Cloning and expression pattern of a hemolin homologue from the diamondback moth, Plutella xylostella

Hemolin has been known to play a key role in insect innate immunity. In an attempt to examine expression pattern of the Hemolin gene in the diamondback moth, Plutellea xylostella, the full-length cDNA of Hemolin was cloned using 5′-RACE PCR technique. The cDNA contained a 5′ untranslated region of 48 nucleotides and a 3′ untranslated region of 198 nucleotides, including a stop codon (TAA) and a poly (A) tail. It consists of 1,401 bp with an open reading frame of 1,245 bp, encoding 414 amino acids. The deduced amino acid sequence of PxHemolin has relatively low identities (35?42%) to various insect Hemolins. However, it has high three-dimensional structural similarity to Hemolin. Interestingly, analysis of spatial expression pattern of PxHemolin shows that it was highly expressed in the Malpighian tubule and the silk gland although it was also detected in fat body and gut. Furthermore, PxHemolin mRNA was highly induced 3 hr after immune-challenging with lipopolysaccharide and was gradually up-regulated after laminarin treatment. These data suggest that PxHemolin may play a role in innate immune responses although it remains to further elucidate the precise biological functions in P. xylostella.     

Cloning,ligand-binding,and temporal expression of ecdysteroid receptors in the diamondback moth,Plutella xylostella

Background

RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.

Results

Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.

Conclusions

Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.     

小菜蛾的耐热性

小菜蛾是世界范围内十字花科蔬菜上的重要害虫.临界高温(critical thermal maxi-mum,CTMax)是昆虫耐热性的常用指标.采用动态加热方法,利用自行组装的装置测定了小菜蛾的临界高温,以此作为其耐热性指标,研究发育阶段、饲养温度、世代、性别和热激对小菜蛾耐热性的影响.结果表明:25℃下饲养的小菜蛾4龄幼虫的CTMax均值为50.31℃,显著高于1龄幼虫(43.03℃)、2龄幼虫(46.39℃)、3龄幼虫(49.67℃)以及雌性成虫(45.76℃)和雄性成虫(47.73℃);不同饲养温度(20、25和30℃)下成虫耐热性无显著差异;30℃下饲养1代、3代及6代的不同世代成虫CTMax也无显著变化;所有处理雌雄成虫的CTMax无显著差异;40℃下45 min热激可使5日龄雄成虫的CTMax值从45.51℃增加到46.49℃.     

小菜蛾碱性磷酸酶基因cDNA片段的克隆及其序列分析

利用DNAman设计简并引物,通过反转录一多聚酶链式反应(RT-PCR)克隆小菜蛾Plutella xylostella(L.)抗性、敏感种群中可能与苏云金芽孢杆菌(Bt)抗性相关的碱性磷酸酶基因。琼脂糖电泳结果显示扩增条带与预期片段长度一致,阳性克隆经测序获得了403bp的基因片断。经blast比较得出所克隆基因属于碱性磷酸酶基因。与敏感种群相比,抗性种群中该基因片段有18个碱基发生变化,有1个氨基酸发生替换(缬氨酸转变为苏氨酸)。研究结果对于进一步研究小菜蛾全基因结构、功能有重要的意义。     

印楝素乳油对小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用。结果表明,其控制作用主要表现在对成虫产卵的显忌避作用。对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害为等方面,印楝素乳油0.005mL/59.0%,使小菜蛾种群趋势指数（I值）降为2.4干扰作用控制指数（IIPC）为0.1079,采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显的控制作用表现在种群发展初期对成虫的忌避作用。     

印楝素乳油x对-小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用.结果表明,其控制作用主要表现在对成虫产卵的显著忌避作用、对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害力等方面.印楝素乳油0.005 mL/L处理对成虫的忌避率为78.3%,对幼虫的选择性和非选择性拒食率分别为71.8%和62.9%,幼虫总取食量是对照的59.0%,使小菜蛾种群趋势指数(I值)降为2.4,干扰作用控制指数(1lpc)为0.1079.采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显著的控制作用表现在种群发展初期对成虫的忌避作用.     

