【Aim】 The main aim of this research was to clone and align the full-length cDNA and promoter sequences of desat4 gene from silkworms, Bombyx mori and B. mandarina, and construct prokaryotic expression vector to obtain a membrane protein Desat4 for further functional study. 【Methods】 Full-length cDNA of the desat4 gene from silkworm was obtained through RACE technique, the encoded protein was expressed in Escherichia coli expression system, and its promoter sequences were cloned based on genome sequences of silkworm. 【Results】 The full-length cDNA of the desat4 gene from B. mori and B. mandarina was 1 717 bp and 1 718 bp, respectively. Their open reading frame (ORF) is 1 059 bp in length and encodes 352 amino acid residues with four transmembrane helixes and three conserved histidine clusters, which are essential for desaturase catalytic activity. The deduced amino acid sequence shares 88.9% similarity to that of the fatty acid desaturase MsexKPSE (GenBank no. CAJ27975) of Manduca sexta. There is no typical TATA-box in promoter sequences, but there is a transcriptional initiator as well as other transcription factor binding sites, including HSF, NIT2, CdxA and so on. The membrane protein Desat4 was expressed in E. coli expression system and solubilized with mild detergent DDM. 【Conclusion】 The full-length cDNA and promoter sequences of desat4 gene from B. mori and B. mandarina were successfully cloned and comparatively analyzed. The membrane protein was expressed in vitro by pET system, thus providing a foundation for further functional study.

Odorant binding proteins (OBPs) in insects play an important role in foraging, courtshipping, multiplying and chemical communication with the environments. Antennalbinding protein (ABP) is one of important groups of OBP family. We analyzed the first reported ABP and ABPX genes in the silkworm Bombyx mori using chromosome mapping and semi-quantitative RT-PCR for further understanding of the expression and function of ABP/ABPX. Chromosome mapping showed that BmABP and BmABPX were respectively organized in chromosome 5 and 26 with different gene structure, suggesting that they probably possess different functions. Analysis of the expression profiles in different tissues of males and females during the embryonic, larval and adult stages revealed that BmABP possessed high expression level in a variety of tissues and organs with no time- and tissue-specificity, while BmABPX possessed conspicuous temporal and spatial expression differences (P<0.05), with the highest relative expression level in the antenna, and lower relative expression levels in most other non-olfactory tissues without significant sex differences. The results suggest that these two genes are more likely to have other functions undiscovered besides olfactory-related functions.

Acting as molecular chaperones, heat shock proteins (Hsps) could help fixing denatured proteins and preventing aggregation of other proteins. In order to better understand the function of molecular chaperones of Hsp families in the parasitoid Pteromalus puparum, Pphsp90, Pphsp70, Pphsc70, Pphsp60, Pphsp40 and Pphsp20 genes from this parasitoid were over-expressed in Escherichia coli. The results showed that the five genes except Pphsp40 were highly expressed and these expressed recombinant Pphsps were soluble and stable under high temperature (80℃). In vivo experiments, Pphsp20 and Pphsp90 significantly increased the survival rate of E. coli cells under high temperature (50℃, 1 h). In vitro protection study verified that only purified recombinant Pphsp20 could reduce the aggregation of luciferase under high temperature. The results suggest that both Pphsp20 and Pphsp90 may be involved in thermo-protective role in E. coli, and Pphsp20 may work independently while Pphsp90 requires the cooperation with other cofactors.

RNA interference （RNAi） has been demonstrated to be used to interfere special life processes by silencing target genes. Therefore, a new countermeasure of RNAi pest control could be developed through exploring effective specific target genes and efficient RNAi techniques. Based on transcriptome data analysis and RACE strategy, two full-length trehalase genes were cloned from the small brown plant hopper (SBPH), Laodelphax striatellus, and were designated as LSTre-1 and LSTre-2, with GenBank accession numbers JQ027050 and JQ027051, respectively. Both LSTre-1 and LSTre-2 have typical characteristics of trehalase genes, and show high sequence identity with trehalase genes in other insects. LSTre-1 is 2 024 bp in length, and its ORF encodes a protein of 602 amino acids including a signal peptide of 25 amino acids and without hydrophobic transmembrane domains. LSTre-2 is 2 619 bp in length, and its ORF encodes a protein of 618 amino acids including a signal peptide of 26 amino acids and two hydrophobic transmembrane domains. Double-stranded RNA （dsRNA） was synthesized based on these two genes and the toxicological effects of RNAi were studied. The results showed that the silencing effect of LSTre-1 dsRNA was higher than that of LSTre-2 dsRNA. However, silencing of these two genes all can not only cause lethal effect by decreasing the mRNA expression and enzyme activity but also prevent the normal growth and development of SBPH. These results imply that SBPH could be controlled through silencing its trehalase genes by feeding dsRNA with proper ways.

