In eukaryotes, transposable elements (TEs) constitute a large fraction of a genome. TEs are usually classified into two classes based on their transposition mechanisms. ClassⅠ elements use an RNA-mediated mode of transposition, while classⅡ elements (transposons) use a DNA-mediated mode of “cut and paste” transposition. ClassⅠ elements are further divided into two subclasses: the elements that are characterized by long terminal repeats (LTR retrotransposons) and the elements that lack terminal repeats (non-LTR retrotransposons). In order to reveal the classification and evolution of LTR retrotransposons, we identified LTR retrotransposons in the domesticated silkworm (Bombyx mori) using de novo and homology search approaches and found 38 families in the silkworm genome, of which 6 families are novel. The sequences of these families constitute 0.64% of the whole genome, which is much less than that previously reported. We also found that 26 of 38 families have EST evidence, implying that they had potential activity. Then RT-PCR was performed to validate the expressions of 11 families (6 families have EST evidence and 5 families have no EST evidence), and the results showed that these 11 families were expressed in some tissues, further supporting their transcriptional activities. Based on these results, we speculated that most of LTR retrotransposon families in the silkworm genome have potential activity. We estimated the insertion time of LTR retrotransposons, and found that most of them were inserted into the silkworm genome within the past million years. A comparison of Ty3/Gypsy superfamily in Drosophila melanogaster, Anopheles gambiae and B. mori showed that this superfamily experienced different expansion patterns. Given the importance of LTR retrotransposon activity in the evolution of other genomes, our results provide some insights into the roles of LTR in insect genome evolution.

 In order to explore the polymorphism of EST-SSR markers in silkworm, we retrieved and analyzed 4 465 expressed sequence tags (ESTs) on linkage group 12 of the silkworm, Bombyx mori. After splicing and jointing, we obtained 581 non-redundant ESTs with the total length about 480 kb. A total of 154 simple sequence repeats (EST-SSRs) were detected in 122 ESTs which accounted for 2.73% of all the investigated ESTs, meaning one EST-SSR per 3.12 kb on average. Trinucleotide repeats and tetranucleotide repeats were the main classes， which accounted for 36.36% and 28.57% of all detected EST-SSRs, respectively. The average length of nucleotide repeats was 16.2 bp, and the maximum was 30 bp. Homologous sequences of 26 EST-SSRs were retrieved in NCBI database, which contained 40 SSRs. Among them, 14 SSRs (35.0%) were located in the 5′-UTR, 11 SSRs (27.5%) in the 3′-UTR and 15 SSRs (37.5%) in the coding sequences (CDSs). Eleven primer pairs were designed for the screened EST-SSRs and 8 of them showed polymorphic bands. The primer ES1204 showed polymorphism among 8 silkworm strains. The results suggest that it is feasible to find SSR markers through searching of silkworm EST databases.

【Aim】 To study the effects of single conserved amino acid residue of pyridoxine 5′-phosphate oxidase (PNPO) from Bombyx mori on its activity. 【Methods】 Lys¹¹¹(AAA) and Ser¹⁶⁰(AGC), two of the most conserved residues, were mutated to Glu (GAA) and Ala (GCC) by using over-lap extension， respectively. The obtained expression plasmids were over-expressed in Escherichia coli Rosetta by IPTG induction, and then the expressed products were purified with affinity chromatography and the activity of the purified PNPO were determined. 【Results】 The molecular mass of the target recombinant protein was ~45.0 kDa. Compared with the wild type PNPO, the activities of the mutants K111E and S160A were reduced by 78.0% and 67.4%, respectively. 【Conclusion】 The results suggest that the residues Lys¹¹¹ and Ser¹⁶⁰ are important to maintain the activity of PNPO. This study confirms the significance of single conserved amino acid residues on the catalytic function of PNPO of B. mori.

