Upregulation of a desaturase is associated with the enhancement of cold hardiness in the onion maggot, Delia antiqua

Cold-acclimated non-diapause pupae, and summer- and winter-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show marked cold hardiness as compared with intact non-diapause pupae. Homeoviscous adaptation of cellular membranes is crucial to enhance the cold hardiness of organisms, and Delta9-acyl-CoA desaturases have been assumed, albeit without experimental evidence in insects, to play a key role in the adaptation. We cloned the cDNA of a desaturase gene (Dadesat) from D. antiqua, which is most likely to encode Delta9-acyl-CoA desaturase. Expression of Dadesat mRNA in the brain, midgut, and Malpighian tubules of cold-acclimated and diapause pupae was upregulated 2-10 fold, correlating well with the increase in cold hardiness. In the pupae with enhanced cold hardiness, palmitoleic and oleic acids, the presumed products of Dadesat, in the phospholipids were significantly increased. These findings suggest that the increase in the expression of Dadesat contributes to enhanced cold hardiness in D. antiqua through the production of these unsaturated fatty acids.     

Chaperonin Contributes to Cold Hardiness of the Onion Maggot Delia antiqua through Repression of Depolymerization of Actin at Low Temperatures

Winter-diapause and cold-acclimated non-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show strong cold hardiness. To obtain insights into the mechanisms involved in the enhancement of cold hardiness, we investigated the expression patterns of genes encoding subunits of chaperonin (CCT) and the morphology of actin, a substrate of CCT, at low temperatures. Quantitative real-time PCR analyses showed the mRNA levels of CCT subunits in pupal tissues to be highly correlated with the cold hardiness of the pupae. While actin in the Malpighian tubules of non-cold-hardy pupae showed extensive depolymerization after a cold treatment, actin in the same tissue of cold-hardy pupae was not depolymerized. Damage to cell membranes became apparent after the depolymerization of actin. Moreover, administration of Latrunculin B, an inhibitor of actin polymerization, to the larvae markedly decreased the cold hardiness of the pupae obtained. These findings suggest that CCT contributes to the cold hardiness of D. antiqua through the repression of depolymerization of actin at low temperatures.     

葱蝇冬滞育蛹的全长cDNA文库的构建

葱蝇Delia antiqua(Meigen)具有冬滞育的特性且与黑腹果蝇Drosophila melanogaster近缘。该研究目的在于构建葱蝇冬滞育蛹的全长cDNA文库,为进一步的冬滞育专化基因筛选、克隆、和表达分析奠定基础。利用RNAiso试剂盒提取葱蝇冬滞育蛹的总RNA,采用SMART技术合成全长cDNA,经SfiⅠ酶切消化后,将cDNA克隆到质粒载体pDNR-LIB。经鉴定,原始文库的滴度为2.4×107cfu/mL。随机挑取15个克隆,经PCR快速鉴定,插入片段大小在0.5～3kb之间,平均大小在1kb左右,重组率达100%。结果表明该研究获得高质量的葱蝇冬滞育蛹cDNA文库。     

葱蝇夏滞育蛹体内DaFOXO1对超氧化物歧化酶基因表达及蛹发育历期的调控作用(英文)