The RNA secondary structure contains more information than its primary structure, and can indicate the phylogenetic relationship more really. Therefore, the complete sequences of mitochondrial L-rRNA genes of Bactrocera cucurbitae and B. correcta were sequenced in this study, and the structural model of L-rRNA genes in Diptera was constructed. The characteristics of L-rRNA secondary structure was analyzed, and preliminary phylogenetic features were inferred based on stem-loop parameters and structural sequences of H45-H47, and structural sequences of L-rRNA genes among 13 families in Diptera. The results showed that the dipterans share the conserved secondary structure of L-rRNA gene; the nucleotide composition and distribution vary within different structure regions of L-rRNA gene; the highest content of completely conserved nucleotides was found in region Ⅳ and Ⅴ; the region Ⅳ and Ⅴ also contains the highest GC content; the AT-skew is negative in region Ⅵ of L-rRNA gene of the majority of dipteran families; the unique bases within individual family and individually conserved nucleotides between families present higher content of G or C. The family Cecidomyiidae exhibits much farther distance with other dipteran families, while Calliphoridae, Tachinidae and Syrphidae share much closer phylogenetic relationship; Tabanidae and Nemestrinidae belong to a small branch; Ceratopogonidae and Culicidae share a large phylogenetic branch. The satisfied phylogenetic result would not be obtained easily using single phylogenetic parameter, and it is thus necessary to infer accurate phylogenesis through combined parameters of different features.

Wolbachia is widely distributed in arthropods and can manipulate the reproduction and fertility of its hosts. Antibiotics can effectively remove Wolbachia from host body. In this study, Wolbachia in Encarsia formosa was removed by feeding wasps with solution of 1, 5 and 10 mg tetracycline hydrochloride/mL sucrose water, and PCR technique was used to detect the completely removing of Wolbachia from female wasps. The numbers of eggs loaded and ovarian tubules of the females were investigated to evaluate the effect of Wolbachia on reproduction of E. formosa. The results indicated that there was no significant difference in the number of ovarian tubules among F0 female of different treatments (P=0.12), whereas there were 6 ovarian tubules in female wasps of Wolbachia free F1, F2 and F3 generations, significantly less than in female wasps of F0 generation (P<0.001). Egg load in F0 generation wasp from which Wolbachia was removed by feeding antibiotics was less than that in the untreated female of F0, but was more than that in F1, F2 or F3 generation (P<0.001). No significant difference in egg load among F1, F2 and F3 generations was found (P=0.59). E. formosa could produce male offsprings after Wolbachia was removed, but no mating behavior was found and females could produce female offsprings without mating. These results suggest that Wolbachia not only affects directly egg load of host but also affects the development of ovarian of host; however, removing of Wolbachia does not change the parthenogenetic reproductive mode of E. formosa.

To scientifically utilize biocides and conserve natural enemies for integrated control of Helicoverpa armigera, the bioefficacy of a mixed biocide BtA in comparison with seven commercial insecticides (abamectin, Bt toxin, beta-cypermethrin, phoxim, chlorfluazuron, fenvalerate and carbosulfan) against H. armigera and its contact toxicity to pupae and female adults of parasitoid Microplitis mediator were bioassayed in the laboratory and compared. Toxicity of these compounds on the 3rd instar larvae of H. armigera by leaf dip bioassays within 72 h in the laboratory showed that BtA had high toxicity (LC₅₀ = 0.7364 mg/mL) in comparison with the other commercial insecticides. Mortalities of the 3rd instar larvae of H. armigera exposed to Brassica rapa var. pekinensis treated with 4 mg/mL of these compounds were significantly different at 24, 48, and 72 h in pot trials (P≤0.05). BtA had no significantly different bioefficacy (P>0.05) in the experimental pots from the other commercial insecticides against H. armigera larvae at 72 h after treatment. After application of BtA, larval mortality of H. armigera increased as time extended. Bioassay of the contact toxicity of these compounds to pupae and female adults of parasitoid M. mediator showed that BtA has low toxicity to pupae and female adults of M. mediator, with the mortality values of 13.82% and 7.33%, respectively, in comparison with fenvalerate and carbosulfan. We so concluded that BtA has a moderate toxicity to the lepidopterous pest, H. armigera, and a relatively low toxicity to its parasitoid, M. mediator.

