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Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1 (TCP-1), which belongs to the heat shock protein 60 (HSP60) family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and qPCR, respectively. The full-length cDNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5'- and 3'-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity (60–99%) with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, qPCR was used to analyze expression levels of Tcp-1 mRNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. qPCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress.

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 cDNA 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 kDa, 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 CcHSP40 in C. chilonis was the Type II HSP40, CcHSP60 was a member of the mitochondrial HSP60 family, and CcHSP90 was a part of cytoplasmic HSP90A 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.

Insects represent a sustainable, protein-rich food source widely consumed in Asia, Africa, and South America. Eating Clanis bilineata tsingtauica Mell is common in the eastern part of China. A comparative characterization of nutrients in the meat and epidermis of C. bilineata tsingtauica was performed in this study. The results showed this insect to be high in nutrients, particularly in the epidermis where protein total was 71.82%. Sixteen different amino acids were quantified in C. bilineata tsingtauica, and the ratio of essential to nonessential amino acids in the epidermis and meat was 68.14% and 59.27%, respectively. The amino acid composition of C. bilineata tsingtauica is balanced, representing a high-quality protein source. Eight minerals were quantified in C. bilineata tsingtauica, including four macro and four trace elements. Fe in the epidermis and Zn in the meat were abundant at 163.82 and 299.31 μg/g DW, respectively. The presence of phytic acid impacted the absorption of mineral elements in food. We also detected phytic acid in C. bilineata tsingtauica. The molar ratio of phytic acid to zinc (PA/Zn) in C. bilineata tsingtauica was very low (3.28) compared to Glycine max and Cryptotympana atrata, which indicated that mineral utilization was high. In conclusion, this study confirms that C. bilineata tsingtauica is a highly nutritious food source for human consumption, and the results provide a basis for further consumption and industrialization of this edible insect.

Searching for new states of matter and unusual quasi-particles in Dirac materials attracts a significant interest in condensed matter physics . Here we investigate the bound state formation in tilted Dirac materials in the presence of the one- and two-dimensional Coulomb potentials . Based on the exact solution of the Dirac equation , we obtain the energy spectrum of the bound states and the corresponding wave functions in the one-dimensional case. It is shown that the lower-energy bound states dive into the continuum below the band gap with the increase of the tilted parameter. The atomic collapse appears when the strength of the Coulomb potential exceeds the critical value. We show that the tilt of Dirac cone facilitates lowing the critical Coulomb potential strength where atomic collapse happens. Most notably, it is found that the bound states are absent for an overtilted Dirac dispersion. For the two-dimensional Coulomb potential, we perform the second-order perturbation theory to study the effect of band tilting on the bound state formation with a single point-like Coulomb impurity. The result indicates that the ground state becomes more unstable near the critical point for a stronger tilt parameter.

Liriomyza trifolii is an invasive leafminer fly that inflicts damage on many horticultural and vegetable crops. In this study, the effects of elevated temperatures on L. trifolii tolerance to insecticides abamectin (AB), monosultap (MO) and a mixture of abamectin and monosultap (AM) were firstly investigated, then five CYP450 genes (LtCYPs) were cloned, and expression patterns and NADPH cytochrome C reductase (NCR) activity in L. trifolii were compared in response to high temperature stress and insecticide exposure. Results showed elevated temperatures induced expression of LtCYP450s, the expression level of LtCYP4g1, LtCYP4g15 and LtCYP301A1 after exposed to different high temperature were significantly up-regulated compared with the control (25 °C), while there was no significant difference in LtCYP4E21 and LtCYP18A1. Under the joint high temperature and insecticide stress, the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1 was significantly higher under elevated temperatures than that of only under AB exposure. For MO and AM exposure, only 40 °C could induce the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1. In general, the LtCYPs expression pattern was correlated with increased NCR activity and decreased mortality in response to insecticide exposure under elevated temperatures. These all demonstrated that insecticide tolerance in L. trifolii could be mediated by high temperature. This study improves our understanding of L. trifolii physiology and offers a theoretical context for improved control that ultimately reduces the abuse of insecticides and decreases exposure to non-target organisms.