【目的】本研究旨在调查葱蝇Delia antiqua夏滞育蛹体内DaFOXO1对超氧化物歧化酶(SOD)基因表达及蛹发育历期的调控作用。【方法】从葱蝇转录组数据中鉴定DaFOXO1下游铜锌超氧化物歧化酶基因DaCu/Zn SOD和锰超氧化物歧化酶基因DaMn SOD;利用生物信息学工具对DaCu/Zn SOD和DaMn SOD的氨基酸序列特征、亚细胞定位和系统发育关系进行分析。通过qRT-PCR方法分析DaFOXO1, DaCu/Zn SOD和DaMn SOD基因在葱蝇夏滞育蛹不同发育阶段的表达特点;进一步分析DaFOXO1基因被干扰后,葱蝇夏滞育蛹中DaCu/Zn SOD和DaMn SOD基因的表达特点、酶活性变化及对葱蝇夏滞育蛹发育历期的影响。【结果】鉴定到的葱蝇DaCu/Zn SOD(GenBank登录号: KR072551)的开放阅读框长459 bp,编码153个氨基酸,预测蛋白分子量为22.4 kD,等电点为6.44,属于细胞质型铜锌超氧化歧化酶;DaMn SOD(GenBank登录号: KR072549)的开放阅读框长648 bp,编码216个氨基酸,预测蛋白分子量为24.4 kD,等电点为8.85,属于线粒体型锰超氧化物歧化酶。氨基酸序列比对结果显示,DaCu/Zn SOD和DaMn SOD与其他10种双翅目昆虫的同源蛋白有75%~94%的氨基酸序列一致性,且具有典型的SOD家族序列特征;系统发育分析显示它们与铜绿蝇Lucilia cuprina同源蛋白形成高支持率的一支。qRT-PCR分析表明,DaFOXO1基因在滞育前期和滞育后期的表达量较高,而在滞育期的表达量低; DaCu/Zn SOD基因在滞育期和滞育后期呈高表达;但DaMn SOD基因在滞育前期和滞育期的表达量最高,在滞育后期次之。干扰DaFOXO1可显著抑制DaCu/Zn SOD和DaMn SOD的基因表达及相应酶活性,并能明显延长夏滞育蛹的滞育期。【结论】结果说明,DaCu/Zn SOD和DaMn SOD是FOXO1信号网络中的重要成员;DaFOXO1对葱蝇夏滞育蛹蛹期有重要调控作用。     

Cold hardiness of Helicoverpa zea (Lepidoptera: Noctuidae) pupae

An insect's cold hardiness affects its potential to overwinter and outbreak in different geographic regions. In this study, we characterized the response of Helicoverpa zea (Boddie) pupae to low temperatures by using controlled laboratory measurements of supercooling point (SCP), lower lethal temperature (LT(50)), and lower lethal time (LLTime). The impact of diapause, acclimation, and sex on the cold hardiness of the pupae also were evaluated. Sex did not significantly affect the SCP, LT(50), or LLTime. However, the mean SCP of diapausing pupae (-19.3°C) was significantly lower than nondiapausing pupae (-16.4°C). Acclimation of nondiapausing pupae to constant temperatures from 10 to 20°C before supercooling also produced a significantly lower SCP than nondiapausing pupae held at 25°C. The LT(50)s of nondiapausing and diapausing were not significantly different, but confirmed that H. zea pupae are chill-intolerant because these lethal temperatures are warmer than the corresponding mean SCPs. Diapausing pupae survived longer than nondiapausing pupae at the same, constant, cold temperatures, a finding consistent with the SCP results. Both of these results suggest enhanced cold hardiness in diapausing pupae. When laboratory results were compared with field temperatures and observed distributions of H. zea in the contiguous United States, the laboratory results corroborated what is currently perceived to be the northern overwintering limit of H. zea; approximately the 40(th) parallel. Moreover, our research showed that areas north of this limit are lethal to overwintering pupae not because of low temperature extremes, but rather the length of time spent at near-zero temperatures.     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.     

寄主植物对棉铃虫越冬蛹抗寒能力的影响

通过对滞育蛹过冷却点的测定 ,初步明确寄主对棉铃虫Helicoverpaarmigera的越冬抗寒性有影响 ,幼虫取食棉花时产生的滞育蛹的过冷却点低于取食玉米的滞育蛹的过冷却点 ,即前者的抗寒能力高于后者 ;但用Bt棉喂养后的棉铃虫滞育蛹抗寒能力下降     

Geographical and diapause-related cold tolerance in the blow fly,Calliphora vicina

