Single base-resolution methylome of the silkworm reveals a sparse epigenomic map

Epigenetic regulation in insects may have effects on diverse biological processes. Here we survey the methylome of a model insect, the silkworm Bombyx mori, at single-base resolution using Illumina high-throughput bisulfite sequencing (MethylC-Seq). We conservatively estimate that 0.11% of genomic cytosines are methylcytosines, all of which probably occur in CG dinucleotides. CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting it has a positive role in gene transcription. We find that transposable elements, promoters and ribosomal DNAs are hypomethylated, but in contrast, genomic loci matching small RNAs in gene bodies are densely methylated. This work contributes to our understanding of epigenetics in insects, and in contrast to previous studies of the highly methylated genomes of Arabidopsis(1) and human(2), demonstrates a strategy for sequencing the epigenomes of organisms such as insects that have low levels of methylation.

Mass rearing of Aphidius gifuensis (Hymenoptera : Aphidiidae) for biological control of Myzus persicae (Homoptera : Aphididae)

The green peach aphid, Myzus persicae, is a major pest of tobacco, Nicotiana tabacum, in Yunnan province, China, where its control still depends on the use of insecticides. In recent years, the local government and farmers have sought to improve the biological control of this tobacco pest. In this paper, we present methods for mass rearing Aphidius gifuensis, a dominant endoparasitoid of M. persicae on tobacco plants in this region. The tobacco cultivar K326 (N. tabacum) was used as the host plant and M. persicae as the host insect. In the greenhouse, we collected tobacco seedlings for about 35 days (i.e., until the six-true-leaf stage), transferred them to 7.5-cm diameter pots, and kept these plants in the greenhouse for another 18 days. These pots were then transferred to an insectary-greenhouse, where the tobacco seedlings were inoculated with five to seven wingless adult M. persicae per pot. After 3 days, the infested seedlings were moved to a second greenhouse to allow the aphid population to increase, and after an additional 4 +/- 1 days when 182 +/- 4.25 aphid adults and nymphs were produced per pot, they were inoculated with A. gifuensis. With this rearing system, we were able to produce 256 +/- 8.8 aphid mummies per pot, with an emergence rate of 95.6 +/- 2.45%; 69% were females. The daily cost of parasite production (recurring costs only) was US$ 0.06 per 1000 aphid mummies. With this technique, we released 109 800 parasitoids in 1998, 196 000 in 1999, 780 000 in 2000, and 5 600 000 in 2001 during a 2-month period each year This production method is discussed with respect to countrywide usage in biological control and integrated control of M. persicae.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (in: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response. (c) 2007 Elsevier Ltd. All rights reserved.

CO2浓度升高对三种树木叶片化学和舞毒蛾幼虫取食及生长的影响

探讨全球气候变化的生物学和生态学效应是当今生态学中的热点，研究大气CO2浓度升高对植物-昆虫相互作用关系的影响具有重要的理论和实践意义。本文使用开顶式气室（Open-top chamber，OTC）在野外条件下研究了CO2浓度升高对三种树木（小青杨、白桦和蒙古栎）叶片化学成分含量的影响，以及树木叶片品质变化对一种广食性森林昆虫（舞毒蛾）幼虫取食、生长发育和取食偏嗜性的影响。得出如下结果：（1）CO2浓度升高对3个受试树种叶片中的营养成分及次生代谢物含量均有显著影响，总体表现为氮含量降低，而碳氮比、非结构性碳水化合物、总酚和缩合丹宁含量增加。叶片中的化学成分含量可随时间发生显著变化，不同树种、甚至同一树种不同冠层高度的叶片对CO2浓度升高的响应强度也是不同的。叶片的干物质含量和比叶重对CO2浓度升高的响应不显著。（2）室内非选择性取食实验、室内选择性取食实验以及上树取食饲养方式下的多龄期取食实验，均发现高浓度CO2处理组内舞毒蛾幼虫的生长发育受到显著抑制。但对四龄舞毒蛾幼虫所进行的短期生物测定并未发现不同CO2浓度处理下幼虫的生长发育速率、对食物的取食率和转化率等昆虫营养指标存在显著差异。（3）叶片品质的降低是导致舞毒蛾幼虫生长发育受抑制的主要原因。但是总体上，CO2浓度升高导致的叶片品质变化并未显著影响幼虫的取食率和取食量。（4）舞毒蛾幼虫对不同叶片种类表现出清晰的取食选择性，这种选择性在其幼龄期就可表现出来。幼虫对小青杨上层叶片有最显著的偏嗜性，对蒙古栎下层叶片有最明显的拒食性。但是CO2浓度升高导致的叶片品质变化对舞毒蛾幼虫的取食选择性和寄主偏嗜行为并未产生显著影响。（5）检测出高浓度CO2处理组内舞毒蛾幼虫虫粪中含有浓度更高的植物次生代谢物质（总酚和缩合单宁），这很可能是昆虫整体生长发育受抑制的重要原因之一。