Cotesia chilonis (Munakata) is the dominant parasitic wasp of the rice pest, Chilo suppressalis (Walker), and is a valuable parasitic wasp for the prevention and control of C. suppressalis. In this study, developmental indicators and expression of Cchsp11.0 (heat shock protein 11.0) and Cchsf (heat shock factor) were compared for C. chilonis at 27 °C and 36 °C. Developmental duration, morphology, emergence rate, and number of C. chilonis offspring were shortened at 36 °C while the ratio of females to males increased. Cchsp11.0 and Cchsf were highly expressed in the 1st instar stage at 36 °C, and Cchsp11.0 expression gradually decreased as C. chilonis matured; Cchsf expression was not correlated with Cchsp11.0 expression. Compared with 27 °C, the expression pattern of Cchsp11.0 and Cchsf was also not consistent, and Cchsp11.0 expression increased significantly at the adult stage. In conclusion, mildly high temperatures impact growth, development and reproduction of C. chilonis and stimulate the expression of Cchsp11.0 and Cchsf, and Cchsp11.0 and Cchsf play different roles in different developmental stages of C. chilonis at normal and high temperature.

Catalase (CAT), one of the key enzymes involved in protecting organisms from reactive oxygen species (ROS), degrades hydrogen peroxide to oxygen and water. In this study, we isolated the catalase gene ( CsCAT ) from the striped stem borer, Chilo suppressalis , a serious pest of rice in Asia. Sequence analysis of the genomic DNA indicated that CsCAT contains a 1023 bp intron and a 1524 bp coding sequence. The predicted CsCAT protein contains 507 amino acids and includes the proximal active site (FXRERIPERVVHAKGXGA) and heme-ligand sequence (RLFSYNDTX) that are conserved in catalase enzymes. Phylogenetic analysis indicated that CAT from Lepidopteran moths could be assigned to one well-supported cluster. qRT-PCR indicated that CsCAT was expressed in insect heads, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, and hemocytes. Expression of CsCAT was highest in the fat body and hemocytes of diapausing larvae, which suggests a potential important role in insect physiology during diapause period. Expression of CsCAT (measured by qRT-PCR) and enzymatic activity of CsCAT were not significantly altered by temperature stress, which indicates that CsCAT may insensitive to temperature.

Small heat shock proteins (sHSPs) are probably the most diverse in structure and function among the various super-families of stress proteins, and they play essential roles in various biological processes. The sweet potato whitefly, Bemisia tabaci (Gennadius), feeds in the phloem, transmits several plant viruses, and is an important pest on cotton, vegetables and ornamentals. In this research, we isolated and characterized three α-crystallin/sHSP family genes (Bthsp19.5, Bthsp19.2, and Bthsp21.3) from Bemisia tabaci. The three cDNAs encoded proteins of 171, 169, and 189 amino acids with calculated molecular weights of 19.5, 19.2, and 21.3 kDa and isoelectric points of 6.1, 6.2, and 6.0, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in Hemiptera and Thysanoptera insects species. All three sHSPs genes from Bemisia tabaci lacked introns. Quantitative real-time PCR analyses revealed that the three BtsHSPs genes were significantly up-regulated in Bemisia tabaci adults and pupae during high temperature stress (39, 41, 43, and 45 °C) but not in response to cold temperature stress (−6, −8, −10, and −12 °C). The expression levels of Bthsp19.2 and Bthsp21.3 in pupae was higher than adults in response to heat stress, while the expression level of Bthsp19.5 in adults was higher than pupae. In conclusion, this research results show that the sHSP genes of Bemisia tabaci had shown differential expression changes under thermal stress.

The highly conserved heat shock protein 70 (HSP70) contributes to survival at a cellular level and greatly enhances stress tolerance in many organisms. In this study, we isolate and characterize Cshsp702, which encodes an inducible form of HSP70 in the rice stem borer, Chilo suppressalis. Cshsp702 does not contain introns; the translational product is comprised of 629 amino acids with an isoelectric point of 5.69. Real-time quantitative PCR revealed that Cshsp702 was expressed at maximal levels in hemocytes and was minimally expressed in the midgut. Expression of Cshsp702 in response to a range of temperatures (−11 to 43 °C) indicated significant induction by extreme cold and hot temperatures, with maximum expression after 2 h at 42 °C. The induction of Cshsp702 in response to the endoparasite Cotesia chilonis was also studied; interestingly, Cshsp702 expression in C. suppressalis was significantly induced at 24 h and 5 days, which correspond to predicted times of C. chilonis feeding and growth, respectively. The potential induction of Cshsp702 as an inflammatory response due to parasitic stress is discussed. In conclusion, Cshsp702 is induced in response to both environmental and biotic stress and plays an important role in the physiological adaptation of C. suppressalis.

