二化螟滞育幼虫的蛋白和核酸含量以及保护酶活性的变化

为了阐明二化螟Chilo supprressalis滞育幼虫的分子特征及滞育期间保护酶活性的变化规律, 本研究应用Trizol法、 总量DNA提取法和蛋白定量试剂盒, 测定了在长光周期16L∶8D和25℃下发育的非滞育老熟幼虫、 在短光周期12L∶12D 和25℃下诱导滞育51 d的幼虫（称为滞育0个月）、 滞育1, 2和3个月幼虫的核酸含量和总蛋白含量; 同时应用试剂盒测定了老熟幼虫、 滞育0, 1和2个月的二化螟幼虫5种保护酶（POD, CAT, SOD, LDH和ATP酶）的活性。结果表明： 滞育幼虫的总RNA含量显著低于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。老熟幼虫的总DNA含量显著高于滞育幼虫（P<0.05）。老熟幼虫的RNA/DNA比值较低, 滞育幼虫的RNA/DNA比值较高, RNA/DNA比值随着滞育时间的推移呈现出先上升后下降的趋势。滞育期大于1个月的幼虫中蛋白含量均显著高于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。二化螟幼虫体内5种保护酶活性随发育阶段不同而存在差异。滞育幼虫中POD, CAT和SOD的活性随滞育时间延长而逐渐增强, 滞育2个月幼虫中的活性最高, 而非滞育老熟幼虫中的活性最低; LDH和ATP酶的活性则相反, 非滞育老熟幼虫中的活性最高, 滞育2个月幼虫中的活性最低。这些结果说明, 总RNA和DNA含量降低、 RNA/DNA比值先升后降、 总蛋白含量升高以及保护酶活性的变化是二化螟幼虫滞育过程中的主要生理特征。     

麦红吸浆虫滞育发生和解除过程中总脂和甘油三酯含量变化

【目的】脂类与昆虫滞育密切相关。本研究旨在明确麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育过程中脂类物质含量的变化规律,探讨麦红吸浆虫滞育与脂类物质变化的关系。【方法】采用香兰素硫酸显色法测定了2008年5月－2010年1月不同时间陕西杨凌养虫圃内麦红吸浆虫滞育前、滞育期及滞育解除后幼虫和蛹体内总脂和甘油三酯含量。【结果】 滞育前幼虫总脂和甘油三酯含量分别为378.12和291.67 μg/mg,显著高于整个滞育期（P<0.05）。滞育年周期中,冬季总脂和甘油三酯含量最高,翌年1月结茧幼虫二者含量分别为335.29 和275.72 μg/mg,显著高于其他季节（P<0.05）;整个滞育期间,裸露幼虫和结茧幼虫总脂和甘油三酯随季节变化趋势相同,但同期结茧幼虫含量高于裸露幼虫;滞育当年与第2年同期幼虫总脂和甘油三酯含量差异不显著（P>0.05）。滞育解除后,总脂和甘油三酯含量随着幼虫的发育和变态逐渐降低,其中中蛹和后蛹显著低于活动幼虫（P<0.05）。【结论】麦红吸浆虫滞育不同时期幼虫及蛹总脂和甘油三酯含量存在明显差异,其滞育与脂类物质的含量密切相关。     

七星瓢虫成虫脂肪体核酸代谢与生殖滞育的关系

本文以微量萤光法测定七星瓢虫成虫繁殖期、滞育期脂肪体核酸、蛋白含量的变化,结果表明:繁殖期雌虫卵巢发育Ⅰ和Ⅱ级时RNA/DNA比值分别为2.47和4.50;卵巢发育至Ⅲ级时RNA/DNA比值出现高峰为8.44,Ⅳ级时降为3.60,产卵后显著下降至1.76。所以脂肪体RNA/DNA比值可做为卵黄蛋白合成的一项指标。 成虫羽化后,以短光照(每日9小时光照,15小时黑暗)诱导成虫滞育,测定预滞育期RNA/DNA比值及蛋白质含量分別为3.16及59微克/毫克脂肪体;进入滞育期则分别降为1.24和14微克/毫克脂肪体;滞育结束后RNA/DNA比值上升为7.04,蛋白含量为133微克/毫克脂肪体。 成虫繁殖期与滞育期不同阶段脂肪体核酸、蛋白量的差异反映卵黄蛋白合成水平。核酸蛋白质含量低可做为滞育出现的信号。而滞育结束后RNA/DNA及蛋白含量的增加,可做为滞育结束的一个生化指标。     