若尔盖湿地3种两栖类的种间竞争及畜牧业对其食性的影响

从空间、时间、食物3个方面研究了若尔盖湿地3种两栖类的种间竞争，运用生态位理论探讨了3种两栖类利用环境资源的能力以及它们之间的共存模式，研究了3种两栖类年周期食性，并考察了畜牧业对3种两栖类食性及食物竞争格局的影响；此外，还通过实验室研究对2种两栖类幼体的种间竞争策略进行了考察。主要结果如下： 1、两栖类的空间资源利用状况：在3种两栖类成体生态位宽度的比较中，岷山蟾蜍（Bufo minshanicus）成体在牧场性质(0.41)、牛粪数量(0.42)、植被盖度(0.20)、地表温度(0.50)、地表湿度(0.51) 5个维度上的生态位宽度最窄；而倭蛙（Nanorana pleskei）成体在牛粪数量(0.81)、植被高度(0.63)、植被盖度(0.47)、小水体距离(0.68) 4个维度上的生态位宽度最宽。在3种两栖类亚成体生态位宽度的比较中，岷山蟾蜍亚成体在牧场性质(0.66)、牛粪数量(0.58)、植被高度(0.64)、小水体距离(0.51)、地表湿度(0.79) 5个维度的上生态位宽度最宽；倭蛙亚成体在牧场性质(0.39)、牛粪数量(0.30)、地表温度(0.18)、地表湿度(0.33) 4个维度上的生态位宽度最窄。高原林蛙（Rana kukunoris）在地表温度（成体：0.62；亚成体：0.56）、地表湿度（成体：0.84；亚成体：0.60）两个维度上具有较大的生态位宽度值，而在小水体距离维度上（成体：0.27；亚成体：0.14）的生态位宽度值则很小。比较3种无尾两栖类在不同生长阶段（成体、亚成体）的生态位宽度，发现高原林蛙和倭蛙的亚成体对栖息环境的要求更高。3种两栖类空间资源利用的相似程度很高，高原林蛙与倭蛙之间的生态重叠度(0.87)较之它与岷山蟾蜍(0.81)的生态位重叠度更大。 2、两栖类的日活动节律：高原林蛙成体、亚成体、岷山蟾蜍亚成体活动的最低气温为0℃、2℃、8℃；岷山蟾蜍和高原林蛙亚成体出现的数量与气温成极显著的正相关（r=0.797, p<0.001；r=0.794, p<0.001），高原林蛙成体出现的数量与气温有一定相关性（r=0.456, p<0.05）；晴天时两栖类的活动性明显高于阴天（p<0.001）；多云转晴天气，高原林蛙和岷山蟾蜍亚成体出现两次日活动高峰，分别为中午12:30左右和下午15:30～16:30之间；多云天气，高原林蛙和岷山蟾蜍亚成体出现两次日活动高峰，分别为9:30～10:30之间和15:30～16:30之间。 3、两栖类的食物资源利用状况：春、秋两季，高原林蛙最主要的食物是蜉金龟科(Aphodiidae)昆虫，相对重要性指数(IRI)最高（春季：35.28%，秋季：28.57%），其次为昆虫的幼虫，以及双翅目的毛蚊科(Bibionidae)、蝇科(Muscidae)、丽蝇科(Calliphoridae)昆虫，秋季，蝗虫是高原林蛙食物组成中的重要部分；岷山蟾蜍最主要的食物是蚂蚁(IRI，春季：85.54%，秋季：49.70%)，其次为蜉金龟科、象甲科(Curculionidae)、步甲科(Carabidae)、粪金龟科(Geotrupidae) 等鞘翅目昆虫；倭蛙春季的最主要食物也是蜉金龟科昆虫(IRI，春季：13.41%)，其次为蚂蚁、毛蚊科昆虫、昆虫的幼虫以及狼蛛科(Lycosidae)。3种两栖类中，倭蛙的食性生态位宽度相对较宽（0.43），而岷山蟾蜍（0.09）和高原林蛙（0.22）的生态位宽度较窄，与春季相比，两栖类在秋季的食谱更宽。以利用食物种类为标准，春季高原林蛙与倭蛙的生态位重叠度(0.40)比它与岷山蟾蜍的生态位重叠度（0.33）更大。 4、畜牧业对两栖类食性及食物竞争格局的影响：以藏牦牛粪为食物或寄居场所的昆虫，如蜉金龟科、粪金龟科、毛蚊科、蝇科、丽蝇科昆虫和某些昆虫幼虫，是3种两栖类食物谱中最主要的组成部分，蜉金龟科昆虫在高原林蛙食谱中的比例更高，高原林蛙可能从畜牧业发展中获得更多的好处，使之在食物竞争方面处于优势地位。与无放牧样地相比，在有放牧样地的中，两栖类食谱中的蜉金龟科昆虫数量更多(有放牧：31.94%；无放牧：21.32%)、出现频率更高(有放牧：76.38%；无放牧：44%)。然而在不同样地上（有放牧/无放牧），两栖类的食物组成无显著性差异(P=0.188)，两栖类的数量(P=0.075)、肥满度(P=0.537)均没有显著差别。 5、两栖类幼体的竞争策略：实验室条件下，通过活动性水平，变态时的体重、增长率和完成变态所需时间考察自然条件下常同水塘分布的中华蟾蜍（Bufo gargarizans）和高原林蛙蝌蚪的竞争策略。结果表明：中华蟾蜍蝌蚪在不同食物资源条件下，所选择的生存策略可能不同，即食物资源充足时，增加活动性获取更多食物，食物资源有限时，降低活动性且提前完成变态；与中华蟾蜍蝌蚪相比，在食物资源有限时高原林蛙蝌蚪获取食物能力可能更强。 This paper presented the study of competition of three amphibians (Rana kukunoris, Nanorana pleskei, Bufo minshanicus) based on spatial, temporal and dietary scales in Zoige wetland. We measured coexistence patterns of three amphibians and analyzed their ability of exploiting resource. Effects of grazing on the diet composition and diet competition of amphibians were analyzed by their diet composition during spring and autumn. Furthermore, we examined the competitive ability of larval common frogs (Rana kukunoris)and common toads(Bufo gargarizans) in a