Physiology of diapause and cold hardiness in overwintering pupae of the apple leaf miner Phyllonorycter ringoniella in Japan

Abstract The apple leaf miner Phyllonorycter ringoniella (Matsumura) (Lepidoptera: Gracillariidae) overwinters as a diapausing pupa. The diapause rate reaches 100% in early October. Diapause intensity decreases gradually from early October and diapause terminates in early February. The fresh body weight of diapausing pupae is 1.6 times that of non-diapausing pupae. The main cryoprotectant in P. ringoniella pupae is trehalose. Three stages are distinguishable as indicated by the correlations between diapause intensity, levels of cold hardiness and the trehalose content: diapause induction occurred in October, diapause development from November to December, and post-diapause quiescence from January to April. During diapause induction, the pupae accumulate low levels of trehalose and do not survive exposure to −15 °C. During diapause development, the pupae gradually accumulate more trehalose and show some ability to survive exposure to −15 °C, but not to −20 °C. During post-diapause quiescence, the pupae accumulate relatively more trehalose and cold hardiness fully develops, but decreases quickly in April. The trehalose content in pupae sampled in December is unaffected by acclimation temperatures in the range 0–30 °C, but decreases in pupae sampled in March after acclimation at temperatures from 5 to 15 °C. These results suggest that overwintering pupae of P. ringoniella have the ability to accumulate trehalose and develop a high level of cold hardiness during diapause development.     

Diapause induction as an interplay between seasonal token stimuli,and modifying and directly limiting factors: hibernation in Chymomyza costata

Larvae of wild type (WT) strain of Chymomyza costata Zetterstedt (Diptera: Drosophilidae) enter diapause (stop developing) in response to short‐day signal at a constant 18 °C, whereas larvae of a non‐photoperiodic‐diapause (NPD) strain do not respond to photoperiodic signalling and continue in larval development irrespective of daylength. The present study shows that WT larvae also respond reliably to thermoperiodic signalling (daily cycles of temperature) under constant darkness, whereas the NPD larvae do not, suggesting that the pathways transducing the environmental token stimuli (photoperiod and thermoperiod) onto the diapause developmental programme might merge functionally in the central biological clock system known to be mutated in NPD strain. Temperature and larval population density modify the output of token stimuli signalling. High temperatures (>24 °C) tend to avert, whereas low temperatures (<18 °C), especially in combination with constant darkness, stimulate diapause induction in WT strain. Overcrowding (>200 larvae per 5 g of larval diet) lengthens the duration of larval development and induces a ‘diapause‐like’ developmental arrest of relatively weak intensity in up to 60% of larvae of both strains. At high temperatures (>30 °C), all WT larvae continue direct development but subsequently die during the pupal stage. Low temperature exposure (<12 °C) causes quiescence in the majority of the larvae of both strains. Starvation blocks development and causes mortality when applied in larvae younger than day 3 of the third instar. Older larvae survive starvation and their photoperiodically‐induced developmental pre‐programming is not affected. Collectively, the results show that diapause induction in C. costata is a result of various interacting effects of multiple environmental factors.     

Physiological and biological patterns of a highland and a coastal population of the European cherry fruit fly during diapause

Adult emergence of univoltine temperate insect species and its synchronization with specific host phenological stages is mainly regulated by obligatory pupal diapause. Although a few studies have investigated the factors affecting diapause intensity, little attention has been paid to the physiological alterations and metabolic regulation that take place during diapause. Here, we describe differences in diapause between a highland and a coastal Greek population of the European cherry fruit fly Rhagoletis cerasi, a major pest of sweet and sour cherries in many European countries. Pupae of both populations were exposed to the environmental conditions prevailing in the two areas and diapause termination was observed under laboratory conditions. The regulation of energetic metabolites during the long pupae stage was examined under both field and laboratory conditions. Differences in diapause intensity revealed that the two populations have adapted to the local geographical and climatic conditions and have different requirements for low temperatures to terminate diapause. The coastal population undergoes a shorter diapause and adults emerge more rapidly, especially in the highland area. The highland population failed to terminate diapause (<40% adult emergence) in the coastal area. Both populations draw on their major energetic reserves (lipids and protein) similarly during diapause. Nevertheless, regulation of carbohydrate and glycogen reserves seems to vary between the populations: major peaks of these stored nutrients occur on different dates in the two populations, suggesting a differential regulation. Differences in diapause intensity imply a genetic differentiation between the two populations. The importance of our findings in understanding the physiological patterns during obligatory diapause of a univoltine insect species, as well as the practical implications for the development of specific phenological models for the European cherry fruit fly are discussed.     