Three geographical strains of the blow fly, Calliphora vicina, were tested for cold tolerance at 0 degrees, -4 degrees and -8 degrees C. Survival to eclosion after 1 to 18 days of cold exposure was greater for diapause-destined larvae than for nondiapause-destined larvae of the two northern strains (Nallikari, Finland 65 degrees N and Edinburgh, Scotland 55 degrees N) but not for the southernmost strain (Barga, Italy 44 degrees N) where no clear differences were apparent. Diapause-destined larvae of the Edinburgh strain were more cold tolerant than those from Nallikari, at both -4 degrees and -8 degrees C, a difference possibly attributable to the long-lasting snow cover in the more northern locality, which might insulate the overwintering soil microclimate. At 0 degrees C, however, Nallikari larvae were more cold tolerant than Edinburgh or Barga. This was also the case for nondiapause-destined larvae, indicating that cold tolerance may occur, in part, independently of the diapause programme. In all three strains diapausing larvae were more cold tolerant than same-age (nondiapausing) pupae. For Nallikari, but not Barga, wandering larvae from short-day exposed flies, therefore initially programmed for diapause, but diverted from the diapause pathway by larval breeding at 19 degrees C, were significantly more cold tolerant than nondiapause larvae from long-day parents, indicating some maternal regulation of larval cold tolerance. There was, however, no evidence for an additional cold hardiness in larvae acclimatised to cold by a gradual reduction of temperature.     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity

When pupae of Delia antiqua were transferred to constant darkness (DD) from light-dark (LD) cycles or constant light (LL), the sensitivity to light of the circadian clock controlling eclosion increased with age. The daily rhythm of eclosion appeared in both non-diapause and diapause pupae only when this transfer was made during late pharate adult development. When transferred from LL to DD in the early pupal stage, the adult eclosion was weakly rhythmic in non-diapause pupae but arrhythmic in diapause pupae. However, the sensitivity of the circadian clock to temperature cycles or steps was higher in diapause pupae than in non-diapause pupae; in the transfer to a constant 20 degrees C from a thermoperiod of 25 degrees C (12 h)/20 degrees C (12 h) on day 10 after pupation or from chilling (7.5 degrees C) in DD, the adult eclosion from diapause pupae was rhythmic but that from non-diapause pupae arrhythmic. In a transfer to 20 degrees C from the thermoperiod after the initiation of eclosion, rhythmicity was observed in both types of pupae. The larval stage was insensitive to the effect of LD cycle initiating the eclosion rhythm. In D. antiqua pupae in the soil under natural conditions, therefore, the thermoperiod in the late pupal stage would be the most important 'Zeitgeber' for the determination of eclosion timing.     

Cold hardiness of the fly pupal parasitoid Nasonia vitripennis is enhanced by its host Sarcophaga crassipalpis

Supercooling points (SCPs) and low temperature survival were determined for diapausing and nondiapausing larvae of the ectoparasitoid Nasonia vitripennis. Neither nondiapausing nor diapausing larvae could survive tissue freezing. The SCP profiles were nearly identical for nondiapause-destined (-27 degrees C) and diapausing larvae (-25 degrees C), but these values were not indicative of the lower limits of tolerance in either type of larvae: larvae were killed by chilling at temperatures well above the SCP. Diapausing larvae could withstand low temperature exposures 3-8 times longer than their nondiapausing counterparts. Low temperature survival was enhanced in diapausing and nondiapausing larvae by their encasement within the puparium of the host flesh fly, SARCOPHAGA CRASSIPALPIS: the LT(50)s determined for nondiapausing and diapausing larvae enclosed by fly puparia were 2-3 times higher than values calculated for larvae removed from the puparia. Additional low temperature protection was gained through acquisition of host cryoprotectants during larval feeding: nondiapausing parasitoid larvae that fed on diapausing flesh fly pupae with high levels of glycerol were able to survive exposure to a subzero temperature 4-9 times longer than wasps reared on nondiapausing fly pupae that contained lower quantities of glycerol. Alanine may also contribute to the cold hardiness of N. vitripennis, as evidenced by the fact that larvae feeding on diapausing fly pupae both contained higher concentrations of alanine and exhibited greater cold hardiness. The results thus demonstrate that several critical features of cold hardiness in the wasp are derived from biochemical and physical attributes of the host.     