The codling moth, Cydia pomonella (Lepidoptera: Tortricidae), is a worldwide quarantine pest and possesses the plasticity in thermotolerance ability. The temperature fluctuations may enhance its tolerance to heat stress. In the present study, we determined the critical threshold of tolerance to high temperature by bioassay method in C. pomonella laboratory population, and applied the techniques of homology-based cloning, RACE and real-time quantitative PCR (RT-qPCR) to elucidate the function of Hsp90 responding to heat stress in C. pomonella. The results of bioassay showed that the mortality of C. pomonella was significantly increased as temperature was elevated and time prolonged. When the 1st-5th instar larvae were exposed to 50℃ and 52℃ for 2, 5 and 10 min, the 3rd instar larvae were found to be the most sensitive and the 5th instar larvae the most resistant. The 1st-4th instar larvae were killed 100% when exposed to 50℃ for 10 min and 52℃ for 5 min, while the survival rate of 5th instar larvae still maintained at 25.0% and 11.1%, respectively. The full-length cDNA of hsp90 gene was obtained from the 5th instar larvae of C. pomonella exposed to 35℃, which is 2 470 bp in length and has an open reading frame (ORF) of 2 148 bp encoding a protein of 716 amino acids with the deduced molecular weight of 82.07 kDa. This gene was registered in GenBank under the accession number JN624775 and designated as Cphsp90. CpHsp90 shares 96% amino acid sequence identity with the Hsp90 from Ostrinia furnacalis and Mamestra brassicae, suggesting that the Hsp90 family is highly conserved. The result of RT-qPCR showed that the Cphsp90 was heat inducible, and the relative expression level of Cphsp90 mRNA was positively correlated with the heat stress when the larvae were exposed to 32-44℃ for 1 h. When the larvae of C. pomonella were exposed to 35℃, Cphsp90 was expressed abundantly in cuticle with the expression level significantly higher than that in the hemolymph, fat body and midgut. The result of the mRNA expression pattern of the Cphsp90 under higher temperature after pretreatment at 35℃ for 3 h indicated that the maximum expression level in the larvae preheated was induced at higher temperature (45℃ for 10 min) compared with the larvae not preheated (40℃ for 10 min). This result further proved that preheat treatment enhanced the thermotolerance of 5th instar larvae. It is so concluded that the positive expression of the Cphsp90 mRNA may play an important role in theromotolerance and the plasticity of C. pomonella.

 Catalase (CAT), one of most important enzymes in organism, plays an essential role in active oxygen metabolism and preventing the formation of free hydroxyl radicals by clearing hydrogen peroxide, superoxide radical and peroxides. In this study, the full-length cDNA of CAT gene from the beet armyworm, Spodoptera exigua, was cloned and characterized by RT-PCR and RACE technique, which is 1 755 bp in length and named as SexiCAT (GenBank accession no. JN051294). The open reading frame (ORF) of SexiCAT is 1 524 bp encoding 507 amino acid residues. The amino acid sequence of SexiCAT shares a significant identity with catelases of Bombyx mori (87%), Drosophila melanogaster (73%), Aedes aegypti (71%), and Tribolium castaneum (70%). The temporal expression profiling revealed that SexiCAT was expressed in different developmental stages of S. exigua at different expression levels, with the highest level in adults, the second highest level in larvae and the lowest level in eggs, and the expression level in adults was 7 times as high as that in eggs. Tissue expression profiling further indicated that in 5th instar larvae of of S. exigua SexiCAT transcript was observed in the integument, midgut, fat body and Malpighian tubules, with the highest expression level in fat body. This study provides a foundation for understanding the functions and applications of CAT as a new target for oxidase blocking agent.