大豆食心虫滞育与非滞育幼虫过冷却能力及其体内主要生化物质含量比较

【目的】本研究旨在明确大豆食心虫Leguminivora glycinivorella滞育与非滞育幼虫体内生化物质含量和保护酶活性的差异,为进一步探索大豆食心虫幼虫滞育调控的分子机制提供依据。【方法】在适温(25℃)环境下,通过控制光周期获得大豆食心虫滞育和非滞育幼虫,分别测定其过冷却点和结冰点、体内主要生化物质(糖类物质、脂类物质和可溶性蛋白)的含量以及3种保护酶[过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物岐化酶(SOD)]的活性并进行比较分析。【结果】在25℃下,大豆食心虫滞育幼虫的过冷却点(-19.20℃)和结冰点(-13.49℃)均低于非滞育幼虫的过冷却点(-16.42℃)和结冰点(-11.22℃),二者过冷却能力差异显著。滞育幼虫体内的总糖、糖原、甘油等能源物质含量均显著高于非滞育幼虫的,滞育幼虫体内总糖含量为非滞育幼虫的2.17倍,甘油含量为非滞育幼虫的1.76倍;但二者体内自由水和可溶性蛋白质含量无显著差异。同时,滞育幼虫POD和CAT活性显著高于非滞育幼虫的,但SOD活力则略低于非滞育幼虫的,无显著差异。【结论】大豆食心虫幼虫由非滞育进入滞育状态过程中,通过调节自身生理代谢使其体内糖类等生化物质含量显著升高,部分保护酶活性显著增强,进而显著提高其过冷却能力以有效应对低温等不利环境条件的来临。     

麦红吸浆虫幼虫滞育期间糖类物质变化

分析研究了麦红吸浆虫Sitodiplosis mosellana (Gehin) 幼虫滞育期间总糖、海藻糖和糖原含量的变化。结果表明： 不同滞育年限幼虫的总糖含量无显著差异。在滞育年周期中,不同时期的滞育幼虫总糖含量明显不同。幼虫老熟入土滞育以前的总糖含量明显高于入土滞育期间。入土滞育过程中,越冬期和春季的总糖含量明显高于夏季和秋季。同期的裸露幼虫与结茧幼虫的总糖含量仅在秋季有一定的差异,其他时间基本相似。结茧幼虫滞育期间的海藻糖含量一直维持较高的水平,说明其滞育期间的糖醇积累型属于海藻糖积累型;秋季与春季糖原含量的增加可能与麦红吸浆虫的滞育解除或滞育强度的变化有一定的关系。     

光周期对三带喙库蚊发育期间体内核酸动态的影响

作者测试了长短光周期处理的三带喙库蚊Culex tritaeniornynchus(Giles)各虫态与滞育及解滞蚊体内RNA与DNA的变化.结果表明:幼虫与蛹核酸含量最高,成虫期变化比较稳定.短光周期可以降低该蚊发育期间核酸的合成作用.雌蚊卵形成期间DNA相对稳定,吸血后卵巢开始发育,RNA含量明显增加,36—48小时达到高峰,然后逐渐下降.10—15日龄的滞育蚊体内DNA比发育蚊低,解滞蚊RNA与DNA含量均比发育蚊为高.新蚊、发育、滞育与解滞蚊间DNA、RNA含量不同,DNA、RNA减少与增加可用作判断滞育发生与解除的一个生化指标.     

中华通草蛉自然越冬成虫在两种光周期下滞育解除过程中的生理生化变化

摘要: 光周期在昆虫滞育中起重要作用, 而昆虫光敏感性差异导致了光在昆虫滞育解除过程中作用机制的多样性。为明确光周期在中华通草蛉Chrysoperla sinica (Tjeder)滞育解除过程中的调控机制, 本研究测定了长光周期（15L∶9D）和短光周期（9L∶15D）条件下, 中华通草蛉自然越冬种群成虫在滞育解除过程中的干重、 含水量、 过冷却点（supercooling point, SCP）、 结冰点（freezing point, FP）及蛋白质和糖原含量变化。结果表明： 雌成虫干重在长光照下处理30 d低于短光照下, 其他处理时间的干重均高于短光照下, 但处理间差异不显著（P>0.05）; 雄成虫在长光照下处理5 d的干重高于短光照下, 其余处理时间的干重均低于短光照下, 其中处理30 d 时差异显著（P<0.05）。雌、 雄成虫含水量总体呈上升趋势, 长光照下上升显著（P<0.05）, 短光照下上升不显著（P>0.05）。雌、 雄成虫的SCP、 FP在短光照下处理不同时间均无显著性差异（P>0.05）, 而长光照下表现出先降后升的趋势且处理间差异显著（P<0.05）。成虫蛋白含量在短光照条件下雄虫处理10 d以及雌虫处理15 d均显著低于长光照（P<0.05）, 其他处理之间没有显著性差异。短光照处理成虫体内糖原含量均高于长光照, 且有显著性差异（P<0.05）。多因素方差分析表明, 不同光周期对中华通草蛉成虫滞育解除过程中的SCP、 FP、 糖原变化均有显著性影响（P<0.05）, 而对干重、 含水量及蛋白质含量影响不显著;性别只对成虫FP与糖原有显著影响（P<0.05）; 处理时间对干重、 含水量、 SCP、 FP、 蛋白及糖原含量均有显著影响（P<0.01）。3种因素的交互作用对SCP、 FP、 蛋白及糖原含量均有显著影响, 对干重及含水量影响不显著。初产卵成虫体内物质含量变化测定发现, 长光照（处理10 d）下雌、 雄成虫体内蛋白和糖原含量极显著低于短光照（处理25 d）下（P<0.01）, 雄成虫体内含水量显著高于短光照下（P<0.05）。结果说明, 中华通草蛉自然越冬成虫在长、 短两种光周期下均逐渐解除滞育, 但在滞育解除过程中呈现出不同的生理生化变化。     

