Control Efficiency of Four Biological Pesticides against Pieris rapae Linne and Their Impacts on Yield and Benefit of Chinese Cabbage

[ Objective] The paper was to screen out ideal biological pesticides, in order to provide guidance for pollution-free control against Pieris rapae Linne. [Method] Taking Chinese cabbage variety Taiyuan Erqing as the test material, the field control efficiencies of four pesticides including PrGV · Bt WP 1 000 times dilution, NPV · Bt SC 750 times dilution, 0.5% azadirachtin EC 750 times dilution, and 0.3% matrine AS 500 times dilution against P. rapae were studied, and their impacts on yield and planting benefits of Chinese cabbage were also determined. Using foliar spray method, the pesticides were sprayed for the first time when the second or third instar larvae of P. rapae first occurred in fields, and sprayed for the second time with the interval of 15 d. The fields sprayed with beta-cypermethrin EC （organic chemical pesticide） and water were set as control. [ Result] NPV · Bt SC 750 times dilution had the best effect after spraying for two times： the control efficiency against P. rapae at 15 d after spraying was 90.11% ; the damage rate of Chinese cabbage was only 0.21%, while the commodity rate reached 100% ; compared with chemical pesticide spraying, the commodity yield （177 262.5 kg/hm2 ） and the income after deducting spraying cost （48 858.5 yuan/hm2 ） were increased by 14.7% and 13.75%, respectively. [ Conclusion] Although biological pesticides are more expensive, they have long persistence and good control effect, resulting in green and safe Chinese cabbage with high commodity rate and yield, and higher eventual economic benefit after deducting spraying cost.

Cloning and Sequence Analysis of lef-3 Gene from Pieris rapae Granulovirus

To better understand the origination and evolution of lef-3 gene from baculovirus genome and determine the relationships between various viruses at molecular level, late expression factor 3 gene （lef-3） fragments of Pieris rapae granulovirus were obtained through conventional PCR method and sequenced after cloned into T-vector. Then, bioinformatics analysis on lef-3 gene and its encoding sequences were conducted by using bid-softs. Four mutations were appeared in the ORF of cloned lef-3 gene, which did not alter the characteristics of amino acids. It was inferred that PiraGV LEF-3 protein contained 399 amino acids, the molecular weight of which was 3.99 kD. Prediction of the LEF-3 advanced structure and homology comparison between other LEF-3 from various baculoviruses showed that the lef-3 gene might encode the single-stranded DNA-binding protein. The result of BLAST revealed that the lef-3 gene only existed in Lepidoptera host for the baculovirus genome, and the evolution analysis illustrated that lef-3 gene could be divided into 3 groups including one granulovirus （GV） group and 2 nucleopolyhedrovirus （NPV） groups. The selection pressure analysis of GV lef-3 gene coding region showed that the majority of lef-3 genes performed negative selection, while the Ka/Ks differed from different lef-3 gene, to some extent, which also performed positive selection. The origination analysis revealed that lef-3 gene of baculovirus might derive from bacteria. The lef-3 gene of PiraGV was cloned successfully and the possible patterns of origination and evolution were speculated through bioinformatics analysis.

Field Efficacy Trials of Different Pesticides against Pieris rapae and Plutella xylostella

Field efficacy trials of different pesticides against Pieris rapae and Plutella xylostella showed that seven pesticides had certain control effects against P.rapae and P. xylostella. 240 g/L Chlorfenapyr SC had the most ideal control effect,with quick effect and long persistence,and the control effects against P. rapae and P. xylostella were 91. 96% and 95. 73% after application for 7 d,respectively. 25% Thiamethoxam WDG 3 000 times dilution had the poorest control effect,and quick effect and persistence were not ideal; the control effects against P. rapae and P. xylostella were 49. 21% and 57. 20% after application for 7 d,respectively. The remaining pesticides had good control effect against both P. rapae and P. xylostella,with certain persistence. Slight injury such as yellowing tender leaves appeared in the area treated with 50% thiocyclam SP,although the injury was reversible,it was still not recommended to use; no other treatments had adverse effects on growth of cabbage.

Enzymatic properties of phenoloxidase from Pieris rapae （Lepidoptera） larvae

The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant （Kin） and maximal velocity （V） of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.

Parasitism of Pieris rapae （Lepidoptera： Pieridae） by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae）： Effects of parasitism on differential hemocyte counts, micro- and ultra-structures of host hemocytes

Parasitism by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae） by using only its associated venom, can suppress the immunal responses of Pieris rapae （Lepidoptera： Pieridae）. However, up to now, current knowledge of the mech- anisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy （TEM）. Five hemocyte types, prohemocytes （PRs）, granulocytes （GRs）, plasmatocytes （PLs）, oenocytoids （OEs） and coagulocytes （COs）, were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.

cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparum

This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length eDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein （1.42% methionine and 18.82% aromatic amino acids）. Reverse transcription- polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum.

Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

Polyphenol oxidase （PPO） is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO a metalloenzyme oxidase, catalyzes the oxidation of o-diphenol to o-quinone. The present paper describes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The results show that benzaldehyde, phydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was estimated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analyses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, phydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyanobenzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other compounds. These results provide a basis for developing PPO inhibition-based pesticides.