To clarify the effect of controlled atmosphere (CA) on stored pests and enzyme activities related to the development of resistance, the effect of CA with enriched CO₂ (75% CO₂, 5% O₂ and 20% N₂ ) on the activities of carboxylesterase (CarE), acid phosphatase (ACP) and glutathione S-transferases (GSTs) from an experimental population of Araecerus fasciculatus (De Geer) were investigated. The results showed that CarE activities were significantly affected by CA with exposure time, increased by 35.41%, 55.02% and 88.98% at 3 h, 6 h and 9 h, respectively, after exposure compared with the control. The CA treatment increased the Michaelis-Menten (M-M) kinetic constant, _Vmax, of CarE by 26.13%, 31.77% and 57.12%, respectively at 3 h, 6 h and 9 h after exposure, but had no effect on the M-M constant, K_m. CA treatments of 3 h, 6 h and 9 h increased the specific activity of ACP by 34.53%, 72.45% and 126.37%, respectively. Compared to the control, the specific activities of GSTs in adults exposed to CA for 3 h, 6 h and 9 h were increased by just 5.40%, 8.40% and 17.59%, respectively. These results suggest that A. fasciculatus can adjust the activity of some detoxification enzymes and the kinetics of CarE-catalyzed reactions under the stress of CA. Under CA conditions, the catalytic activity of some detoxification enzymes towards their substrates were enhanced, potentially providing the ability for the pest to adjust its metabolic activity against extreme conditions. This study provides insights into insect responses to CA treatment and the mechanism of CA resistance or tolerance.

To clarify the physiological and biochemical mechanism of the effect of transgenic cry1Ab/cry1Ac rice on larvae of Sesamia inferens (Walker), the effects of feeding transgenic Bt rice on the activities of three protective enzymes (SOD, CAT and POD) in S. inferens larvae were studied by assaying enzyme activities in the 3rd and 5th instar larvae which were fed with stems of transgenic and non-transgenic rice (control) for different time. The results showed that SOD activity in the 3rd instar larvae fed with Bt rice for 24 h was significantly higher than that of the control (increased by 43.44%), but declined to the minimum at 48 h after feeding. The POD activity in the 3rd larvae fed with Bt rice reached the maximum at 24 h after feeding, and significantly higher than that of the control （increased by 29.22%）, but declined to the minimum at 48 h after feeding which was significantly lower than that of the control. The CAT activity in the 3rd larvae fed with Bt rice for 4 h increased by 30.33%, and decreased by 27.01% at 48 h after feeding compared with that of the control. The SOD activity in the 5th instar larvae was significantly higher than that of the control after the larvae was fed on transgenic rice for 4 h, but declined to the minimum at 36 h after feeding (decreased by 31.62% compared with that of the control). The POD activity reached the maximum and increased by 73.20% compared with that of the control after the larvae was fed on Bt rice for 8 h, but declined to the minimum at 36 h after feeding. The CAT activity reached the maximum and increased by 75.73% after the larvae was fed with transgenic rice for 4 h and decreased by 7.55% at 48 h after feeding compared with that of the control. The resistance level of the 3rd instar larvae was lower than the 5th instar larvae when treated by transgenic Bt rice for the reason that the 3rd instar larvae had lower defense capability. The results suggest that in the initial stage of feeding, the enzyme activity in larvae increases in order to protect larvae from being damaged by Bt toxalbumin. The enzyme activities in larvae rapidly descend and the metabolism of larvae is disturbed due to feeding transgenic rice with extension of feeding time, causing the poisoning symptoms and even death of larvae.

Sugar diets can prolong longevity of synovigenic parasitoids. To understand the differences in the nutritional function of naturally occurring sugars to synovigenic parasitoids, the longevity and oogenesis of Diglyphus isaea (Walker), a predominant parasitoid of Liriomyza leafminer provided respectively with glucose, fructose, sucrose, trehalose and melezitose, were compared in this experiment. The results showed that the longevity of female wasps feeding on sugars was prolonged significantly (P<0.0001). Longevity of wasps feeding on fructose was significantly longer than that feeding on glucose, sucrose, trehalose and melezitose, respectively (P<0.0001). However, no differences were observed in the longevity of wasps feeding on glucose, sucrose and trehalose (P>0.4234), which was, however, significantly longer than that feeding on melezitose (P<0.0001). The longevity was positively related with body size when female wasps fed on different sugars. Newly emerged females did not bear the third grade eggs (mature eggs) in their ovaries. When water was available, the number of the third grade eggs reached the maximum at 24 h after eclosion, while all of the third grade eggs were reabsorbed at 48 h. When the parasitoids fed on the five kinds of sugars, the number of the third grade eggs and the total number of eggs including immature and mature eggs significantly increased and then declined gradually. Meanwhile, the peak time of the total number of eggs varied among the different treatments, but the maximums of both the third grade eggs and total eggs had no significant differences among female wasps feeding on different sugars (P>0.05). The results provide a basis for mass-rearing indoors and mass-releasing in fields of the parasitoid D. isaea.