Physiology of diapause and cold hardiness in the overwintering pupae of the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae) in Japan

The fall webworm Hyphantria cunea Drury, which was accidentally introduced to Japan in 1945, overwinters on the ground in pupal diapause. Diapause termination, as indicated by the respiration rate and the period required for adult emergence, began in March and ended in April. Cold hardiness (the ability to survive exposure to -15 degrees C) decreased linearly with diapause development from November to the following April under field conditions. Cold hardiness of diapause pupae (DP) decreased as the acclimation temperature decreased from 15 to -10 degrees C, whereas cold hardiness of non-diapause pupae (NDP) remained high as the acclimation temperature decreased from 5 to -5 degrees C. However, H. cunea in Japan can survive exposure to -5 degrees C for two weeks, whether it is in a diapause or non-diapause state. Trehalose was the main sugar detected in the body, but its level was less than 0.8%. Trehalose levels increased in field-collected pupae from January to March. DP accumulated less trehalose than NDP, as the acclimation temperature was decreased from 5 to -5 degrees C. The alanine content in field-collected pupae increased from November to February. Both diapause and low temperature caused an accumulation of alanine. These results suggest that under field conditions, overwintering pupae of H. cunea in Japan do not accumulate high levels of sugars and polyols and do not develop a high level of cold hardiness. Furthermore, DP do not accumulate high levels of sugars and polyols and their ability to survive exposure to -15 degrees C is not greater than that of NDP. The physiological and biochemical bases of diapause in H. cunea from Japan are discussed.     

Cold hardiness of diapausing and non-diapausing pupae of the European grapevine moth, Lobesia botrana

Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) is a key pest of grapes in Europe. It overwinters as a pupa in the bark crevices of the plant. Supercooling point (SCP) and low temperature survival was investigated in the laboratory and was determined using a cool bath and a 1 °C min^?1 cooling rate. Freezing was fatal both to diapausing and non‐diapausing pupae. SCP was significantly lower in diapausing male (?24.8 °C) and female (?24.5 °C) pupae than in non‐diapausing ones (?22.7 and ?22.5 °C, respectively). Sex had no influence on SCP both for diapausing and non‐diapausing pupae. Supercooling was also not affected by acclimation. However, acclimation did improve survival of diapausing pupae at temperatures above the SCP. Survival increased as acclimation period increased and the influence was more profound at the lower temperatures examined. Diapausing pupae could withstand lower temperatures than non‐diapausing ones and lethal temperature was significantly lower than for non‐diapausing pupae. Freezing injury above the SCP has been well documented for both physiological stages of L. botrana pupae. Our findings suggest a diapause‐related cold hardiness for L. botrana and given its cold hardiness ability, winter mortality due to low temperatures is not expected to occur, especially in southern Europe.     

葱蝇热激蛋白DaHSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析（英文）

【目的】低分子量（12~43 kDa）热激蛋白(sHSPs)具有抗逆应答的功能,滞育是昆虫抵抗不良环境的特殊发育形式,但sHSPs在昆虫滞育发育过程中的作用仍不清楚。本研究克隆和特征化葱蝇Delia antiqua sHSP基因,并研究它在夏滞育和冬滞育发育过程中的表达模式,为阐明sHSPs在滞育发育上的功能奠定基础。【方法】通过RACE-PCR方法克隆了葱蝇HSP23基因,通过相似性比较分析了其特征、结构域及与双翅目代表性同源基因的系统发育关系;采用实时荧光定量PCR研究了该基因在葱蝇冬滞育蛹和夏滞育蛹发育过程中的表达情况,通过表达的差异比较揭示了该基因与滞育发育的关系。【结果】克隆出了葱蝇HSP23基因,命名为DaHSP23（GenBank登录号：HQ392521.1）,其cDNA全长序列为904 bp,编码186个氨基酸,推测蛋白分子量为20.9 kDa,等电点为6.42。该基因的编码蛋白与其他双翅目昆虫的sHSPs有超过66%的氨基酸序列一致性,与已报道的其他双翅目昆虫的滞育相关HSP23基因同源。基因组测序显示该基因无内含子。DaHSP23基因在葱蝇非滞育蛹的发育过程中一直保持在较低的水平,各发育阶段间的表达量不存在显著差异。但在冬滞育和夏滞育蛹中,该基因从滞育起始期开始逐渐显著升高表达,到滞育维持期的中后期达到峰值,在滞育终止期逐渐降到较低的水平。【结论】DaHSP23基因在葱蝇冬滞育和夏滞育发育过程中明显上调表达,但存在差异,它在滞育期的调控可能是种专化的。DaHSP23可能在葱蝇两种类型的滞育上起重要作用。     

