Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera:Thripidae)

Heat shock protein 70（HSP70） is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length c DNAs of HSP70s gene（Fohsc704 and Fohsc705） were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of c DNA. Real-time PCR was used to analyze the HSP70 expression patterns. The c DNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids（aa） with a calculated molecular weight of 75 and 54 k Da, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported Fo HSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.

茶黑刺粉虱与樟刺粉虱(半翅目:粉虱科)形态特征的比较观察

通过比较研究茶黑刺粉虱Aleurocanthus camelliae Kanmiya&Kasai和樟刺粉虱Aleurocanthus cinnamomi Takahashi形态特征,有助于更好地区分这两种刺粉虱种类。采集这两种粉虱标本,通过体视显微镜和扫描电镜对茶黑刺粉虱和樟刺粉虱不同龄期形态结构进行了系统观察和比较研究;同时将伪蛹制作玻片标本观察。茶黑刺粉虱和樟刺粉虱卵、1龄若虫和2龄若虫形态结构比较相似,但是伪蛹形态不同,刺的数量及分布有较大的区别。茶黑刺粉虱中央区隆起,尤其是管状孔区域更明显,位于一个凸起的瘤突上;亚缘区上有10~11对刺排列,樟刺粉虱亚缘区具有刺14对,其中头胸部5对,腹部9对。茶黑刺粉虱管状孔隆起,亚心形或近圆形,盖瓣心形,几乎充塞了整个管状孔区域;樟刺粉虱管状孔半圆形,盖瓣半圆形,充塞管状孔一半区域。两种刺粉虱成虫整体上比较相似,复眼、胸部和腹部及前翅基部为橙黄色;前后翅及身体上的白色粉状物都是由后足将腹部蜡盘分泌物覆盖上的,茶黑刺粉虱的粉状物比樟刺粉虱要多。前后翅上有不同形状的白色斑纹,左右翅斑相连,但是樟刺粉虱前翅后缘全白,而茶黑色粉虱前翅后缘仅有部分白斑。本研究结果有助于更好地区分樟刺粉虱和茶黑刺粉虱,为农林生产过程中准确鉴定和防治提供一定的参考资料。

Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis(Walker)

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.

No evidence for an effect of Wolbachia on mtDNA variation and evolution in natural populations of Sesamia inferens(Lepidoptera: Noctuidae)

Wolbachia are widespread maternally-inherited endosymbiotic bacteria that infect numerous arthropods.This study represents a thorough survey of the Wolbachia infection patterns in the pink stem borer,Sesamia inferens(Walker),an important rice pest in China,based on nucleotide comparisons for the surface protein(wsp)and cell division protein(ftsZ)genes.The effects of Wolbachia on mtDNA variation and evolution of S.inferens were also investigated.Although we identified six genetically diverse strains,we found infections to be infrequent,with only 7.8%of hosts infected,and identified geographical differences in infection rates between southern and northern populations.Nucleotide indexes(haplotype diversity(Hd),nucleotide diversity(π)and number of variable sites(S)of mtDNA in infected populations were not significantly lower or higher than that in the uninfected populations.Furthermore,there was no association between Wolbachia infection status and phylogeny of mtDNA haplotypes.Analysis of molecular variance(AMOVA)showed that significant differentiation mainly existed within groups rather than among the groups.Additionally,using Tajima’s D and Fu’s F values,the mtDNA genes did not deviate significantly from neutral evolution.Taken together these results indicate that currently there were no effects of Wolbachia infection on host mtDNA variation and evolution in S.inferens.