laboratory experiment, and analyzed their competitive strategies respectively. The results were as follows: 1 .The status of using spatial resource Niche breadths of B. minshanicus adults on 5 dimensional axes including character of pasture(0.41), number of yaks dung(0.42), vegetation coverage(0.20), temperature (0.50)and humidity(0.51) of ground surface were narrower than adults of R. kukunoris and N. pleskei. Niche breadths of B. minshanicus subadults were broader than R.kukunoris subadults and N.pleskei subadults on 5 dimensional axes including character of pasture (0.66), number of yaks dung (0.58), vegetation height (0.64), distance to small waterbodies (0.51), humidity of ground surface (0.79). Niche breadths of N. pleskei subadults were the narrowest in three anurans subadults on 4 dimensional axes including character of pasture (0.39), number of yaks dung (0.30), temperature (0.18) and humidity (0.33) of ground surface, niche breadths of N. pleskei adults were the broadest in three anurans adults on 4 dimensional axes including number of yaks dung (0.81), vegetation height (0.63) and coverage(0.47), distance to small waterbodies(0.68).Comparatively, niche breadths of R. kukunoris were broader on the two microclimate factors including temperature(adults：0.62；subadults：0.56) and humidity (adults：0.84；subadults：0.60)of ground surface, but was narrow on distance to small waterbodies(adults：0.27；subadults：0.14). Strategies for using habitat resource of adults and subadults of the three species anuran were different. Generally, subadults of R. kukunoris and N. pleskei needs better habitat condition. It was quite similar that three anurans exploited spatial resource, Niche overlap between R. kukunoris and N. pleskei (0.87) was greater than that between R. kukunoris and B.minshanicus(0.81). 2.Daily activity rhythm R. kukunoris audlts were active when air temperatures were as low as 0℃, R. kukunoris subadults were active at 2℃, B.minshanicus subaudlts were active at 8℃. Positive correlation was found between activities of amphibians and air temperature, Subadults of R.kukunoris, (r=0.797, p<0.001), Subadults,of,B.minshanicus, (r=0.794, p<0.001), andbadults,of,R.kukunoris(r=0.456, p<0.05).Amphibians were more active during sunny days than cloudy days. In cloudy turning into sunny, R. kukunoris and B.minshanicus subadults had two active peak: at noon about 12:30 and 15:30～16:30 pm; in cloudy, R. kukunoris and B.minshanicus subadult had two active peak too : 9:30～10:30am,15:30～16:30pm. 3.Diet analysis Aphodiidae was the most commonly consumed food item by R. kukunoris based on index of relative