Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator (Hymenoptera: Braconidae). The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head (without antennae), thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Effects of lead stress on Vg expression in the beet armyworm over five successive generations

Heavy metals have been found to be endocrine disruptors in invertebrates.Lead is one of the most widespread elements of contamination,but there has been no research about the effects of lead stress on vitellogenin(Vg) gene expression in insects exposed to lead over multiple generations.In this paper,the effects of different concentrations of lead(0,0.3,4.8 and 76.8 mg kg~(–1)) on the expression of Vg in the beet armyworm over five successive generations were studied.The results showed that lead stress had significant effects on Vg expression in a dose-dependent manner.For females at the larval and adult stages,as lead concentration increased,Vg expression was significantly inhibited; for males at these two developmental stages,Vg expression was induced and increased as lead concentration increased.In addition,with the increase over stressed generations,inhibited effects for females and induced effects for males at the larval and adult stages became increasingly more obvious.However,at the pupal stage,Vg expression in the two genders was different from that at the larval and adult stages.The results indicate that lead stress can upregulate Vg expression in males which should be a useful indicator for environmental risk assessment.     

Role of TRP channels and HSPs in thermal stress response in the aphid parasitoid Aphelinus asychis(Hymenoptera:Aphelinidae)

Aphelinus asychis is an important aphid endoparasitoid. Under field and greenhouse conditions, high temperature is one of the factors limiting the application of A. asychis for biological pest control. To explore the potential role of transient receptor potential(TRP) channels and heat shock proteins(HSPs) in this process, we identified 11 genes encoding TRP channels and nine genes encoding HSPs. Three proteins(AasyTRPA5, AasyPyrexia, AasyPainless) that belong to transient receptor potential ankyrin(TRPA) subfamily and nine HSPs are involved in the response to high temperature. We also investigated the survival of A. asychis and the response of the identified TRP channels and HSPs to high temperature. The results showed that the maximum temperature that allowed A. asychis survival was approximately 41℃; females had higher survival rates than that of the males at 40 and 41℃. Short-term heat-shock resulted in increased expression of Aasyshsp in males, and Aasyhsp40, Aasyhsp68, Aasyhsp70-4, Aasyhsp70-5 and Aasyhsp90 were upregulated and then downregulated, whereas Aasyhsp70-3 was upregulated at 41℃. Moreover, Aasyhsp40 and Aasyhsp90 showed higher expression levels in females, while Aasyshsp and Aasyhsp70-3 presented opposite expression patterns. At temperature above 35℃, expression of AasyPyrexia in females was significant higher than that in males, whereas AasyPainless and AasyTRPA5 presented higher expression in males at 40 and 41℃, respectively. Altogether, these results indicate that protection against thermal stress in A. asychis is coordinated by TRP channels and HSPs. These findings provide a basis for understanding the potential mechanism of A. asychis in response to high temperatures.     

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut（Arachis hypogaea L.） Seeds

Phosphoenolpyruvate carboxylase(PEPC;EC 4.1.1.31) catalyses phosphoenolpyruvate(PEP) to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase(PK;EC 2.7.1.40) catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes(AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5) from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties(E11,Hebeigaoyou,Naihan 1,and Huayu 26).It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging(DAP);whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.     

Hoxc13 Expression Pattern in Cashmere Goat Skin During Hair Follicle Development

Hoxc13 has an important role in controlling hair formation. In this study, we examine the Hoxc13 RNA expression pattern of skin during embryo development. The result indicated that changes of the Hoxe13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In interpreting these results, we investigated whether the regulation motifs is in Hoxc13 intron, which is a 5.4 kb fragment. To blast with other mammals, we found a very conservative region in all mammal animals and two regions in livestock, such as cow, sheep, horse, dog, and so on, which are not in other Hox genes. We have examined putative pre-miRNA in this region, providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hoxc13 gene expression.     

Expression of Inflammatory Factors in Bovine Reproductive Tract During Follicular and Luteal Phases

In order to improve reproduvtive efficieny and understand reproduvtive defense mechanism, the oviduct, uterine horn and uterine body of bovine were used to detect the changes of inflammatory factors and the relationship between estrogen and progesterone receptor protein during estrous cycle by real-time PCR and Elisa method. The results showed that interleukin-4(IL-4), interleukin-6(IL-6), interleukin-10(IL-10), interleukin-1α(IL-1α) and interleukin-1β(IL-1β) were expressed in cow oviduct, uterine horn and uterine body. In the follicular phase and the luteal phase, m RNA expression of five inflammatory factors in the uterine horn and uterine body was higher than that in the oviduct. In the follicular phase, IL-10 was highly expressed in the uterine horn and uterine body, IL-4 was highly expressed in the uterine horn, uterine body and oviduct. Additionally, in the luteal phase, IL-6 and IL-1β were highly expressed in the uterine horn, uterine body and oviduct, and the highest expression of IL-1β was observed in the uterine horn. The levels of Estrogen Receptor(ERα) protein in the oviduct, uterine horn and uterine body significantly increased in the follicular phase. The levels of Progesterone Receptor(PR) protein in the same portions of the reproductive tract in the luteal phase were significantly higher than those in the follicular phase. IL-4 and IL-10 in the cow reproductive tract might play a major role in the follicular phase, while IL-6 and IL-1β might play a major role in the luteal phase. The expression of five inflammatory factors was not directly regulated by ERα and PR.     

