Depending on different apoptosis pathways, the effector Cscaspase-3 in Chilo suppressalis exposed to temperature and parasitic stress was induced.

Apoptosis is a form of programmed cell death (PCD) that is largely triggered by caspases through both the mitochondria-dependent and mitochondria-independent pathways. The rice stem borer, Chilo suppressalis, serves as an economically important pest of rice, which is often suffered by temperature and parasitic stress under natural conditions. In the present study, effector Cscaspase-3 encoding caspase was obtained from the rice pest Chilo suppressalis. CsCaspase-3 possesses p20 and p10 subunits, two active sites, four substrate-binding sites, and two cleavage motifs. Real-time quantitative PCR showed that Cscaspase-3 was expressed at maximal levels in hemocytes; furthermore, transcription was most highly in female adults. Expression of Cscaspase-3 was induced by hot and cold temperatures, with the highest expression at 39 °C. Cscaspase-3 expression was also significantly induced at 10 h, 2 d, 5 d, and 7 d of parasitism. Flow cytometry results showed that both temperature and parasitism trigger apoptosis, but only parasitism induces apoptosis via the mitochondrial apoptosis pathway in C. suppressalis. RNAi-mediated silencing of Cscaspase-3 expression reduced C. suppressalis survival at -3 °C. This study provides a foundation for further studies of caspases in insects during biotic and abiotic stress.

Orthodontic-surgical treatment for severe skeletal class II malocclusion with vertical maxillary excess and four premolars extraction: A case report.

BACKGROUND: Patient satisfaction with facial appearance at the end of orthodontic camouflage treatment is very important, especially for skeletal malocclusion. This case report highlights the importance of the treatment plan for a patient initially treated with four-premolar-extraction camouflage, despite indications for orthognathic surgery. CASE SUMMARY: A 23-year-old male sought treatment complaining about his unsatisfactory facial appearance. His maxillary first premolars and mandibular second premolars had been extracted, and a fixed appliance had been used to retract his anterior teeth for two years without improvement. He had a convex profile, a gummy smile, lip incompetence, inadequate maxillary incisor inclination, and almost a class I molar relationship. Cephalometric analysis showed severe skeletal class II malocclusion (A point-nasion-B point = 11.5°) with a retrognathic mandible (sella-nasion-B point = 75.9°), a protruded maxilla (sella-nasion-A point = 87.4°), and vertical maxillary excess (upper incisor to palatal plane = 33.2 mm). The excessive lingual inclination of the maxillary incisors (upper incisor to nasion-A point line = -5.5°) was due to previous treatment attempts to compensate for the skeletal class II malocclusion. The patient was successfully retreated with decompensating orthodontic treatment combined with orthognathic surgery. The maxillary incisors were repositioned and proclined in the alveolar bone, the overjet was increased, and a space was created for orthognathic surgery, including maxillary impaction, anterior maxillary back-setting, and bilateral sagittal split ramus osteotomy to correct his skeletal anteroposterior discrepancy. Gingival display was reduced, and lip competence was restored. In addition, the results remained stable after 2 years. The patient was satisfied with his new profile as well as with the functional malocclusion at the end of treatment. CONCLUSION: This case report provides orthodontists a good example of how to treat an adult with severe skeletal class II malocclusion with vertical maxillary excess after an unsatisfactory orthodontic camouflage treatment. Orthodontic and orthognathic treatment can significantly correct a patient's facial appearance.

Chilo suppressalis heat shock proteins are regulated by heat shock factor 1 during heat stress.

Heat shock factor 1 (HSF1) functions to maintain cellular and organismal homeostasis by regulating the expression of target genes, including those encoding heat shock proteins (HSPs). In the present study, the gene encoding HSF1 was cloned from the rice pest Chilo suppressalis, and designated Cshsf1. The deduced protein product, CsHSF1, contained conserved domains typical of the HSF1 family, including a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal transactivation domain. Real-time quantitative PCR showed that Cshsf1 was highly expressed in hemocytes. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsf1 was most highly expressed in male adults. RNAi-mediated silencing of Cshsf1 expression reduced C. suppressalis survival at high temperatures. To investigate the regulatory interactions between Cshsf1 and Cshsps, the promoters and expression patterns of 18 identified Cshsps in C. suppressalis were analysed; four types of heat shock elements (HSEs) were identified in promoter regions including canonical, tail-tail, head-head, and step/gap. The expression of Cshsp19.0, Cshsp21.7B, Cshsp60, Cshsp70 and Cshsp90 was positively regulated by Cshsf1; however, Cshsp22.8, Cshsp702, Cshsp705 and Cshsp706 gene expression was not altered. This study provides a foundation for future studies of HSF1 in insects during thermal stress.

The regulation of inhibitor of apoptosis proteins (IAPs) during the apoptosis of Cotesia chilonis.

Inhibitor of apoptosis proteins (IAPs) are crucial components of apoptosis that perform vital roles in the regulation of caspase activity in organisms. In this study, two IAPs genes were identified from Cotesia chilonis, the dominant parasitic wasp of Chilo suppressalis. CcIAP1 gene is a typical IAP and contains two BIR domains and a RING domain, whereas CcIAP gene is an atypical IAP1 only containing two BIR domains. Phylogenetic analysis indicated that CcIAP1 and CcIAP were grouped with other Hymenopteran IAPs and IAP1 in C. suppressalis. Real-time quantitative PCR revealed that CcIAP1 and CcIAP genes were both highly induced at -6°C and 30°C, and expression was highest at the third instar stage. The expression of CcIAP1 and CcIAP genes were significantly induced during parasitism of C. suppressalis, and the 7-d time point resulted in the highest expression levels for both genes, in which was an advanced stage of larval development of C. chilonis. RNAi experiments showed that CcIAP1 gene was the key IAP in the regulation of apoptosis of C. chilonis and its host. In conclusion, CcIAP1 and CcIAP correlate with the development of C. chilonis and their responses to temperature stress.