Proteome changes in the plasma of Pieris rapae parasitized by the endoparasitoid wasp Pteromalus puparum

Parasitism by the endoparasitoid wasp Pteromalus puparum causes alterations in the plasma proteins of Pieris rapae. Analysis of plasma proteins using a proteomic approach showed that seven proteins were differentially expressed in the host pupae after 24-h parasitism. They were masquerade-like serine proteinase homolog (MSPH), enolase (Eno), bilin-binding protein (BBP), imaginal disc growth factor (IDGF), ornithine decarboxylase (ODC), cellular retinoic acid binding protein (CRABP), and one unknown function protein. The full length cDNA sequences of MSPH, Eno, and BBP were successfully cloned using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the transcript levels of MSPH and BBP in the fat bodies of host pupae were inducible in response to the parasitism and their variations were consistent with translational changes of these genes after parasitism, while the transcript levels of Eno and IDGF were not affected by parasitism. This study will contribute to the better understanding of the molecular bases of parasitoid-induced host alterations associated with innate immune responses, detoxification, and energy metabolism.

商陆等3种植物提取物对菜粉蝶的拒食作用研究(英文)

[Objective] The research aimed to study antifeedant activity of Phytolacca acinosa Roxb., Setaria viridis （L.） Beauv and Viola yedoensis Makino extracts against Pieris rapae. [Method] Activity material was extracted from S. viridis （L.）, P. acinosa and V. yedoensis using acetone cold soak method, and non-selective antifeedant activity of extracts to Pieris rapae larva was determined by using lobular plate addition method. [Result] The results showed that the acetone leaching agent of P. acinosa had most obvious antifeedant effects on Pieris rapae. The antifeedant rate were 74.53% and 82.34% at 24 and 48 h respectively. With the concentration increasing, the antifeedant effect of P. acinosa extracts increased. The antifeedant rate of 0.050 g/ml treatment was the highest, being 74.53% and 82.34% at 24 and 48 h. [Conclusion] P. acinosa could be studied and utilized as potential botanical insecticide.

Molecular characterization of three Hsp90 from Pieris and expression patterns in response to cold and thermal stress in summer and winter diapause of Pieris melete

Heat shock proteins （Hsps） have been linked to stresses and winter diapause in insects, but whether they are components of summer diapause is still unknown. In this study, complementary DNAs of lisp90 from Pieris melete, Pieris rapae and Pieris canidia named PmHsp90, PrHsp90 and PcHsp90, respectively, were cloned and sequenced. The deduced amino acid sequence consisted of 718 amino acid residues with a putative molecular mass of 82.6, 82.6 and 82.7 kDa, respectively. The amino acid sequences contained all of the five conserved signature motifs in the Hsp90 family and a bHLH protein folding activity region. The differential expression pattern of PmHsp90 in response to summer diapause and winter diapause, which are related to heat/cold stress, was investigated. Cold stress induced Hsp90 up-regulation in summer and winter diapause pupae, but not in non-diapause individuals. Heat shock up-regulated PmHsp90 gradually with an increase in temperature in summer diapause, and PmHsp90 was rapidly up-regulated in winter diapause. After 30 min heat shock at 39℃, substantial up-regulation of PmHsp90 transcript levels were observed both in summer and winter diapause. However, in non-diapause a relatively stable expression was found under different durations of 39℃ heat shock. Compared to the optimal treatment of 18℃ for diapause development, a high temperature acclimation of 31 ℃ induced PmHsp90 up-regulation in summer diapause, whereas a low temperature acclimation of 4℃ induced up-regulation in winter diapause. The current results indicate that Hsp90 may play an important role in response to heat/cold stress both in summer and winter diapause.

Transcriptional responses of Brassica nigra to feeding by specialist insects of different feeding guilds

Plants show phenotypic changes when challenged with herbivorous insects. The mechanisms underlying these changes include the activation of transcriptional responses, which are dependent on the attacking insect. Most transcriptomic studies on crucifer-insect interactions have focused on the model plant Arabidopsis thaliana, a species that faces low herbivore pressure in nature. Here, we study the transcriptional responses of plants from a wild black mustard （Brassica nigra） population to herbivores of different feeding guilds using an A. thaliana-based whole-genome microarray that has previously been shown to be suitable for transcriptomic analyses in Brassica. Transcriptional responses of B. nigra after infestation with either Pieris rapae caterpillars or Brevicoryne brassicae aphids are analyzed and compared. Additionally, the insect-induced expression changes of some individual genes are analyzed through quantitative real-time polymerase chain reaction. The results show that feeding by both insect species results in the accumulation of transcripts encoding proteins involved in the detoxification of reactive oxygen species, defensive proteins and glucosinolates and this is correlated with experimental evidence in the literature on such biochemical effects. Although genes encoding proteins involved in similar processes are regulated by both insects, there was little overlap in the induction or repression of individual genes. Furthermore, P. rapae and B. brassicae seem to affect different phytohormone signaling pathways. In conclusion, our results indicate that B. nigra activates several defense-related genes in response to P rapae or B. brassicae feeding, but that the response is dependent on the attacking insect species.