The silkworm Bombyx mori is an egg-diapause insect. There are no morphological changes, organ development and tissue differentiation during diapause stage. However, its physiological metabolism is still in progress. In order to further research the diapause mechanism, the change in activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), pyruvate kinase (PK, EC 2.7.1.40), acetylcholine esterase(AchE, EC 3.1.1.7) and lactate dehydrogenase (LDH, EC 1.1.1.28) in the early embryonic stage of diapause eggs, diapause eggs with instant hydrochloric acid (HCl) soaking and non-diapause eggs were detected in this study. The results showed that the activities of SOD, CAT, PK and AChE in the HClsoaked eggs, from 1 d to 7 d, increased from 56 517.00 U/g to 81 986.94 U/g, 14.98 U/g to 106.90 U/g, 25.19 U/g to 181.70 U/g, and 17.88 U/g to 287.86 U/g, respectively, while that of LDH decreased from 169.96 U/g to 122.82 U/g. In the non-diapause eggs during the same period, the activities of SOD and LDH decreased from 86 417.99 U/g to 66 024.19 U/g and 169.07 U/g to 135.02 U/g, respectively; the activities of CAT, PK and AchE, however, increased from 1.47 U/g to 44.37 U/g, 20.56 U/g to 92.09 U/g, and 21.40 U/g to 99.17 U/g, respectively. In the diapause eggs, the activities of SOD and AChE were stable; the CAT activity was raised but LDH activity was decreased with the embryonic development; and the PK activity was raised in the first four days of the embryonic development and remained stable afterwards. Understanding the change of related enzyme activities among diapause, non-diapause and HCl-soaked silkworm eggs in the embryonic development will help to reveal the diapause mechanism.

In order to investigate the effects of Pteromalus puparum venom on the encapsulation and phagocytotic capacities of granulocytes (GRs) and plasmatocytes (PLs) in its host, Pieris rapae, we first purified these two types of host hemocytes using Na₂-EDTA treatment and nylon wool method, respectively. Then, with the method of cell culture in vitro we also studied the different roles of GRs and PLs in host encapsulation and phagocytosis, and the effects of parasitoid venom targeting to these two cellular responses. The results indicated that both GRs and PLs took part in the host encapsulation. The encapsulation performed by GRs was stronger than by PLs. The highest rate was presented in encapsulation reaction mediated by the mixture of GRs and PLs. The host cell-free plasma did not significantly influence the encapsulation performed by GRs and PLs, respectively. The encapsulation capacities of GRs and PLs were both markedly inhibited by the parasitoid venom in a dose-dependent manner. Additionally, in the host, both GRs and PLs possessed the phagocytotic capacity. The phagocytotic capacity of GRs was much stronger than that of PLs. Parasitoid venom also noticeably suppressed the phagocytosis performed by GRs and PLs, respectively, also in a dose-dependent manner. The results suggest that both GRs and PLs in the host, P. rapae, play key roles in the host cellular responses, and P. puparum venom can significantly decrease host cellular responses including encapsulation and phagocytosis performed by GRs and PLs.

Compound eye is the main vision organ of insect, which plays a significant role in their foraging, finding mate and habitat, learning, memory and so on. In this study, the external morphology and internal microstructure of the compound eyes of Harmonia axyridis ab. conspicua were observed by using scanning electron microscope and paraffin section. The results indicated that: (1) the compound eye is oval, and located on the lateral upside of the head. There is a nick on the compound eye, which is near to antennal socket. The surface of the ommatidia is smooth and not covered with corneal nipples. The compound eye of the female and male consists of 705 and 691 ommatidia, respectively. (2) The ommatidia located in the central region of the compound eye are typically hexagonal, while those located in the periphery of the compound eye are often of irregular, pentagonal, and even squarish shapes. (3) The ommatidium consists of corneal lens, crystalline cone, 8 retinula cells, rhabdom, basement membrane and pigment granules. The crystalline cone is composed of 4 cells. Among the 8 retinula cells, 6 cells are in the periphery and the other 2 are in the centre. (4) The microstructure of compound eye of H. axyridis ab. conspicua is significant different between dark and light adaptation. In light adaptation, most of the pigment granules are distributed between the crystalline cone and rhabdoms; the peripheral rhabdom is in ring form, and its inner and outer sides are covered with the pigment granules. In dark adaptation, the pigment granules move longitudinally and are distributed on the lateral upsides of the crystalline cone and rhabdoms; the peripheral rhabdoms twist appearing as irregular polygons, and only its outer sides are covered with the pigment granules. This study reveals that the compound eyes of H. axyridis ab. conspicua are of the type of apposition eye, and they adapt to the change of light and dark by the mechanism of pigment granules moving longitudinally and rhabdoms twisting.
 