In order to clarify the inheritance of wing forms in three species of rice planthoppers including Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus, selection and cross breeding experiments were carried out using rice seedlings under the same condition (25℃, 14L∶10D). The results showed that the wing forms of three planthoppers exhibited a strong selection response, and the pure line of macropterous form was the most vulnerable to be selected in S. furcifera, the moderate difficult in L. striatellus, and the most difficult in N. lugens. For the three planthoppers, 95%-100% of females and males were brachypterous (B) and macropterous (M), respectively, when the cross breeding group M♂×B♀ has been selected for three to five generations. The great part of wing form data from N. lugens selection and cross breeding experiments could be explained well using the sex-influenced inheritance trait controlled by a pair of alleles on euchromosome, and the macroptery was dominant in males whereas the brachyptery was dominant in females. However, the phenotype of wings in N. lugens was also controlled by other factors beside the genotype. The wing forms of offsprings from B♂×B♀ and M♂×M♀ were very similar when their parents came from M♂×M♀, and the same result was found with the parents B♂×B♀. Few brachypterous males appeared in S. furcifera and L. striatellus under the selection conditions. Genetic control of S. furcifera and L. striatellus was similar, mainly determined by two pairs of alleles, of which one is located in a sex chromosome controlling males’ wing forms with the macropetery dominant, while the other is located in a euchromosome controlling females’ wing forms with the brachyptery dominant. It is so concluded that the difference of dominant and recessive genes in females and males and the inconsistency between phenotype and genotype are the very important genetic mechanism for planthoppers to cannily control their wing forms under different conditions.

The genetic diversity of eight characteristic geographical populations of Plutella xylostella from China was investigated with ISSR method using fifteen selective primers in this study in order to clarify the genetic diversity of different geographic populations of P. xylostella. The results showed that total 395 ISSR loci, of which 89.11% are polymorphic, were obtained, and the characteristic ISSR pattern for each P. xylostella individual was detected. The proportion of polymorphic bands (P), Shannon’s index (I) and Nei’s gene diversity index (H) of P. xylostella displayed a high degree of genetic diversity both at the species level (P=89.11%, H=0.2706, I=0.4286) and at the population level (P=88.80%, H=0.2759, I=0.4349). The genetic diversity of P. xylostella was higher in areas of southern China (Yunnan and Hubei) than in areas of northern China (Beijing, Tianjin, Shandong, Heilongjiang, and Gansu). The intra-population genetic variation was the highest in Nankou of Beijing, and the lowest in Haikou of Hainan and Lanzhou of Gansu. In additional, 5.66% genetic variations are from intra-population and 94.34% from inter-population (Nm=8.3399). No obvious genetic differentiation exists among the eight characteristic geographical populations of P. xylostella in this study. The results provide a valuable basis for the pesticide resistance control and the integrated management of the field population of P. xylostella.

A standard photographic map of Simulium (Wilhelmia) xingyiense Chen and Zhang was constructed from larval salivary gland polytene chromosomes and is described herein. Polytene chromosomes from wild larvae collected from three populations in Guiyang, Guizhou Province, China were analyzed. By using an upgraded method of preparation, the polytene chromosomes of the larval salivary glands of S. (W.) xingyiense were observed and analysed. The results indicated that this species has three pairs of chromosomes (2n=6) which are arranged from the longest to the shortest. Chromosome Ⅰ is metacentric while the chromosome Ⅱ and III are submetacentric. The nucleolar organizer is situated near the centromeric band of the short arm of chromosome Ⅰ. The Balbiani ring and double bubble are located about the middle of short arm of chromosome Ⅱ. The pericentric heterochromatin of all chromosome arms forms an evident chromocenter. High inversion polymorphism is observed, with 0.64 inversions per individual. Thus, the polytene chromosomes of S. (W.) xingyiense are species-specific. Inversion is one of the characteristics of polytene chromosomes of S. (W.) xingyiense.

As one group of old social insects, termites face serious threats from a variety of potential entomopathogenic microbes during their long life history. This article gives a brief review of the mechanisms by which termites resist the infection and spreading of entomopathogenic microbes in terms of physiological immunization and social behaviors, such as cellular and humoral immune responses as well as other altruistical behaviors like allogrooming, so as to provide necessary information for the studies on termite immunity strategies. At present, several antimicrobial compounds have been identified from cephalic gland and sternal gland secretions of termites. Several antimicrobial peptides such as termicin and spinigerin and antimicrobial proteins like transferrin and lysozymes as well as the social behaviors including allogrooming and trophallaxis turn out to play important roles in protecting termites from being infected by entomopathogenic microbes. Researchers have clarified preliminarily the physicochemical properties of antimicrobial peptides and confirmed that controlling termites with entomopathogenic microbes by means of RNA interference and other molecular biological methods is feasible and promising. However, it needs further study to clarify the evolution of antimicrobial peptides, the interactions between various antimicrobial peptides and proteins as well as the interactions between physiological immunity strategies and intra-colonial social behaviors.