The western flower thrips, Frankliniella occidentalis, is an important invasive pest with a strong tolerance for extreme temperatures; however, the molecular mechanisms that regulate thermotolerance in this insect remain unclear. In this study, four heat shock protein genes were cloned from F. occidentalis and named Fohsp90, Fohsc701, Fohsc702 and Fohsp60. These four Hsps exhibited typical characteristics of heat shock proteins. Subcellular localization signals and phylogenetic analysis indicated that FoHsp90 and FoHsc701 localize to the cytosol, whereas FoHsc702 and FoHsp60 were located in the endoplasmic reticulum and mitochondria, respectively. Analysis of genomic sequences revealed the presence of introns in the four genes (three, four, seven, and five introns for Fohsp90, Fohsc701, Fohsc702 and Fohsp60, respectively). Both the number and position of introns in these four genes were quite different from analogous genes in other species. qRT-PCR indicated that the four Fohsps were detected in second-stage larvae, one-day-old pupae, and one-day-old adults, and mRNA expression levels were lowest in larvae and highest in pupae. Fohsc701 and Fohsc702 possessed similar expression patterns and were not induced by cold or heat stress. Expression of Fohsp60 was significantly elevated by heat, and Fohsp90 was rapidly up-regulated after exposure to both cold and heat stress. Exposure to -8°C had no effect on expression of the four Fohsps; however, expression of Fohsp90 and Fohsp60 was highest after a 2-h incubation at 39°C. Furthermore, cold and heat hardening led to significant up-regulation of the four Fohsps compared to their respective controls. Collectively, our results indicate that the four FoHsps contribute to insect development and also function in rapid cold or heat hardening; furthermore, FoHsp90 and FoHsp60 contribute to thermotolerance in F. occidentalis.

In quantitative real-time PCR (qRT-PCR), data are normalized using reference genes, which helps to control for internal differences and reduce error among samples. In this study, the expression profiles of eight candidate housekeeping genes, 18S ribosomal (18S rRNA), elongation factor (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein L17 (RPL17), histone 3 (H3), arginine kinase (AK), amd β-Actin (ACTB), were evaluated in the parasitic wasp Cotesia chilonis in response to different temperatures. Specifically, the performance and stabilities of these genes were compared in adult wasps maintained in a growth condition at 27°C (normal storage conditions) and in adults obtained from pupae refrigerated at 4°C for five days (cold storage conditions). Data were analyzed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The optimal numbers and stabilities of reference genes varied between the two temperature treatments (27°C and 4°C). In samples stored at normal developmental temperature (27°C), the requirement for normalization in response to low temperature exposures was three genes (18S, H3, AK), whereas normalization in response to high temperature exposures required only two reference genes (GAPDH, ACTB). In samples stored at cold temperature (4°C), for low temperature exposures two reference genes (RPL17, RPL10) were required for standardization, while following high temperature exposures three reference genes (18S, H3, ACTB) were needed. This study strengthens understanding of the selection of reference genes before qRT-PCR analysis in C. chilonis. The reference genes identified here will facilitate further investigations of the biological characteristics of this important parasitoid.

Heat shock proteins (HSPs) participate in diverse physiological processes in insects, and HSP70 is one of the most highly conserved proteins in the HSP family. In this study, full-length cDNAs of three HSP70 genes (Lthsc70,Lthsp701, andLthsp702) were cloned and characterized fromLiriomyza trifolii, an important invasive pest of vegetable crops and horticultural crops worldwide. These three HSP70s exhibited signature sequences and motifs that are typical of the HSP70 family. The expression patterns of the threeLthsp70s during temperature stress and in different insect development stages were studied by real-time quantitative PCR.Lthsp701was strongly induced by high- and low-temperature stress, butLthsc70andLthsp702were not very sensitive to temperature changes. All threeLthsp70s were expressed during insect development stages, but the expression patterns were quite different. The expression ofLthsc70andLthsp702showed significant differences in expression during leafminer development;Lthsc70was most highly expressed in female adults, whereasLthsp702was abundantly expressed in larvae and prepupae.Lthsp701expression was not significantly different among leafminer stages. These results suggest that functional differentiation within theLtHSP70 subfamily has occurred in response to thermal stress and insect development.