 In order to screen entomopathogenic fungi strains with high virulence against the new pest Basilepta melanopus on Camellia oleifera, the growth rate and sporulation of different Beauveria bassiana and Metarhizium anisopliae strains were investigated, and bioassay evaluation of B. bassiana and M. anisopliae against B. melanopus adults was carried out through dipping in spore suspension. The results showed that the growth rate and sporulation of different fungi strains were significantly different. The four strains, i.e., MaYTTR-04, BbFZ-17, MaZPTR-01 and BbTK-01, had higher growth rate and sporulation than other strains. The cumulative mortality of B. melanopus adults was gradually increased with time after inoculation with B. bassiana and M. anisopliae. The cumulative mortality of B. melanopus adults reached 100% at 7 d after inoculation with B. bassiana, while the mortality of adults at 14 d after inoculation with MaZPTR-01 and MaYTTR-04 were 80.3% and 78.8%, respectively. The cadaver rate of adults inoculated with B. bassiana was significantly higher than that inoculated with M. anisopliae. The cadaver rates of BbTK-01 and BbFZ-17 were highest, being 85.7% and 75.8%, respectively. The median lethal time (LT₅₀) values of B. bassiana strains BbXJ-01, BbFZ-17 and BbTK-01 were shortest, being 3.0, 3.3 and 3.4 d, respectively. However, the LT₅₀ values of the two M. anisopliae strains, MaZPTR-01 and MaYTTR-04, were 6.0 d and 6.2 d, respectively. The results of bioassay showed that B. bassiana strains had higher virulence to B. melanopus adults than M. anisopliae, especially the two strains, BbTK-01 and BbFZ-17, had higher lethality and infection rate than other strains, and at the same time, they had the characteristics of rapid growth and high sporulation, suggesting that the two strains have great potential in biocontrol of B. melanopus adults.

 To understand the thermoperiodic response of the cotton bollworm, Helicoverpa armigera (Hübner), the effects of thermoperiod on larval development and pupal diapause in 4 different geographic populations (Guangzhou population, 23.08°N, 113.14°E; Yongxiu population, 29.04°N, 115.82°E; Tai’an population, 36.15°N, 116.59°E; and Kazuo population, 41.34°N, 120.27°E) were systematically investigated under the photoperiod of L12∶D12. The results showed that at the same photophase temperature there were no significant differences in the larval duration (P＞0.05) between 20T (thermophase, 20℃)∶ 5C (cryophase, 5℃) (12 h photophase temperature∶12h scotophase temperature) (the rest in the same analogy) and 20T∶9C， 22T∶5C and 22T∶9C， 25T∶5C and 25T∶9C， and 28T∶5C and 28T∶9C, respectively, in 4 different populations. However, at the same or near average temperature, the larval duration at  20T∶9C (an average temperature of 14.5℃) was significantly higher than that at 22T∶5C (an average temperature of 13.5℃) (P＜0.05); the larval duration at 20T∶20C (a constant temperature of 20℃) was significantly higher than that at 28T∶9C (an average temperature of 18.5℃) (P＜0.05), suggesting that the larval development is influenced strongly by the photophase temperature. At the same or near average temperature, the diapause rates in 4 different populations at 20T∶9C were higher than those at 22T∶5C, and the diapause rates at 25T∶ 20C (an average temperature of 22.5℃) were higher than those at 28T∶9C (an average temperature of 18.5℃); the diapause rates in 4 different populations at 20T∶20C  were significantly higher than those at 28T∶5C (an average temperature of 16.5℃) (P＜0.05). However, there were significant differences (P＞0.05) at the same photophase temperature between 20T∶5C and 20T∶9C， 22T∶5C and 22T∶9C， 25T∶5C and 25T∶9C， and 28T∶5C and 28T∶9C, respectively, suggesting that the diapause induction is influenced strongly by the photophase temperature. At the same thermoperiod, the larval duration and diapause rate were significantly different among different geographic populations. The larval duration and diapause rate were positively correlated with latitude of habitat, suggesting that sensitivity to temperature is gradually enhanced with a decrease in latitude of habitat.