Changes of biogenic amine levels in haemolymph during diapausing and non-diapausing status in Pieris brassicae L

During the larval development of Pieris brassicae, photoperiod status induces either direct (non-diapausing) development or diapausing development. Biogenic amines such as dopamine and serotonin and their derivatives may be implicated in the physiological control and adaptation of these insects to different photoperiods. High performance liquid chromatography coupled with electrochemical detection was used to measure biogenic amine concentrations in insect haemolymph in different photoperiod conditions. At the end of the higher photosensitivity phase, dopamine levels were two-fold higher in diapausing (0.914 pmol/microl+/-0.025) as compared to non-diapausing insects (0.415 pmol/microl+/-0.02; P<0.001). Globally, dopamine catabolites are found in higher levels in non-diapausing insects (in prepupa, near seven times more for NADA, and 25 times more for DOPAc), thus indicating a higher dopamine turn-over. Serotonin levels were detected only at the beginning of this instar in diapausing insects but not in direct development insects. During pupal life, dopamine levels were significantly higher in diapausing than in non-diapausing insects (in 3-day pupae, dopamine was 28 times higher), and remained at high levels throughout diapausing life. At the beginning of diapause, serotonin levels were higher in diapausing insects than in non-diapausing insects (in 3-day pupae, serotonin was 13 times higher). These levels decreased in the middle of diapause period, and then increased just before its breaking. Dopamine and serotonin levels presented the typical kinetics of diapause development and may be used as an indicator of the evolution of diapause status.     

Drosophila americana Diapausing Females Show Features Typical of Young Flies

Diapause is a period of arrested development which is controlled physiologically, preprogrammed environmentally and characterized by metabolic depression that can occur during any stage of insect development. Nevertheless, in the genus Drosophila, diapause is almost always associated with the cessation of ovarian development and reproductive activity in adult females. In this work, we show that, in D. americana (a temperate species of the virilis group), diapause is a genetically determined delay in ovarian development that is triggered by temperature and/or photoperiod. Moreover, we show that in this species diapause incidence increases with latitude, ranging from 13% in the southernmost to 91% in the northernmost range of the distribution. When exposed to diapause inducing conditions, both diapausing and non-diapausing females show a 10% increase in lifespan, that is further increased by 18.6% in diapausing females, although senescence is far from being negligible. ActinD1 expression levels suggest that diapausing females are biologically much younger than their chronological age, and that the fly as a whole, rather than the ovarian development alone, which is phenotypically more evident, is delayed by diapause. Therefore, diapause candidate genes that show expression levels that are compatible with flies younger than their chronological age may not necessarily play a role in reproductive diapause and in adaptation to seasonally varying environmental conditions.     

滞育昆虫营养物质的积累、转化与调控

滞育是昆虫度过不良环境的一种生存适应策略,营养物质的利用、积累和转化影响昆虫的滞育。滞育期间,昆虫体内发生一系列生理生化变化,包括脂类、碳水化合物和氨基酸等内源营养物质呈特异性地积累或转化,保障了滞育个体在逆境中存活及滞育解除后发育的能量需求。外源营养物质对昆虫滞育的影响较复杂,其种类、丰度、质量能通过"食料-寄主-天敌"营养关系进行传递,影响昆虫体内营养物质的积累和转化,改变昆虫的耐寒性、滞育率等,制约昆虫的滞育深度、滞育存活率。滞育昆虫营养物质积累、利用机制复杂多样,胰岛素信号通路-叉头转录因子以及激脂激素的调控效应在滞育诱导及营养物质转化中起到了关键的调控作用,但滞育的系统性调控仍需进一步深入研究。     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata

Dry weight (D.W.), protein, RNA, and DNA have been determined in the blowfly, Lucilia sericata, throughout all stages of normal development in long and short photoperiod regimes and during larval diapause. During normal development protein/D.W. levels fluctuate markedly during the larval and puparial stages, increased levels being correlated with the synthesis of new cuticle, etc. prior to ecdysis and the histogenesis of adult tissues prior to emergence. Protein levels remain relatively high and constant during adult life to senescence. RNA/D.W. levels are highest in first instar larvae but decline rapidly during larval development until just before puparium formation. Sharp increases are found prior to pupation and then again prior to adult emergence. In the adult stage, the levels decline steadily throughout the life span. DNA/D.W. levels are very low in the egg but rach their highest levels in early first instar larvae. They then decline during larval development, with small increases being found prior to puparium formation and adult emergence. Adult levels remain relatively constant throughout the life span. The ratio has extremely high values in the egg, indicating the high degree of synthetic activity that takes place during embryogenesis. There is a steady decline in values during adult life to senescence in both sexes, suggesting that physiological ageing in L. sericata is accompanied by a decrease in protein synthesis potential.During larval diapause all parameters, with the exception of the ratio, are maintained at low and constant levels, reflecting the fact that diapause is a period when synthetic and mitotic activity are minimal. The great variations in levels, however, indicate that individuals within a group of larvae can terminate diapause spontaneously at 24°C and return to the normal processes of morphogenesis.     

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.     

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.