光周期和温度对草地螟滞育诱导的影响

草地螟Loxostege sticticalis以老熟幼虫滞育越冬。在室内通过人工诱导的方法对其滞育的光周期和温度诱导条件进行了研究。结果表明:草地螟是一种典型的长日照发育型种类。光周期、温度及其交互作用均对草地螟滞育诱导具有重要影响, 其中光周期起主导作用, 温度伴随着光周期起作用。对幼虫滞育诱导最有效的光周期是L12∶D12; 随着温度的升高, 临界光周期呈缩短趋势（18℃除外）。18, 22, 26和30℃条件下幼虫滞育的临界日长依次为13.97, 14.48, 13.92 和12.88 h。光敏感实验揭示:21℃时草地螟对光照反应最敏感时期为幼虫孵化后的11～17 d（约5龄幼虫）, 但孵化后7～11 d（约4龄幼虫）的短光照积累对提高滞育率也有重要作用, 可以将滞育率从40.0%提高到90.0%。     

麦红吸浆虫滞育发生和解除过程中保护酶活力动态

采用保护酶活性测试盒分别测定了麦红吸浆虫滞育前、滞育期及滞育解除后过氧化物酶（POD）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）等3种保护酶的活力.结果表明：幼虫从老熟到进入滞育的初期,3种保护酶的活力均呈下降趋势.滞育年周期中,SOD和CAT活力对环境温度的反应相同,即低温促进其活力升高,高温导致其活力下降;POD活力与环境温度和滞育发育有关;整个滞育期间,裸露幼虫和结茧幼虫3种保护酶的活力随季节变化趋势相同,但同期的裸露幼虫活力略高于结茧幼虫;不同滞育年限幼虫3种保护酶的活力差异不显著.滞育解除后,3种保护酶的活力均随生长发育进程逐渐升高.     

麦红吸浆虫幼虫不同滞育时期体内山梨醇含量及山梨醇脱氢酶基因表达水平变化

【目的】本研究旨在明确麦红吸浆虫Sitodiplosis mosellana(Géhin)滞育进程中山梨醇含量及山梨醇脱氢酶基因(SmSDH)mRNA表达水平的变化规律,探讨麦红吸浆虫滞育与山梨醇含量及SmSDH表达水平的关系。【方法】分别采用比色法和实时荧光定量PCR方法,对2013年5月-2014年3月不同时间陕西杨凌养虫圃内麦红吸浆虫滞育前后及滞育期幼虫体内山梨醇含量及SmSDH2a和SmSDH2b mRNA表达水平进行分析。【结果】麦红吸浆虫幼虫滞育前山梨醇含量最高(为9.41μg/mg);滞育期间,12月份含量最高(为8.50μg/mg);滞育解除后山梨醇含量为3.25~3.49μg/mg,显著低于滞育前和整个滞育期(P0.05)。山梨醇脱氢酶基因SmSDH2a及SmSDH2b在滞育进程中mRNA表达水平变化趋势相同,且与滞育强度递减趋势一致,即进入滞育后表达水平显著降低(P0.05)并一直稳定在较低水平,滞育解除后逐渐升高并达到最高。滞育期间,同期不同滞育状态幼虫之间山梨醇含量和SmSDH2a表达水平差异不明显,但裸露幼虫SmSDH2b表达水平始终高于结茧幼虫。【结论】山梨醇和SmSDH参与麦红吸浆虫滞育调控,与滞育维持和解除密切相关。     