Biological control of the diamondback moth, Plutella xylostella: A review

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive cosmopolitan insect pests of brassicaceous crops. It was the first crop insect reported to be resistant to DDT and now, in many crucifer producing regions, it has shown significant resistance to almost every synthetic insecticide applied in the field. In certain parts of the world, economical production of crucifers has become almost impossible due to insecticidal control failures. Consequently, increased efforts worldwide have been undertaken to develop integrated pest management (IPM) programs, principally based on manipulation of its natural enemies. Although over 130 parasitoid species are known to attack various life stages of DBM, most control worldwide is achieved by relatively few hymenopteran species belonging to the ichneumonid genera Diadegma and Diadromus, the braconid genera Microplitis and Cotesia, and the eulophid genus Oomyzus. DBM populations native to different regions have genetic and biological differences, and specific parasitoid strains may be associated with the specific DBM strains. Therefore, accurate identification based on genetic studies of both host and parasitoid is of crucial importance to attaining successful control of DBM through inoculative or inundative releases. Although parasitoids of DBM larvae and pupae are currently its principal regulators, bacteria-derived products (e.g., crystal toxins from Bacillus thuringiensis) and myco-insecticides principally based on Zoophthora radicans and Beauveria bassiana are increasingly being applied or investigated for biological control. Viruses, nematodes and microsporidia also have potential as biopesticides for DBM. When an insect pest is exposed to more than one mortality factor, there is the possibility of interactions that can enhance, limit, or limit and enhance the various aspects of effectiveness of a particular control tactic. This paper reviews the effectiveness of various parasitoids and entomopathogens against DBM, interactions among them, and their possible integration into modern IPM programs.     

云南弥渡小菜蛾发生及防治

通过田间调查 ,查明弥渡县小菜蛾年发生 1 0～ 1 2代 ,发生危害期有 9个月 ,一般有两个发生高峰期 ,即 4～ 6月和 9～ 1 0月 ,春季危害比秋季重 ;其发生影响因素主要有温度、降雨、十字花科蔬菜种植面积以及寄生蜂的种类和数量。针对小菜蛾发生规律及影响因素 ,提出了相应的防治措施。     

小菜蛾在温带地区越冬研究进展

小菜蛾是世界性重要害虫。在热带和亚热带地区冬季的十字花科植物上能正常发育繁殖,可见到各种虫态。但在温带冬季十字花科植物不能生长的地区,小菜蛾的越冬成为一个重要生态学问题。综述了亚洲、北美洲和欧洲小菜蛾越冬的研究进展。小菜蛾在日本的北海道、本州岛的北陆和东北大部分地区不能越冬,越冬北限相当于冬季积雪覆盖时间为60d的区域;在中国,小菜蛾不能在寒冷的东北地区越冬,在长江中下游以南地区冬季可见各虫态,但越冬北限尚不清楚;小菜蛾在冬天气候温和的韩国以及澳大利亚东南部继续发生;北美洲的加拿大西部和安大略地区大量的试验证明小菜蛾不能成功越冬,在美国南部小菜蛾冬季可正常发生,北部小菜蛾的越冬尚未见系统研究报道,但确认春季从南部运输的受小菜蛾感染的甘蓝等种苗是美国北部的重要虫源。小菜蛾在欧洲各地越冬的系统研究未见报道,没有证据表明在英国小菜蛾会发生有显著意义的越冬。目前小菜蛾越冬研究主要采用冬季直接试验观察和基于耐寒性试验的越冬预测两种方法。冬季直接试验观察法包括:(1)利用人工饲养的小菜蛾在田间各种潜在的越冬场所的越冬试验;(2)在秋播、野生或残留的十字花科植物上进行冬季种群的系统抽样调查;(3)越冬前后在前茬为十字花科植物的田块广泛搜寻普查小菜蛾的存活个体。基于耐寒性试验的越冬预测法:在获取小菜蛾越冬场所温度的基础上设计低温处理模式,试验低温处理后小菜蛾的存活率及后续发育和生殖。将试验数据和各地气温或小气候相结合,对小菜蛾在的越冬可能性进行推断。     

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth,Plutella xylostella

Calreticulin (CRT) plays pivotal roles in Ca²⁺ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full-length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly-A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.     

小菜蛾化学生态学研究现状与展望

本文综述了几种植物吸引或阻抑小菜蛾成虫产卵或幼虫取食的效应。完整的、机械损伤的和菜粉蝶为害的甘蓝类蔬菜释放出数十种醇、醛、酯、酮、硫化物、羧酸类、异硫氰酸酯类和萜烯类挥发性化合物。机械损伤的甘蓝和小菜蛾、菜粉蝶及蜗牛为害的甘蓝释放的挥发物引诱菜粉蝶绒茧蜂。小菜蛾为害甘蓝释放的挥发物引诱菜蛾绒茧蜂。小菜蛾性信息素基本成分是顺 -1 1 -十六碳烯醛和顺 -1 1 -十六碳烯乙酸酯。当顺 -1 1 -十六碳烯醛、顺 -1 1 -十六碳烯乙酸酯和顺 -1 1 -十六碳烯醇以 5∶5∶0 1或顺 -1 1 -十六碳烯醛、顺 -1 1 -十六碳烯乙酸酯、顺 -1 1 -十六碳烯醇和顺 -9-十四碳烯乙酸酯按70∶3 0∶1∶0 0 1比例制成的诱芯诱蛾效果较好。宜深入探究寄主植物—小菜蛾—菜蛾绒茧蜂间的通讯机制 ,开发高效诱芯。     

Inheritance of resistance and cross resistance pattern in indoxacarb-resistant diamondback moth Plutella xylostella L.