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.     

葱蝇海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达

海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase, TPS）是昆虫海藻糖合成途径中的关键酶之一。本研究通过对葱蝇Delia antiqua海藻糖-6-磷酸合成酶基因的克隆、 序列分析及滞育相关表达的分析, 旨在证明该基因在能源合成以及抵御高温和低温环境方面发挥重要作用, 为进一步弄清葱蝇滞育分子机制提供理论依据。根据葱蝇抑制消减杂交文库中的EST序列信息, 设计特异性引物, 并通过RACE技术克隆了葱蝇海藻糖-6-磷酸合成酶基因全长cDNA, 命名为DaTPS1 （GenBank登录号： JX681124）, 其全长为2 904 bp, 开放阅读框2 448 bp, 编码815个氨基酸, 推测其相对分子质量为91.2 kD, 等电点为5.96。生物信息学分析表明, 该基因编码的氨基酸序列具有两个保守结构域, 与其他物种TPS具有较高的同源性, 其中和黑腹果蝇Drosophila melanogaster亲缘关系最近, 氨基酸序列一致性为92.1%; 其蛋白质三维结构有15条大的α螺旋和11股反向平行的β链折叠。RT-PCR分析表明, DaTPS1在葱蝇非滞育、 夏滞育和冬滞育期蛹中都有表达, 但是非滞育期各时期表达量基本没有变化, 而在夏滞育和冬滞育蛹的滞育前期表达量较高, 滞育保持期表达量较低, 滞育期后期表达量又有所升高。推断在葱蝇蛹夏滞育和冬滞育期前期, TPS1开始催化合成较多的海藻糖以提高滞育期抵御不良环境的能力, 滞育保持期蛹的新陈代谢降低, 所需能量较少, 所以TPS1处于低水平表达状态, 而滞育期结束后, 蛹生长发育逐渐恢复, 所需能量有所增加, TPS1的表达量再次升高。本研究对揭示昆虫TPS在能量代谢通路中的作用及昆虫滞育的分子机理具有一定的科学意义。     

桔小实蝇自然种群蛹和越冬成虫的耐寒性

本文通过过冷却点和低温耐寒能力的测定,研究了我国桔小实蝇Boctrocera dorsalis (Hendel)不同季节种群和地理种群蛹的耐寒性规律。结果表明：桔小实蝇自然种群蛹的低温存活力有明显的季节规律,冬前种群的耐寒性显著强于夏季种群。如在0℃低温下暴露2天,夏季种群基本全部死亡,而冬前种群存活率仍为61.4%。2005-2006年不同月份桔小实蝇1日龄蛹过冷却点差异明显,以2月份均值最高,为-9.26℃,8月份最低,为-15.20℃;且2月份桔小实蝇蛹个体过冷却点值出现明显的分化现象,分别在-15℃左右和-6℃左右出现两个明显的聚集区。所研究的5个桔小实蝇地理区域种群没有出现明显的耐寒性分化。结合本文结果,文中还就桔小实蝇自然种群耐寒性影响因素进行了讨论。     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.