大斑芫菁滞育幼虫在滞育不同阶段体内糖类和醇类含量的变化

在以卵滞育的昆虫中昆虫滞育时的生理代谢特点已经得到了大量研究。本文对以末龄幼虫（5龄）滞育的大斑芫菁Mylabris phalerate（Pallas）在不同滞育阶段体内糖类和醇类代谢的特征进行了研究。结果表明： 滞育个体血淋巴中的海藻糖含量高于非滞育个体,且随滞育时间的加大逐渐升高,滞育5个月时达到最大值,为5.61 μmol/mL。糖原的含量随滞育的进程逐渐减少,滞育初期（0.5个月）为0.72 mg/mL,到滞育末期（5个月）时仅为0.1 mg/mL。滞育个体脂肪体中的海藻糖含量都高于非滞育个体,滞育1个月时为非滞育个体的3倍,至滞育末期时达非滞育个体的5倍,为2.5 μmol/g脂肪体。糖原含量总体变化趋势是随滞育时间的加大逐渐减少,滞育早期和中期都高于非滞育个体。在滞育过程中血淋巴积累的小分子多元醇主要为甘油,其次是山梨醇;而在脂肪体中主要为甘油,其次是甘露醇,少量积累山梨醇：表明大斑芫菁滞育幼虫主要积累的是海藻糖和一些小分子多元醇。滞育幼虫在准备滞育时储备了大量糖原,这些糖原可能为滞育期间海藻糖、山梨醇和甘油的代谢提供了原料。     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

Factors affecting the natural duration of diapause and post-diapause development in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae)

Sesamia nonagrioides responds to photoperiod for diapause termination at high temperature with a Type II response curve, and only photoperiods longer than 12:12 terminate diapause. However, these photoperiods never occur in the field when diapausing larvae are competent to terminate diapause. Under a temperature similar to the natural field temperatures diapause terminates spontaneously in approximately 4 months, which ensures that the larvae reach the middle of winter without pupation. S. nonagrioides larvae pupate after going through a specific number of light-dark cycles or days: the required day number (RDN) for diapause completion. This RDN could be modulated by temperature but more research is necessary to clarify this point. In post-diapause development, when a suitable temperature threshold is considered, the absolute accumulation of heat is more important than whether the temperature received is fluctuating or constant. The temperature threshold for diapause and post-diapause development was lower than the temperature threshold of larvae in continuous development. This is important for adjusting phenological models in S. nonagrioides and in other species, and may explain why in many cases adults appear in the field when the supposed temperature threshold for development has not been attained.     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

Desiccation resistance in pre-diapause, diapause and post-diapause larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae)

Desiccation resistance was examined in pre-diapause, diapause and post-diapause larvae of the spruce budworm, Choristoneura fumiferana (Clemens), in terms of passive water evaporation under three desiccation conditions: freeze-drying, desiccant-drying at 2 degrees C and desiccant-drying at 18 degrees C. Diapausing second instar larvae and post-diapause non-feeding second instar larvae showed strongest desiccation resistance: a significant amount of water was retained after repeated drying under desiccating conditions, while pre-diapause first instar larvae and post-diapause feeding second instar larvae lost almost all their water content after one or two drying cycles. A hibernaculum covering had no effect on water evaporation. While dead larvae tended to lose significantly more water than their living counterparts, particularly among first instar larvae, such an impact was much weaker among diapausing second instar larvae. Desiccation resistance was lost when post-diapause second instar larvae were allowed access to water while the level of desiccation resistance was maintained or enhanced when the larvae did not have access to water. These results are discussed in the context of overwintering ecology of the species and possible mechanisms for the desiccation resistance are also discussed.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

Regulation of larval diapause by juvenile hormone in the European corn borer,Ostrinia nubilalis

The effects of juvenile hormone (JH) on the larval diapause of the European corn borer, Ostrinia nubilalis, were investigated. The larval period was prolonged and the development of gonads was retarded if non-diapause larvae were treated with JH. Pupation of diapausing larvae was accelerated by allatectomy. It was concluded that the diapause of this borer is largely regulated by JH.     

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.     

SOME PHYSIOLOGICAL CHARACTERISTICS IN DIAPAUSE LARVAE OF ASIAN CORN BORER, OSTRINIA FURNACALIS (LEPIDOPTERA:PYRALIDAE)

Abstract  The physiological and biochemical characteristics of the diapause larvae of Asian corn borer Ostrinia furnacalis , were investigated. The molecular weight of diapause associated protein in hemolymph was determined. The development of male reproductive system, the body water content, the tolerance to suffocation and the rates of respiration of the diapause and non-diapause larvae were compared. The results revealed that the diapause larvae have the following characteristics: 1. There is a diapause associated protein in heamolymoph; its molecular weights of two polypeptide chains are 72,000 and 84,000 Dal. respectively. 2. The male reproductive organ is underdeveloped. 3. The body water content is lowered. 4. A much higher tolerance to suffocation is exhibited after submergence under water at room temperature. 5. A low rate of respiration prevailed.