 To explore the potential factors that influence host plant selection of Ophraella communa for oviposition on common ragweed (Ambrosia artemisiifolia), a consecutive field survey of 60 plant patches for egg deposition was made in the field.  Hurdle models were fitted to analyze factors that influence probability of oviposition and the amount of eggs deposited on plants. The results showed that: (1) Oviposition increased with progress of the season, feeding damage level, plant height, and patch size in open environment, but decreased with plant patch size, blossom, seasonal progress in blossom, and feeding damage level with progress of the season and after blossom. (2) Number of eggs deposited on plants increased with progress of the season, feeding damage level, open environment, feeding damage level at the blossom stage, and plant height at the blossom stage, but decreased with blossom, and feeding damage level with progress of the season. (3) The increase of eggs on plants with progress of the season was more distinct during vegetative growth stage than blossom stage, and constantly linear in open environment, but linear at first and then asymptotic in shading environment. The study suggests that the factors influencing oviposition selection are not identical to those affecting quantity of egg deposition, and the influence is exercised more through interactions between factors than single factor alone.  

 To understand if the feeding behavior and capacity of Agasicles hygrophila changed after the insect has been introduced to China for more than 20 years, the controlling effects of all stages (larvae and adults) at different infestation densities on alligator weed, Alternanthera philoxeroides, were quantitatively detected in the laboratory, and in the detection tests the experimental larvae and adults were obtained from adults collected in fields and reared with A. philoxeroides. The results showed that the 1st instar larvae of A. hygrophila preferred to feed the top buds and tender leaves. A. philoxeroides plants still produced new leaves and axillary buds, and its biomass, plant height and number of stem nodes increased at the infestation densities of both 0.2 and 1 1st instar larvae of A. hygrophila per plant. The biomass, numbers of leaves and axillary buds exhibited negative growth at the infestation density of 5 1st instar larvae of A. hygrophila per plant. All measured parameters including biomass, plant height, and the numbers of leaves, stem nodes and axillary buds exhibited negative growth at the infestation densities of 10 1st instar larvae of A. hygrophila per plant. The 2nd instar larvae of A. hygrophila preferred to feed on the top bud and tender leaves and also fed on tough foliages and stems. Forty percent of experimental A. philoxeroides plants died at 7 d after infestation at the density of 10 2nd instar larvae of A. hygrophila per plant. The 3rd instar larvae fed both leaves and stems, and then pupated in the stems at the later stage. Fifty-two percent of experimental A. philoxeroides plants died at 7 d after infestation at the density of 10 3rd instar larvae per plant,  and the number of stem nodes of surviving plants markedly decreased. The adults could continuously eat any tissues of the weed for 24 h. At the infestation density of 0.2 adults per plant, the numbers of leaves and axillary buds of the experimental plants revealed negative growth. At the infestation density of 5 adults per plant, the biomass, plant height, and the numbers of leaves, stem nodes and axillary buds of the experimental plants exhibited remarkably negative growth. A. hygrophila at the infestation density of 10 adults per plant had a better controlling effect on the alligator weed than that at the infestation density of 5 adults per plant.