Aquaporins are integral membrane proteins some of which form high capacity water-selective channels, promoting water permeation across cell membranes. In this study, we isolated the aquaporin transcript (CsDrip1) of Chilo suppressalis, one of the important rice pests. CsDrip1 included two variants, CsDrip1_v1 and CsDrip1_v2. Although CsDrip1_v2 sequence (>409 bp) was longer than CsDrip1_v1, they possessed the same open reading frame (ORF). Protein structure and topology of CsDrip1 was analyzed using a predicted model, and the results demonstrated the conserved properties of insect water-specific aquaporins, including 6 transmembrane domains, 2 NPA motifs, ar/R constriction region (Phe69, His194, Ser203, and Arg209) and the C-terminal peptide sequence ending in “SYDF.” Our data revealed that the Xenopus oocytes expressing CsDrip1 indicated CsDrip1 could transport water instead of glycerol, trehalose and urea. Further, the transcript of CsDrip1 expressed ubiquitously but differentially in different tissues or organs and developmental stages of C. suppressalis. CsDrip1 mRNA exhibited the highest level of expression within hindgut and the third instar larvae. Regardless of pupae and adults, there were significantly different expression levels of CsDrip1 gene between male and female. Different from at low temperature, the transcript of CsDrip1 in larvae exposed to high temperature was increased significantly. Moreover, the mRNA levels of CsDrip1 in the third instar larvae, the fifth instar larvae, pupae (male and female), and adults (male and female) under different humidities were investigated. However, the mRNA levels of CsDrip1 of only female and male adults were changed remarkably. In conclusions, CsDrip1 plays important roles in maintaining water homeostasis in this important rice pest.

Heat shock proteins (HSPs) play important roles in the adaptation of organisms to environmental changes. In this study, we explore the role of HSPs during heat stress in Sesamia inferens (Walker) (Lepidoptera: Noctuidae), an important pest of rice. In order to understand the mechanistic basis of ecological adaptability and stress tolerance, we analyzed the expression profiles of genes encoding five S. inferens heat shock proteins (Sihsps) across temperature gradients by real-time quantitative PCR. Our results indicated that genes encoding ribosomal protein S13 (RPS13) and β-actin (ACTB) were the most stable reference genes under high temperature stress. The mRNA levels of Sihsp19.6, Sihsp20.6 and Sihsp83 were significantly up-regulated by heat stress; however, the expression of Sihsp21.4 and Sihsc70 did not vary significantly. The massive increase in the expression of Sihsp19.6 and Sihsp20.6 by thermal stress suggests a critical role in preventing cellular damage to S. inferens. Moreover, the threshold temperatures (39 °C for Sihsp19.6, Sihsp20.6 and Sihsp83) may partly explain the more southern distribution of S. inferens. Our results also support the contention that the absence of introns may favor the expression of stress-responsive genes (e.g. Sihsp19.6, Sihsp20.6 and Sihsp83 in the present study).

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads, Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

The pink rice stem borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae), is a major rice pest in China and elsewhere in Asia. While low winter temperatures are a major environmental constraint on the survival of most insect species, the mechanism of S. inferens’ cold tolerance in winter remains unknown. In this study, we elucidated the cold tolerance characteristics of S. inferens collected in the field from Oct 2012 to Apr 2013. The cold tolerance of overwintering larvae was found to vary significantly. Maximum S. inferens cold tolerance was observed in larvae collected on 30 Jan 2013. However, the SCP (supercooling points) of larvae did not vary insignificantly, with a mean of -6.80 °C. Before 9 Mar 2013, larval water content stabilized at the mean low level of 63.5 %, but subsequently rose significantly, to 75.2%. Low molecular weight sugars and polyols, closely related to freeze tolerance strategy, increased from low levels to their peaks in Jan (glycerol, 359.8 μg/g; trehalose, 20.5 mg/g; fructose, 69.8 μg/g; glucose, 377.3 μg/g; myo-inositol, 59.6 μg/g), after which levels declined. This study demonstrates that the Yangzhou population of S. inferens is freeze-tolerant.

Power system visualization technology is essential in improving the power system automation and human-computer interaction environment. The power of information visualization technology can display a lot of information in a computer-generated image, and help system personnel to process and analysis of numerous grid data more efficiently. It provides a more intuitive operating platform for system operators.

In this study, we report the complete mitochondrial genome (mitogenome, mtDNA) of Aposthonia borneensis and compare it with another sequenced webspinner, Aposthonia japonica. The A. borneensis mitogenome is smaller than A. japonica, but the size of each gene and the A + T content of protein-coding genes (PCGs) are almost identical in the two mitogenomes. Among the PCGs, atp6 shows the highest evolutionary rate and cox1 the lowest. The mtDNA map in A. borneensis is similar to Drosophila yakuba, but distinctly different from A. japonica, which has extensive rearrangement. Phylogenetic analyses dated the divergence time of the two webspinners at ca. 103 Ma. We speculate that the most recent common ancestor (MRCA) of A. borneensis and A. japonica was divided into several geographic groups during the Pangea breakup. Geographic isolation between the Japanese islands and the continental southeastern Asia resulted in the divergent evolution of A. borneensis and A. japonica, thus generating mtDNA structural variations between the two species. Based on the phylogenetic analyses and specific distributional features, the genus Aposthonia was supported as non-monophyly, and we speculate that both highly rearranged and relatively conserved mitogenomes exist in other webspinners.