Leaf-dip assay of Plutella xylostella against indoxacarb showed that the concentration that produced 50% mortality (LC₅₀) of indoxacarb ranged from 20.1 to 11.9 ppm, with highest in Nasik and lowest levels in Coimbatore strains. In selection studies, the LC₅₀ of indoxacarb was 18.5 ppm at generation 1 (G1), which increased to 31.3-fold (167.8 ppm) resistance after ten exposed generations (G10) as compared to unexposed. The LC₅₀ of quinalphos was 74.4 ppm, which increased to 10.0-fold (631.5 ppm) resistance after G10. The LC₅₀ of cypermethrin resistant strain resulted in an 11.5-fold increase in resistance after G10. In P. xylostella , heritability (h²) after ten generations of selection was estimated at 0.4. The number of generations required for tenfold increase in LC₅₀ (1/R) were 6.7. The response to indoxacarb selection in P. xylostella was 0.2 and the selection differential was estimated as 0.4. The phenotypic standard deviation was 0.2. Reciprocal crosses between indoxacarb-resistant and susceptible strains showed that the inheritance of indoxacarb resistance was autosomal. The degree of heritability (D_LC) (0.4, 0.4) indicated incomplete recessive inheritance of indoxacarb resistance.     

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).     

小菜蛾鞣化激素基因克隆及其功能分析

鞣化激素是调节昆虫表皮骨化和翅膀发育的一种神经激素, 尽管已经在许多不同种昆虫上克隆了鞣化激素基因, 但是关于小菜蛾 Plutella xylostella鞣化激素及其基因的研究至今未见报道。本研究克隆了两个小菜蛾鞣化激素基因Pxbursα和Pxbursβ （GenBank 登录号分别为KF498645和KF498646）全长cDNA, 其序列长度分别为537 bp和360 bp, 与已报道的其他昆虫的鞣化激素氨基酸序列一致性分别为51%~68% 和37%~57%。实时定量PCR分析发现Pxbursα和Pxbursβ均在蛹期表达量高, 而在幼虫期和成虫期的表达量低。以Pxbursα部分序列的双链RNA（dsRNA）饲喂小菜蛾4龄末期幼虫, 发现蛹期Pxbursα的表达受到了显著抑制, 小菜蛾的发育停滞在蛹期而无法正常羽化, 并最终死亡。由此推测, 小菜蛾鞣化激素基因在蛹期的大量表达对其生长发育和羽化具有重要的作用。     

浙江临海小菜蛾成虫数量季节消长规律

在浙江临海相隔60km的2个十字花科蔬菜种植区,于2002~2005年利用性诱剂诱捕器每日对小菜蛾Plutella xylostella(L.)雄成虫进行诱集并计数。对两地成虫数量季节消长与种群基数、气温、雨量和日照等因子的相关性做了统计分析,建立了5个预测模型。应用这些模型可预测全年36个旬期的雄成虫数量变化,从而可预测春季高峰期并对全年的发生量和为害程度进行预警,这对提高当地小菜蛾监测预警与综合防治水平具有实践意义。     

小菜蛾对几种常用药剂的敏感性测定

采用叶片浸溃法,在2001年10月和2002年10月分别测定了福州上街蔬菜基地的小菜蛾,对10种杀虫剂的敏感性。结果表明,上街菜区小菜蛾对灭多威、杀虫双、辛硫磷、毒死蜱、杀灭菊酯和功夫菊酯等农药极不敏感,已不能有效控制小菜蛾的危害。而小菜蛾对氟啶脲、阿维菌素、氟虫腈和虫螨腈比较敏感。通过两次毒力测定比较(2002年10月的LC50;2001年10月的LC50),结果表明,大多数杀虫剂敏感性变化较小。但小菜蛾对阿维菌素敏感性明显下降,毒力倍数达6．65倍,对氟虫腈毒力倍数相差1．48倍。此外,2002年10月小菜蛾对功夫菊酯的LC50为2001年10月的2．02倍。用浸叶法和浸溃法测定多杀菌素、氟虫腈与虫螨腈推荐浓度对小菜蛾幼虫的毒杀作用。结果表明3种药剂浸叶法处理效果均较理想。