The environmental heterogeneity caused by topographical diversity is an important mechanism underlying the formation and maintenance of bio-geographic pattern of diversity distribution at the micro-scale, and the prerequisite for the difference in species richness. With the help of GIS and S-Plus, the GAM model was used to study topographic indexes influencing the distribution of grasshopper from July to August, and the relationship between the regional locust richness and terrain complexity was also studied based on the quantitative analysis of topographic variation characteristics of the upper reaches of the Heihe River on the northern slope of the Qilian Mountains. The results showed that in total 13 species (including 3 149 individuals) belonging to 3 families and 10 genera were collected in 36 samples. The order that the richness of grasshopper was influenced by topographical factors was elevation>slope>aspect>profile>plane>position. Grasshopper distribution was almost balanced in the plane of curvature and the gradient profile curvature, and presented a two parabola distribution in elevation, slope and the whole position of slope gradient, showing a decreasing trend. On the regional distribution, there were higher richness of grasshoppers in the whole regions, but they were mainly located in the region with altitudes of 2 600-2 700 m, and mainly concentrated in the northwest and west slopes, which was consistent with actual observation. The relationship between grasshopper richness and terrain factors as well as the grasshopper distribution indicates that the redistribution of water and heat conditions due to topographical factors caused the diversification and fragmentation of grasshopper distribution patterns.

【Aim】 To identify the differential expression of pyridoxal kinase (PLK), which is a key enzyme related to VB₆ metabolism, in different tissues of Bombyx mori. 【Methods】 The recombinant plasmid was transformed to Escherichia coli Rosetta for induction, expression and purification, and then the polyclonal antibody was prepared. The expression of PLK at different developmental stages, in different tissues of day-3 5th instar larva and in the tissues of cuticle, head and Malpighian tubules on different days of the 5th instar was analyzed by Western blot. The transcription level of PLK gene in different tissues was analyzed by real-time PCR. 【Results】 The highest expression level of PLK was found in the 5th instar larva. The expression order in various tissues of day-3 5th instar larva from high to low was spermary, Malpighian tubules, cuticle, midgut, head, ovary, fat body and silk glands. The transcriptional analysis of PLK gene on different days of the 5th instar showed that the biggest difference in expression level was in cuticle, and slightly higher on the early days in head and Malpighian tubules. Realtime PCR results showed that PLK gene was highly expressed in spermary, and moderately expressed in Malpighian tubules and head. 【Conclusion】 The  differential expression of PLK gene at different developmental stages and in different tissues of the 5th instar larva further reveals the importance of PLK related to VB6 metabolism in B. mori.

 In order to research the interaction between the small brown planthopper (Laodelphax striatellus Fallén, SBPH) and rice stripe virus (RSV), yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) SBPH populations was constructed. SBPH infected with RSV was maintained in our laboratory. The mRNA was isolated and purified from high-viruliferous populations, and used as the template to synthesize the double-strand cDNAs. The cDNAs were ligated with three-frame adapter and purified by the cDNA size fractionation columns. By using homologous recombination reaction, the cDNA entry library was prepared and then recombined into Gateway compatible vector pGADT7-DEST to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained 3.68×10⁷ independent clones, and the titer of the amplified library was 2.62×10¹⁰ cfu/mL. The recombination rate was above 95%, and the average size of inserts was above 1 kb in the cDNA library. The results demonstrate that the library database meets the requirements of the standard cDNA library. The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and RSV.

To search out a rapid, sensitive and specific method for detection of Nosema bombycis and DNA extraction, both TaqMan probe real-time PCR method and SYBR Green real-time PCR method were established and optimized compared with traditional PCR method. In addition, four DNA extraction methods have been evaluated for the efficiency for traditional PCR and real-time PCR. The results showed that the germination solution of N. bombycis could be directly used for real-time PCR and traditional PCR, without the DNA extraction procedure. Moreover, the direct method had much higher sensitivity than other extraction methods in the real-time PCR and traditional PCR detection. The sensitivity of the four methods from high to low was in the following order: direct method, phenol-chloroform extraction method, animal tissue DNA extraction kit and plant tissue DNA extraction kit method. TaqMan probe method and SYBR Green method had similar sensitivity in the detection, up to 10² Nb/mL in detecting the germination solution of N. bombycis, and both had higher sensitivity than traditional PCR. Real-time PCR technique combining with the DNA-extraction-skip skill makes a simple, sensitive and specific method for detection of N. bombycis. This study would be helpful for quarantine and control of pebrine.