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from −11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to −3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.

Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR protocol for functional genomics research in L. trifolii.

Aim To investigate the absorption characteristics of the total alkaloids from Mahoniae Caulis (TAMC) through the administration of monterpene absorption enhancers or protein inhibitors. Method The absorption behavior was investigated in an in situ single-pass intestinal perfusion (SPIP) assay in rats. Results The intestinal absorption of TAMC was much more than that of a single compound or a mixture of compounds (jatrorrhizine, palmatine, and berberine). Promotion of absorption by the bicyclic monoterpenoids (borneol or camphor) was higher than by the monocyclic monoterpenes (menthol or menthone), and promotion by compounds with a hydroxyl group (borneol or menthol) was higher than those with a carbonyl group (camphor or menthone). The apparent permeability coefficient ( Papp ) of TAMC was increased to 1.8-fold by verapamil, while it was reduced to one half by thiamine. The absorption rate constant ( Ka ) and Papp of TAMC were unchanged by probenecid and pantoprazole. Conclusion The intestinal absorption characteristics of TAMC might be passive transport, and the intestinum tenue was the best absorptive site. In addition, TAMC might be likely a substrate of P-glycoprotein (P-gp) and organic cation transporters (OCT), rather than multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP). Compared with a single compound and a mixture of compounds, TAMC was able to be absorbed in the blood circulation effectively.

The striped stem borer, Chilo suppressalis, is one of the most serious pests of rice, causing extensive damage and yield loss. This report is a study on the expression patterns of three heat shock protein genes of C. suppressalis. Results indicated that hsp90, hsp70, and hsp60 all expressed within eight tissues (organs) sampled from fifth-instar larvae, with the highest levels being found in the hindgut and the lowest levels in the foregut. Interestingly, an abundance of all three hsps were observed in the head. We also examined heat shock protein expression in different life stages and sexes of rice stem borer. The hsp90 and hsp70 also appeared correlated with the developmental processes of C. suppressalis. The results showed that the transcripts of hsp70 varied remarkably in different developmental stages. The hsp60 demonstrated significantly higher expression in female than male adults. All three hsps of C. suppressalis could be induced by temperature stress, especially heat stress. For example, the highest increase for hsp90, hsp70, and hsp60 was at 42, 36, and 36°C, respectively. All three hsps were more sensitive to heat than cold.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a valuable tool for estimating gene expression; however, the validity is largely dependent on the selection of stable reference genes. The suitability of various reference genes for qRT-PCR analysis was evaluated in, Chilo suppressalis (Walker). The ΔCt method, geNorm, NormFinder, and BestKeeper were used to evaluate the suitability of nine candidate reference genes for normalizing gene expression in larval tissues and organs and during high and low temperature stress. The ΔCt method, geNorm, and NormFinder produced similar stability rankings; H3, UBI, and EF1 were the most stable reference genes for monitoring gene expression in larval tissue and organs, and EF1, TUB, and AK were the optimal genes for thermal stress. However, for thermal stress, RPS11 was the most stable gene based on BestKeeper. To validate these recommendations, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. Hsp60 transcript levels showed significant differences when normalized to the most versus least stable reference genes. These results further confirm the importance of testing reference genes using the selected experimental parameters. The reference genes identified in the present study will improve the quality of gene expression data obtained for C. suppressalis and will facilitate future studies aimed at understanding the biology of this important insect pest.

Water chestnut beetle (Galerucella birmanica Jacoby) is a pest of the water chestnut (Trapa natans L.). To analyze the phylogeny and biogeography of the beetle and provide evidence for the origin of T. natans in China, we conducted this by using three mitochondrial genes (COI, COII and Cytb) and nuclear ITS2 ribosomal DNA of G. birmanica. As for mtDNA genes, the beetle could be subdivided into three groups: northeastern China (NEC), central-northern-southern China (CC-NC-SC) and southwestern China (SWC) based on SAMOVA, phylogenetic analyses and haplotype networks. But for ITS2, no obvious lineages were obtained but individuals which were from NEC region clustered into one clade, which might be due to sequence conservation of ITS2. Significant genetic variation was observed among the three groups with infrequent gene flow between groups, which may have been restricted due to natural barriers and events in the Late Pleistocene. Based on our analyses of genetic variation in the CC-NC-SC geographical region, the star-like haplotype networks, approximate Bayesian computation, niche modelling and phylogeographic variation of the beetle, we concluded that the beetle population has been lasting in the lower, central reaches of the Yangtze River Basin with its host plant, water chestnut, which is consistent with archaeological records. Moreover, we speculate that the CC-NC-SC population of G. birmanica may have undergone a period of expansion coincident with domestication of the water chestnut approximately 113,900-126,500 years ago.