importance (IRI) during spring (35.28%) and autumn (28.57%) in Zogie wetland. Besides Aphodiidae, larval insect, dipterans such as Bibionidae, Muscidae, Calliphoridae also were important food item for R. kukunoris, in autumn, locust was one of important food item for R. kukunoris. The most important food item for B.minshanicus during spring (IRI:85.54%) and autumn (IRI:49.70%) was ants, following, was coleopterans, such as Aphodiidae, dung beetle. Aphodiidae (IRI:13.41%) were the most important consumed food item by N. pleskei during spring too, following, was ants and Bibionidae. Dietary breadth of N. pleskei (0.43) were greater than R. kukunoris (0.22) and B. minshanicus (0.09). As a whole, Dietary breadth of amphibians during aurumn were greater than spring. Based on prey item, dietary overlap between R. kukunoris and N. pleskei (0.40) was greater than that between R. kukunoris and B.minshanicus (0.33) during spring. 4.Effects of grazing on the diet composition and diet competition of amphibians Amphibians are an important part of the pasture ecosystems as prey and predator. In Zogie wetland, major diet of amphibians was closely associated with dung of yaks, for example, Aphodiidae, Bibionidae, Muscidae, dung beetle. Dung of yaks was major diet and habitat of these insects. Proportion of Aphodiidae was higher in diet composition of R. kukunoris than N. pleskei and B.minshanicus, with development of pasturage, R. kukunoris may have a diet competitive advantage over N. pleskei and B.minshanicus. Number of Aphodiidae in diet composition of amphibians was higher in samples with grazing (31.94%) than in those without grazing (21.32%). Occurrence Frequency of Aphodiidae in diet composition of amphibians was higher in samples with grazing (76.38%) than in those without grazing (44%). However, There was not significantly different on diet composition (P=0.188), and number (P=0.075) and the relative fatness (P=0.537) of amphibians between grazing samples and without grazing. 5.Competitive strategies of amphibian larvae I examined the competitive ability of larval toads (Bufo gargarizans) and frogs (Rana kukunoris) which co-occur in the nature pond by activity level, the growth rate and mass at metamorphosis and larval period in a laboratory experiment. The results suggest: In laborary, B.gargarizans adapted himself to different food level by changing activity. At high food level, B. gargarizans increased activity to gain more diet. At low food level, B. gargarizans decreased activity and achieved early metamorphosis. When food resource was limit, R. kukunoris could gain more food than B. gargarizans.