Molecular cloning and expression patterns of two small heat shock proteins from Chilo suppressalis(Walker)

Small heat shock protei ns(sHSPs)are a very complex protei n superfamily that increase in sect temperature tolera nee.In order to deeply understand the function and role of sHSPs in Chilo suppressalis(Walker),this study isolated and identified two CsHSP genes lacking introns from C.suppressalis,Cshsp23.9 and Cshsp27.3.The cDNA full-length of Cshsp23.9 and Cshsp27.3 were 909 and 1036 bp encoding 220 and 242 amino acids,respectively.Alignment with homologs and phylogenetic analysis indicated Cshsp23.9 and Cshsp27.3 were two new types of Cshsps in C.suppressalis.Real-time quantitative PCR(qPCR)revealed that Cshsp23.9 had the highest relative expression in hindgut compared with other tissues(head,epidermis,foregut,midgut,fat body,Malpighian tubules,and hemocytes),while Cs/?sp27.3 expressed the highest in fat body con tent.Whe n exposed to thermal stress from-11 to 43°C for 2 h,two genes showed differe nt expression patterns.Cshsp23.9 did not respond to low temperature,but could be up-regulated by high temperature and the highest expression temperature was at 36°C.Cshsp27.3 could only be induced by mild temperature,with the highest expression at 15 and 30°C.In conclusion,Cshsp23.9 and Cshsp27.3 existed in different tissues/organs of C.suppressalis,and played different important roles in C.suppressalis to resist temperature stress and regulate physiological activities.

Genes encoding heat shock proteins in the endoparasitoid wasp,Cotesia chilonis,and their expression in response to temperatures

Five genes encoding heat shock proteins（HSPs）, Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90, were cloned and sequenced from Cotesia chilonis using RT-PCR and RACE. The c DNA sequences of Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90 were 1 265, 2 551, 2 094, 2 297 and 2 635 bp in length, respectively, with a molecular weight（MW） of 39.1, 60.6, 71.45, 70.19 and 82.92 k Da, respectively. The predicted amino acid sequences of these proteins showed high similarities with published HSPs of other insects in Hymenoptera. Analysis of genomic DNAs indicated that Cchsp40, Cchsp60, Cchsp70 and Cchsp90 lacked introns, but Cchsc70 contained an intron. The results also suggested that CcH SP40 in C. chilonis was the Type II HSP40, Cc HSP60 was a member of the mitochondrial HSP60 family, and Cc HSP90 was a part of cytoplasmic HSP90 A family. Expression patterns varied in the five Cchsps in response to temperature. Expression of Cchsp40 and Cchsp60 was induced significantly by cold but not heat stress. Cchsp70 and Cchsc70 showed similar response to the thermal stress and could be induced by both cold and heat, but their expression levels were consistently lower than that of Cchsp40 and Cchsp60. Cchsp90 could be induced by heat stress and mild cold, but not cold stress. In addition, the results demonstrated Cchsc70 might be constitutive and inducible protein that was expressed during normal cell functioning and also up-regulated in response to stressful stimuli while Cchsp70 was solely inducible protein induced by temperature changes. Overall, results generated from this study could significantly advance the understanding of Cchsps in response to temperature and provide important biological information for C. chilonis insects that reared under different temperatures.

Genetic Variations Among Liriomyza sativae Blanchard Populations Indicated by β-tubulin Gene Sequences

To analyze the genetic differentiation of the host- and geo-populations of Liriomyza sativae Blanchard, the β-tubulin gene of 5 host-populations and 6 geo-populations of L. sativae was sequenced. Subsequently, the sequences were analyzed by biosoftware DNAStar and MEGA, and the phylogenetic trees constructed. The results obtained by the two softwares were similar, that is, the sequences of β-tubulin gene were more than 98% homologous, among the host- and geo-populations of L. sativae, with only 8 variable sites, but no insertions and deletions were detected. It seems that differentiations in β-tubulin gene among the host- and geo-populations of L. sativae are related to the hobby to hosts and the geographical distributions, respectively.

Insect heat shock proteins and their underlying functions

Considering huge number of insect species in the world,studies of heat shock proteins on insects are still very limited.Special focus of＂Insect heat shock proteins and their underlying functions＂provides a comprehensive knowledge for the given topics,which focuses on the heat shock proteins from four insect species：i）Five heat shock proteins（HSPs）from Cotesia chilonis were identified,and their expressional patterns under different temperatures were examined.