The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is an important, widely distributed pest of rice, Oryza sativa L., in China. However, information is lacking about postdiapause levels of cold resistance during periods of normal development. Our results indicate that field larvae of C. suppressalis collected in March 2010 had very low survival if exposed to -21°C. When exposed to test temperatures for 1 h, the LTemp50 and LTemp90 values of C. suppressalis were -18.8 and -20.8°C, respectively. LTime50 and LTime90 values at -16°C (5.1 and 9.6 h) were unexpectedly lower than those at -17°C (7.1 and 18.3 h). For larvae collected in August, mortality at -18°C (with 2-h exposure) was 98% but was only 8% at -6°C (2-h exposure). Maximum cold tolerance of C. suppressalis was observed in larvae collected on 15 January 2011. These data suggest that cold hardiness in C. suppressalis is independent of the diapause state of the insect.

Western flower thrips (WFT), Frankliniella occidentalis, is an invasive worldwide pest that causes great economic loss. Temperature plays an important role in shaping insect distributions. In this study, we report the survival rates of 3-day-old WFT exposed to rapid changes in extreme high and low temperatures. Temperature, exposure time, and gender all significantly affected the survival of adult WFT, with the lowest survival associated with more extreme temperatures and/or longer exposures. The temperature required to kill 50% of exposed WFT individuals (LT50) decreased with extended exposure time, but females were more tolerant to extreme temperature than males. Investigation of rapid cold or heat hardening suggested that a short prior exposure to a sub-lethal low or high temperature increased WFT survival during a subsequent exposure to a lethal temperature. Tolerance of extreme temperatures and an ability to undergo rapid hardening are of great ecological relevance in determining the geographic distribution of WFT, allowing it to survive better in temporary bouts of extreme temperature stress. Our findings provide useful information on the environmental limits on the distribution of WFT, which have implications for control of this pest.

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.

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°C. Mean survival rates of the larvae which exposed to either 0 or 5°C for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups (P

The striped stem borer, Chilo suppressalis (Walker), is an important pest of rice in China and other parts of the world. To further explore the population genetic structure and genetic differentiation of C. suppressalis populations found on rice in China, we amplified 432 bp fragments of the cytochrome b (cyt b) gene for 44 C. suppressalis populations. Nineteen variable sites in the mtDNA gene were observed, and 16 haplotypes were identified. Nucleotide diversity (π) and haplotype diversity (h) ranged from 0.00274 to 0.00786 and 0.72297 to 0.87604, respectively, while genetic structure analysis found significant genetic differentiation to be present among the five regions in China - northern China (NC), northeastern China (NEC), central China (CC), southern China (SC) and southwestern China (SWC) - where C. suppresalis was collected. In addition, molecular variance (AMOVA) showed that a relatively high proportion (57.6%) of the total genetic variance was attributable to variation within the populations. N(m) and F(ST) analyses suggested that the differentiation was not significantly different between NEC and NC, CC and SC, and SC and SWC regions, but was significant between NEC and CC, SC and SWC regions, corresponding well with the geographical distribution of the sampled populations. Phylogenetic analysis divided the populations into two indistinct clades: a NEC-NC-CC clade and a CC-SC-SWC clade, while CC region acted as a transition zone between north and south China, a finding different from previous work.

To assess the clinical features and therapeutic effect of free thin anterolateral thigh flap for the reconstruction of intraoral defects.The clinicopathologic data of 34 cases with oral carcinoma were obtained from Institute and Hospital of Stomatology, Nanjing University Medical School, from December 2008 to December 2011. These 34 patients underwent the simultaneous tumor resection and intraoral defects reconstruction with free thin anterolateral thigh flaps. The defects were located at tongue, buccal, mouth floor, and so on. The subcutaneous fat thickness of the flap at the site of the perforator is usually measured by ultrasound before the operation. If the thickness of the subcutaneous fat at the site of the perforator exceeded 1.5 cm, the patient was excluded from the study.There were 16 male and 18 female patients. The mean age was 55.4 years. Among the 34 patients, 26 flaps with musculocutaneous perforators and 8 flaps with septo-cutaneous perforators were used. Partial flap necrosis occurred in 2 patients.The free thin anterolateral thigh flap is the ideal soft tissue flap for the intraoral defects reconstruction. Satisfactory functional results can he achieved at recipient area with minimal morbidity at the donor site area.