Molecular cloning and characterisation of prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and its transcription injected by Vibrio anguillarum

The prophenoloxidase(ProPO) gene was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of prophenoloxidase gene consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), encoding 686 amino acids. Phylogenetic analysis revealed that it belongs to insect-type invertebrate prophenoloxidase gene family. To understand ProPO reaction for pathogeny's challenge in shrimp, the expressions of ProPO in different tissues were studied by real-time PCR after challenged by Vibrio anguillarum. The results showed that the expression level of ProPO gene in haemocytes was highest among three studied tissues including haemocytes, lymphoid organ and hepatopancreas. The time-course change of ProPO mRNA levels in challenge experiment showed that ProPO mRNA transcripts had the biggest change extent in lymphoid organ.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

A toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interteukin-IR (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5 h post-Vibrio anguillorum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum. (c) 2008 Elsevier Ltd. All rights reserved.

Phylogenetic analyses of some genera in Oedipodidae (Orthoptera : Acridoidea) based on 16S mitochondrial partial gene sequences

Based on the 16S mitochondrial partial gene sequences of 29 genera, containing 26 from Oedipodidae and one each from Tanaoceridae, Pyrgomorphidae and Tetrigidae (as outgroups), the homologus sequences were compared and phylogenetic analyses were performed. A phylogenetic tree was inferred by neighbor-joining (NJ). The results of sequences compared show that: (i) in a total of 574 bp of Oedipodidae, the number of substituted nucleotides was 265 bp and the average percentages of T, C, A and G were 38.3%, 11.4%, 31.8% and 18.5%, respectively, and the content of A+T (70.1%) was distinctly richer than that of C+G (29.9%); and (ii) the average nucleotide divergence of 16S rDNA sequences among genera of Oedipodidae were 9.0%, among families of Acridoidea were 17.0%, and between superfamilies (Tetrigoidea and Acridoidea) were 23.9%, respectively. The phylogenetic tree indicated: (i) the Oedipodidae was a monophyletic group, which suggested that the taxonomic status of this family was confirmed; (ii) the genus Heteropternis separated from the other Oedipodids first and had another unique sound-producing structure in morphology, which is the type-genus of subfamily Heteropterninae; and (iii) the relative intergeneric relationship within the same continent was closer than that of different continents, and between the Eurasian genera and the African genera, was closer than that between Eurasians and Americans.

Effects of azadirachtin on hemolymph protein expression in Ostrinia furnacalis (Lepidoptera : Crambidae)

The botanical insecticide azadirachtin affects a variety of biological processes. Our early work indicated that protein level and type are significantly influenced by azadirachtin in pupae of Osttiniafumacalis (Guenee) (Lepidoptera: Crambidae) because a correlation exists between protein content and azadiraebtin concentration. By use of proteomic techniques, we analyzed changes in hemolymph protein expression of 48-h-old pupae in O. furnacalis induced by azadirachtin treatment. After feeding by third instars on an artificial diet containing 10 ppm azadirachtin until pupation, 48-b-old pupae were collected, and hemolymph protein samples were prepared. They were separated by two-dimensional polyacrylamide gel electrophoresis, and six proteins were significantly affected by azadiracbtin treatment compared with an untreated control. Two of these proteins were identified by database searching with peptide mass fingerprinting by using matrix-assisted laser desorption/ time-of-flight mass spectrometry after in-gel trypsin digestion. They belong to the insect apolipophorin-III and phospboribosyltransferase family, respectively. These two proteins may function on lipid metabolism in insect hemolymph. Furthermore, fat body is the center of synthesis and secretion of hemolymph proteins. We suggest that the azadirachtin exerts its insecticidal effects on the fat body of O. furnacalis by interfering with protein expression related to hemolymph lipid metabolism.