柑橘大实蝇非典型气味受体基因的克隆、序列分析及组织表达模式

旨在探究柑橘大实蝇（Bactrocera minax）非典型气味受体（odorant receptor co-receptor,Orco）基因的分子特征和组织表达模式,通过对前期获得的柑橘大实蝇成虫转录组数据分析,利用RT-PCR技术克隆柑橘大实蝇Orco基因,并对该基因进行生物信息学分析,采用实时荧光定量PCR技术检测其在柑橘大实蝇雌雄成虫不同组织中的表达谱。克隆获得柑橘大实蝇Orco基因并命名为BminOrco,该基因开放阅读框全长1 431 bp,编码476 个氨基酸,含有7 个跨膜结构域,氨基端位于膜内,羧基端位于膜外;预测该蛋白等电点为8.69,分子质量53.41 ku,无信号肽,亲水性总平均值为0.296,是疏水蛋白。同源性比对和系统树分析结果显示,BminOrco氨基酸序列与鞘翅目、双翅目、鳞翅目、半翅目、膜翅目和直翅目昆虫具有很高的同源性,其中与黑腹果蝇（Drosophila melanogaster）的DmelOrco同源性最高,达86.42%。实时荧光定量PCR分析显示,柑橘大实蝇Orco基因在触角中的表达量显著高于其他组织,在雌成虫触角中的表达量显著高于雄成虫;在其他组织中微量表达,比较而言,在头部（不含触角）的表达量略高于在胸、腹、足和翅中的表达量。     

Ultrastructure of the sensilla on antennae and mouthparts of larval and adult Plutella xylostella (Lepidoptera: Plutellidae)

Plutella xylostella (L.) (Plutellidae) is an important agricultural pest throughout the world. In this study, the morphology of antennal and mouthpart sensilla in the larvae and adults of P. xylostella (L.) was observed by using a scanning electron microscope. The larval antennae possess six sensilla basiconica, two sensilla chaetica and one sensillum styloconicum. Larval mouthparts possess six types of sensilla: sensilla chaetica, sensilla digitiformia, sensilla epipharyngeal, sensilla basiconica, sensilla styloconica and sensilla placodea. In the adult, seven types of sensilla are found on the antennae in males and six types of sensilla (sensilla basiconica absent) occur in females. Sexual dimorphism is also found in the number and size of these sensilla on the antennae of adults. We describe for the first time the five types of sensilla on the mouthparts of the adult of P. xylostella. This study provides useful information for further research into the function of these sensilla, and better understanding the behavioral mechanisms involved in pest control.     

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.     

大灰象甲非典型气味受体基因SvelOrco的克隆及组织表达谱分析

旨在克隆大灰象甲Sympiezomias velatus的Orco基因，明确其分子特征及其组织表达谱。通过分析成虫触角转录组数据并利用RT-PCR克隆出大灰象甲Orco基因，对大灰象甲Orco蛋白进行结构分析和系统发育分析，并采用qPCR测定Orco基因在大灰象甲成虫不同组织中的表达量。克隆获得大灰象甲Orco基因并命名为SvelOrco(GenBank登录号：OM417062),其开放阅读框长1 449 bp,编码482个氨基酸。预测SvelOrco蛋白的分子质量为53.49 ku,等电点为5.66,主要由α-螺旋和无规则卷曲组成，有7个跨膜结构域。系统发育分析表明大灰象甲与云杉八齿小蠹Ips typographus亲缘关系最近。qPCR结果显示：SvelOrco主要在大灰象甲成虫触角上表达，雄虫触角表达量最高，是雌虫触角表达量的1.22倍，SvelOrco在翅部也有少量表达，在头、胸、腹和足的表达量极低。     