Diapause is a complex and dynamic process. Chilo suppressalis, an important rice pest in Asia enters facultative diapause as larvae. Our results demonstrated in Yangzhou, China, diapause was initiated between September 4 and 12, 2010. After diapause termination, C. suppressalis remained in quiescence in the field for as long as three months. The average time between collection of field larvae of C. suppressalis and their pupation decreased as the season progressed from fall to next spring. Unexpectedly, the pupated ratio of female to male in the initiation of diapause was 0.22. The abundance of hsp90, hsp70, hsp60 and CsAQP1 all peaked on January 8 or 15, 2011. Nitric oxide (NO) is a secondary messenger that is positively correlated with the diapause of C. suppressalis. Among several geographically separated populations of C. suppressalis, there are no significant differences in the mRNA levels of hsp70, hsp60 or CsAQP1. National Basic Research and Development Program of China 2012CB114100 National Natural Science Foundation of China 31371937 Ph.D. Programs Foundation of the Ministry of Education of China 20113250110008

Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases. In this study, we isolated the caspase gene (Cscaspase-1) of Chilo suppressalis (one of the leading pests responsible for destruction of rice crops). It possesses the open reading frame (ORF) of 295 amino acids including prodomain, large subunit and small subunits, and two cleavage sites (Asp23 and Asp194) were found to be located among them. In addition to these profiles, Cscaspase-1 contains two active sites (His134 and Cys176). Genomic analysis demonstrated there was no intron in the genome of Cscaspase-1. The Cscaspase-1 transcripts were found in all tissues of the fifth instar larvae, and higher levels were found in the midgut, hindgut and Malpighian tubules. Examination of Cscaspase-1 expression in different developmental stages indicated low constitutive levels in the eggs and early larvae stages, and higher abundances were exhibited in the last larvae and pupae stages. The relative mRNA levels of Cscaspase-1 were induced by heat and cold temperatures. For example, the highest increase of Cscaspase-1 transcription was at −3 °C and 36 °C respectively. In a word, Cscaspase-1 plays a role of effector in the apoptosis of C. suppressalis. It also correlates with development, metamorphosis and thermotolerance of C. suppreassalis.

Small heat shock proteins (sHSPs) are the most diverse but also the most poorly known family of molecular chaperones, and they play essential roles in various biological processes. The striped stem borer, Chilo suppressalis (Insecta: Lepidoptera: Pyralidae), is one of the most serious pests of rice, causing extensive damage and yield loss. In this study, we isolated and characterized five members of the sHSPs family—Cshsp19.8, Cshsp21.4, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b—from C. suppressalis. The cDNAs of these genes encoded proteins of 177, 187, 191, 191, and 191 amino acids with isoelectric points of 7.0, 5.6, 6.1, 6.3, and 6.3, respectively. While Cshsp19.8, Cshsp21.5, and Cshsp21.7b had no introns, Cshsp21.4 and Cshsp21.7a contained one and two introns, respectively. Structural analysis indicated that all five Cshsps possessed conserved arginine and a V/IXI/V motif, which is related to hydrophobic characteristics of sHSPs. The five heat shock proteins can be classified into two main groups: an orthologous type (Cshsp21.4 and Cshsp21.7a) and a species-specific type (Cshsp19.8, Cshsp21.5, and Cshsp21.7b). Real-time quantitative PCR analyses revealed that Cshsp19.8, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b all exhibited their highest expression levels within Malpighian tubules or the hindgut, while such levels were found in the head for Cshsp21.4. The expression of Csshsps at different developmental stages revealed that the mRNA levels of Cshsp19.8, Cshsp21.4, Cshsp21.5, and Cshsp21.7b peaked in adults, whereas the highest level of Cshsp21.7a was observed in first instar larvae. Cshsp19.8 and Cshsp21.7b were both upregulated dramatically by heat and cold, and Cshsp21.5 could be induced by cold stress. Neither Cshsp21.4 nor Cshsp21.7a responded to heat or cold. These results demonstrated that different Csshsps play distinctive roles in the regulation of the physiological activities in C. suppressalis.

