有相关性r=0.125,相关程度微弱;幼虫种群与成虫有相关性r=0.253,但相关程度微弱。【结论】滇中菜区常年成片种植十字花科蔬菜,一年4造、周年复种的种植制度对小菜蛾的种群发生有促进作用,区域模式相对稳定是造成小菜蛾种群周年活动频繁危害严重的主要原因之一。     

Effect of temperature level on thermal acclimation in Large White growing pigs

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.     

Development and survival of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) as a function of temperature: effect on the number of generations in tropical and subtropical regions

The diamondback moth, Plutella xylostella (L.), is the most important pest of brassicaceous crops worldwide. Since temperature is the major abiotic factor influencing insect development and thermal requirements may vary among insect populations, it is important to know the effect of temperature on development and survival of a subtropical strain of P. xylostella. Development and survival of the diamondback moth was evaluated under seven constant temperatures ranging from 10°C to 35°C. Development was completed between 10°C and 32.5°C, but at 35°C all individuals died in the larval stage. Data were fitted to one linear and five nonlinear models. Considering as criteria the goodness of fit and the ability to estimate parameters of biological significance, the models Briere-1 and Briere-2 were the most adequate to describe the relationship between temperature and development of P. xylostella. The linear model demonstrated that P. xylostella required 312.5 degree-days above a lower threshold of 6.3°C to complete development. The degree-day model showed that the number of diamondback moth generations in the tropical region of Brazil is nearly twice the number in the subtropical region of the country. This result explains, at least in part, the higher population levels of this species in the tropical region of Brazil, and also demonstrates that P. xylostella is tolerant to a wide range of temperatures (6.1-32.5°C). Therefore, temperature cannot be considered a limiting factor for the occurrence of diamondback moth throughout the year in most regions of Brazil.     

Insecticidal, Antifeedant and Growth Inhibitory Activities of Efrapeptins, Metabolites of the Fungus Tolypocladium

This study shows for the first time that efrapeptins secreted by Tolypocladium have antifeedant and growth inhibitory properties. In both choice and no-choice assays against Plutella xylostella and Phaedon cochleariae there were significant differences between consumption of treated and untreated leaf discs with DC 50 values (concentration resulting in a deterrency index 50%) of 25 and 69 ppm for P. xylostella and P. cochleariae, respectively. Reduced feeding resulted in significant weight loss of larvae of both species exposed for 24 h to efrapeptin-treated leaves. The effect of the toxin persisted even after the larvae were transferred to fresh, untreated leaves, reduced larval and pupal weights, retarded larval and pupal development and delayed adult emergence. The effects were more pronounced in Phaedon than Plutella. This study shows that efrapeptins have direct contact toxicity but appear to be more efficacious against insects contacting treated leaves. Toxicity in both topical and leaf dip assays was dose-related with larvae of P. xylostella being more susceptible to the insecticidal activity of efrapeptins than P. cochleariae.     

昼夜变温幅度对小菜蛾不同发育阶段生活史性状的影响

【目的】昼夜变温幅度对昆虫的发育、存活、寿命、繁殖等核心生命活动有非常重要的影响。以往研究主要以恒温为主,温度设置不符合自然界中昼夜温度波动变化,无法明确温度波动幅度与恒温之间的生物效应差别。【方法】本研究采用了二步变温,模拟了不同的昼夜温度波动幅度(25±2℃,25±4℃,25±6℃,25±8℃,25±10℃和25±12℃)与相应恒温25℃,研究了不同变温幅度对十字花科世界性害虫小菜蛾Plutella xylostella不同生活史阶段中发育、存活、寿命与繁殖的影响。【结果】结果表明,变温幅度对小菜蛾不同阶段的发育、存活、寿命与繁殖影响存在显著差异。25±2℃,25±4℃和25±6℃对小菜蛾生活史性状的影响与恒温(25℃)相似,但25±10℃和25±12℃却产生了显著的负面影响。【结论】我们发现,较大的昼夜变温幅度显著影响小菜蛾不同阶段的发育、存活、寿命与繁殖,而适宜夜低温在一定程度上修复了日高温胁迫对小菜蛾的不利影响;并且认为昼夜变温幅度作为影响昆虫核心生命活动一种重要决定因素,必需纳入到昆虫种群数量预测模型中,才能真实地反映自然界中复杂变温模式对昆虫生态学效应的影响,才能提高昆虫田间发生预测预报的准确性。     