捕食性天敌叉角厉蝽生长发育、繁殖及各虫态形态特征观察

【目的】明确叉角厉蝽[Eocanthecona furcellata(Wolff)]从卵到成虫发育历期及繁殖能力,比较叉角厉蝽各虫态的主要形态鉴别特征,为叉角厉蝽的扩繁及生防潜能开发提供理论支持。【方法】在室内条件下,以云南元江采集并在室内扩繁6代以上的叉角厉蝽为研究对象,在培养皿中采用单头饲养的方法测定叉角厉蝽生长发育指标(卵孵化率、若虫存活率、卵到成虫的发育历期),在饲养盒中雌雄虫配对共培养测定其成虫繁殖力特征。运用显微照相系统比较叉角厉蝽各虫态的主要形态特征。【结果】叉角厉蝽从卵到成虫的平均发育历期为29.00±1.41 d,卵历期为8.00±0.21 d,若虫期为19.76±0.12 d,1~5龄若虫平均发育历期为3.24~5.38 d,卵孵化率为92.91%,若虫存活率为80.21%,雌、雄成虫的平均寿命分别为35.40±1.96和37.40±2.65 d;叉角厉蝽雌成虫交配后,一生平均产卵次数为4.73±2.01次,最多9次,最少2次,平均产卵前期为7.87±2.75 d,产卵期持续14.33±5.13 d,雌成虫与雄成虫交配单次平均产卵量为59.46±15.77粒,未交配雌成虫单次平均产卵量为29.34±15.31粒且为无效卵,最终无法孵化。叉角厉蝽卵为矮杯形或圆筒形,具卵盖,边缘有10~12根刺状精孔突,有金属光泽;1~5龄若虫体色红黑相间,若虫触角4节。1龄若虫触角第2和第3节末端有明显白色环,喙暗红色,接近体长,无翅芽;2龄若虫头部前段扁平突出,触角节间环变为红色,喙黑色;3龄若虫触角第2和第3节末端红色环明显,喙长为体长的一半;4龄若虫喙长为体长的一半,胸部刻点明显,出现翅芽,并延伸至胸部末端;5龄若虫喙暗红色,短于体长一半,中足和后足胫节中部出现白斑,翅芽延伸至腹部第3节;成虫体色黄褐与黑褐混杂相间,密布刻点,触角5节,喙黄褐色,最后一节黑色,喙长短于体长的一半,前胸背板侧角呈剑叉状突出,雄虫腹部近三角形,雌虫腹部卵圆形。【结论】明确了叉角厉蝽的发育历期和繁殖能力,证实叉角厉蝽无孤雌生殖现象。农业生产中可以喙的颜色、长度及翅芽发育作为叉角厉蝽主要形态鉴别特征。     

中华蜜蜂NPC2基因家族克隆及表达模式分析

【背景】 作为我国本土重要的资源昆虫,中华蜜蜂（Apis cerana cerana,简称中蜂）对我国初冬季低温开花植物的传粉具有重要的生态价值,其传粉习性与嗅觉系统密切相关。前期对中蜂采集蜂高、低温处理后的触角转录组数据分析发现,与昆虫嗅觉相关的尼曼匹克C2型蛋白（Niemann-Pick type C2 protein,NPC2）基因家族在低温时表达量上升。【目的】 以中蜂NPC2家族基因为研究对象,克隆并分析其结构特征和表达谱以及高、低温处理下表达量的差异,为深入研究中蜂NPC2基因家族在中蜂低温适应的嗅觉感受功能提供参考。【方法】 基于中蜂高、低温转录组测序结果,利用RT-PCR克隆获得中蜂NPC2基因ORF序列,进行系统进化树分析和三维结构预测,然后通过实时荧光定量PCR分析中蜂NPC2基因在不同发育时期、组织的时空表达谱,以及高、低温时的表达量变化。【结果】 获得4个中蜂NPC2基因——AcNPC2a、AcNPC2b、AcNPC2c、AcNPC2d的ORF全长,分别为447、480、459和465 bp,编码148、159、152和154个氨基酸,预测蛋白分子量为16.12—18.53 kD,等电点分别为7.98、7.57、6.56和6.34。进化树分析显示AcNPC2与意大利蜜蜂的NPC2同源序列亲缘关系最近,与其他膜翅目昆虫NPC2也有一定相似性。实时荧光定量PCR结果显示,AcNPC2a在新出房蜂的腹部表达量最高,其次是在哺育蜂的腹部以及幼虫期;AcNPC2b在新出房蜂的胸部表达最高,在采集蜂的头部、胸部和后足也有表达;AcNPC2c在哺育蜂和采集蜂的触角中呈高丰度表达;AcNPC2d在采集蜂的头部表达量最高。经低温处理后,4个AcNPC2基因在采集蜂触角中的表达量均有所上升,但差异不显著。【结论】 AcNPC2具有NPC2蛋白的保守结构,其基因家族成员在中蜂的时空表达谱中呈现多样性,其中AcNPC2c在触角中呈高丰度表达,表明其与中蜂嗅觉感受功能关系密切。AcNPC2基因家族在低温时采集蜂触角中表达量均有所上升,表明该基因家族有可能参与中蜂的低温适应性,或与初冬季访花行为有关。