To assess the clinical features and therapeutic efficacy of anterolateral thigh (ALT) flaps for the intraoral defects reconstruction.The clinicopathologic data of 67 cases with oral tumors were obtained from School of Stomatology, Nanjing University Medical Center from Dec. 2008 to Dec. 2010. All the patients underwent the simultaneous tumor resection and intraoral defects reconstruction with free anterolateral thigh flaps. The defects included the tongue, buccal, gingival, mouth floor, and so on. The descending branch of lateral femoral circumflex artery was anastomosed to the external maxillary artery or superior thyroid artery; the vein was anastomosed to the common facial vein or external jugular vein. The flaps were divided into three types: musculocutaneous ALT flap, fasciocutaneous ALT flap and thinned ALT flap.There were 38 male patients and 29 female. The anterolateral thigh flaps included 35 musculocutaneous flaps, 17 fasciocutaneous flaps and 15 thinned flaps. The success rate was 98.5% (66/67). Partial necrosis happened in one case with diabetes, which healed after debridement and dressing. 1 flap was totally necrosis. Double venous anastomosis was performed in 41 flaps, and one venous anastomosis was performed in 26 flaps. 8 patients required operative exploration in the perioperative period including 6 flaps with thrombotic events (5 flaps were complete survival after the salvages, and 1 flap was failure) , 1 flap with hematoma, and 1 flap with twisting of perforator. The follow-up period ranged from 2 to 24 months( mean, 8.7 months). The result was satisfied. The donor sites were closed directly in all patients, and the wounds healed uneventfully.The free anterolateral thigh flap is an ideal soft tissue flap for the intraoral defects reconstruction with good functional result at recipient area and less morbidity at the donor site.

Heat shock protein 60 is an important chaperonin. In this paper, hsp60 of the stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), was cloned by RT-PCR and rapid amplification of cDNA end (RACE) reactions. The full length cDNA of hsp6 degrees Consisted of 2142 bp, with an ORF of 1719 bp, encoding 572 amino acid residues, with a 5'UTR of 158 bp and a 3'UTR of 265 bp. Cluster analysis confirmed that the deduced amino acid sequence shared high identity with the reported sequences from other insects (77%-86%). To investigate whether hsp60 in C. suppressalis responds to thermal stress, the expression levels of hsp60 mRNA in larval haemocytes across temperature gradients from 31 to 39 degrees C were analysed by real-time quantitative PCR. There was no significant difference for hsp60 expression from 28 to 31 degrees C. he temperatures for maximal induction of hsp60 expression in haemocytes was close to 36 degrees C. Hsp60 expression was observed by using flow cytometry. These results revealed that thermal stress significantly induced hsp60 expression and Hsp60 synthesis in larval haemocytes, and the expression profiles of Hsp60 at the mRNA and protein levels were in high agreement with each other from 33 to 39 degrees C.

To investigate the application of titanium mesh and free forearm flap for reconstruction of maxillary defect resulted from tumor resection.From 2004 to 2008, 19 cases with maxillary tumor underwent tumor resection. The defects were reconstructed immediately with titanium mesh for bony defects and free forearm flap for oral mucosa defects.16 cases achieved satisfactory functional and cosmetic results. The speech assessment was good without oronasal reflux. The titanium mesh was exposed and infected in 3 cases. Then the meshes were taken out and the defects were covered with the forearm flap to close the oronasal fistula. The midface was slightly depressed with no functional morbidity.It is simple and practical to reconstruct maxillary defect with titanium mesh and free forearm flap. Both the functional and cosmetic results are satisfactory.

Aimed to understand the mechanisms of cold-resistance of overwintering Chilo suppressalis (Walker) larvae at physiological and biochemical levels, the supercooling point (SCP) and the contents of water, ash, elements, fat, fatty acid, glycrol, total sugar, and protein in the larvae samples collected at different time were determined. The results showed that the SCP and the contents of free water, dissociated fat, total sugar, and protein in the overwintering larvae decreased first and increased then, whereas the contents of bound water, ash, combined fat, K, Na, Mg, Fe, Ni and Cr, and glycerol decreased after an initial increase. The components of fatty acids in overwintering larvae had some changes, but the main components were still 9-hexadecenoic acid, 9-hexadecanoic acid, and 9-octadecenoic acid. The contents of 9-hexadecenoic acid and 9-hexadecanoic acid decreased first and increased then, while that of 9-octadecenoic acid was in adverse. The dynamic changes of the contents of test substances could reflect the cold-resistance of overwintering C. suppressalis larvae.

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.