Temperature niche shift observed in a Lepidoptera population under allochronic divergence

A process of adaptive divergence for tolerance to high temperatures was identified using a rare model system, consisting of two sympatric populations of a Lepidoptera (Thaumetopoea pityocampa) with different life cycle timings, a 'mutant' population with summer larval development, Leiria SP, and the founder natural population, having winter larval development, Leiria WP. A third, allopatric population (Bordeaux WP) was also studied. First and second instar larvae were experimentally exposed to daily-cycles of heat treatment reaching maximum values of 36, 38, 40 and 42 °C; control groups placed at 25 °C. A lethal temperature effect was only significant at 42 °C, for Leiria SP, whereas all temperatures tested had a significant negative effect upon Leiria WP, thus indicating an upper threshold of survival c.a. 6 °C above that of the WP. Cox regression model, for pooled heat treatments, predicted mortality hazard to increase for Leiria WP (+108%) and Bordeaux WP (+78%) in contrast to Leiria SP; to increase by 24% for each additional °C; and to decrease by 53% from first to second instar larvae. High variability among individuals was observed, a population characteristic that may favour selection and consequent adaptation. Present findings provide an example of ecological differentiation, following a process of allochronic divergence. Results further contribute to a better understanding of the implications of climate change for ecological genetics.     

Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp, Pteromalus puparum in response to environmental stresses

Six heat shock protein (HSP) genes from five HSP families in the parasitoid, Pteromalus puparum, were evaluated for their response to temperature (-15 ~ 3°C , and 30 ~ 42°C for 1 h), heavy metals (0.5 ~ 5 mM Cd(2+) and Cu(2+) for 24 h and 60 h), and starvation (24 h). Compared with other insect HSPs, all conserved motifs are found in P. puparum HSPs, and they are very similar to those of the recently sequenced ectoparasitoid Nasonia vitripennis. The temporal gene expression patterns indicated that these six HSP genes were all heat-inducible, of which hsp40 was the most inducible. The temperatures for maximal HSP induction at high and low temperature zone were 36 or 39°C and -3°C, respectively. In the hot zone, all HSP genes have the same initial temperature (33°C) for up-regulation. Low concentrations of Cd(2+) for a short-term promoted the expression of all HSP genes, but not high concentrations or long-term treatments. Cu(2+) stress for 24 h increased expression of nearly all HSP. Four HSP genes changed after starvation. We infer that all six HSP genes are sensitive to heat. This may help understand the absence of P. puparum during the summer and winter. The expression profiles of six HSP genes in P. puparum under heavy metal stress indicates that HSP is a short-term response to cellular distress or injury induced by Cd(2+) and Cu(2+).     

Effects of different concentrations of enterotoxigenic and verotoxigenic E. coli on boar sperm quality

The presence of bacteria in boar semen causes economic losses in artificial insemination (AI) centers, as a consequence of alterations on boar sperm quality. For this reason, the effects of different concentrations of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) on boar sperm quality were determined in this study, by conducting two experiments. The first one consisted of assessing these effects on boar sperm quality after incubating the inoculated doses at 37°C for a 96-h period, whereas the second inoculated doses were stored at 15°C during 11 days. In both experiments, the infective concentrations ranged from 10(8)cfu mL(-1) to 10(2)cfu mL(-1); the negative control being a non-inoculated dose. Twenty-four hours after inoculation, we checked by PCR for the presence of bacteria in all tubes. Sperm quality (sperm motility, sperm viability and sperm morphology) was assessed at 24h, 48h, 72h and 96h after inoculations in the first experiment (37°C), and after 3, 5, 7, 9 and 11 days in the second (15°C). Whereas no changes were observed in sperm morphology in both experiments, the percentages of progressive motile spermatozoa dramatically diminished after 24h of incubation at 37°C, the effect being more detrimental at the highest infective concentration of microbes. Moreover, a significant decrease in the percentage of viable spermatozoa in the tube inoculated with the highest concentration (10(8)cfu mL(-1)) was detected after 24h of incubating contaminated doses at 37°C. After 48h of incubation, the presence of infective concentrations of ETEC and VTEC from 10(8)cfu mL(-1) to 10(3)cfu mL(-1) resulted in a significant diminution in the percentage of viable spermatozoa. These results suggest that ETEC and VTEC PCR analyses should be done in doses destined for AI to minimize the use of doses with diminished sperm quality due to the presence of bacteria and to avoid the potential spread of infective diseases.     

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N?-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N?-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.