Multimode Gesture Recognition Algorithm Based on Convolutional Long Short-Term Memory Network.

Gesture recognition utilizes deep learning network model to automatically extract deep features of data; however, traditional machine learning algorithms rely on manual feature extraction and poor model generalization ability. In this paper, a multimodal gesture recognition algorithm based on convolutional long-term memory network is proposed. First, a convolutional neural network (CNN) is employed to automatically extract the deeply hidden features of multimodal gesture data. Then, a time series model is constructed using a long short-term memory (LSTM) network to learn the long-term dependence of multimodal gesture features on the time series. On this basis, the classification of multimodal gestures is realized by the SoftMax classifier. Finally, the method is experimented and evaluated on two dynamic gesture datasets, VIVA and NVGesture. Experimental results indicate that the accuracy rates of the proposed method on the VIVA and NVGesture datasets are 92.55% and 87.38%, respectively, and its recognition accuracy and convergence performance are better than those of other comparison algorithms.

Species Identity Dominates over Environment in Driving Bacterial Community Assembly in Wild Invasive Leaf Miners.

The microbiota of invasive animal species may be pivotal to their adaptation and spread, yet the processes driving the assembly and potential sources of host-microbiota remain poorly understood. Here, we characterized microbiota of four Liriomyza leaf miner fly species totaling 310 individuals across 43 geographical populations in China and assessed whether the microbiota of the wild leaf miner was acquired from the soil microbiota or the host plant microbiota, using high-throughput 16S rRNA sequencing. Bacterial communities differed significantly among four leaf miner species but did not mirror host phylogeny. Microbiota diversity in the native L. chinensis was significantly higher than in three invasive leaf miners (i.e., L. trifolii, L. huidobrensis, and L. sativae), yet the microbial community of the invasive species exhibited a more connected and complex network structure. Structural equation models revealed that host species identity was more important than environmental factors (e.g., geography, climate, or plants) in shaping microbiota composition. Using neutral and null model analyses, we found that deterministic processes like variable selection played a primary role in driving microbial community assembly, with some influence by stochastic processes like drift. The relative degree of these processes governing microbiota was likely correlated with host species but independent of either geographical or climatic factors. Finally, source tracking analysis showed that leaf miners might acquire microbes from their host plant rather than the soil. Our results provide a robust assessment of the ecological processes governing bacterial community assembly and potential sources of microbes in invasive leaf miners. IMPORTANCE The invasion of foreign species, including leaf miners, is a major threat to world biota. Host-associated microbiota may facilitate host adaption and expansion in a variety of ways. Thus, understanding the processes that drive leaf miner microbiota assembly is imperative for better management of invasive species. However, how microbial communities assemble during the leaf miner invasions and how predictable the processes remain unexplored. This work quantitatively deciphers the relative importance of deterministic process and stochastic process in governing the assembly of four leaf miner microbiotas and identifies potential sources of leaf miner-colonizing microbes from the soil-plant-leaf miner continuum. Our study provides new insights into the mechanisms underlying the drive of leaf miner microbiota assembly.

Nutritional Properties of Larval Epidermis and Meat of the Edible Insect Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

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.

Transcriptional regulation of small heat shock protein genes by heat shock factor 1 (HSF1) in Liriomyza trifolii under heat stress.

Small heat shock proteins (sHSPs) function as molecular chaperones in multiple physiological processes and are active during thermal stress. sHSP expression is controlled by heat shock transcription factor (HSF); however, few studies have been conducted on HSF in agricultural pests. Liriomyza trifolii is an introduced insect pest of horticultural and vegetable crops in China. In this study, the master regulator, HSF1, was cloned and characterized from L. trifolii, and the expression levels of HSF1 and five sHSPs were studied during heat stress. HSF1 expression in L. trifolii generally decreased with rising temperatures, whereas expression of the five sHSPs showed an increasing trend that correlated with elevated temperatures. All five sHSPs and HSF1 showed an upward trend in expression with exposure to 40 ℃ without a recovery period. When a recovery period was incorporated after thermal stress, the expression patterns of HSF1 and sHSPs in L. trifolii exposed to 40 °C was significantly lower than expression with no recovery period. To elucidate potential interactions between HSF1 and sHSPs, double-stranded RNA was synthesized to knock down HSF1 in L. trifolii by RNA interference. The knockdown of HSF1 by RNAi decreased the survival rate and expression of HSP19.5, HSP20.8, and HSP21.3 during high-temperature stress. This study expands our understanding of HSF1-regulated gene expression in L. trifolii exposed to heat stress.

Transcriptomic analysis of pre-diapause larvae of Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) in natural populations.

Chilo suppressalis Walker is a devastating pest of rice in Asia and exhibits facultative diapause in the larval stage. Most prior experiments on diapausing and non-diapausing C. suppressalis were conducted in the laboratory. In this study, transcriptome analyses were performed on pre-diapausing larvae collected from field populations of C. suppressalis and compared to laboratory populations. Among 2674 differentially expressed genes (DEGs), 32 DEGs related to pre-diapause and 239 universally expressed genes were screened; these were primarily enriched in "neuroactive ligand-receptor interaction", "lysosome" and "glycerolipid metabolism" in KEGG pathway analysis. With respect to clusters of orthologous genes (COG), DEGs were assigned to "posttranslational modification, protein turnover, chaperones", "carbohydrate transport and metabolism", and "secondary metabolite biosynthesis, transport and catabolism" categories. Further analysis also revealed that a key "circadian clock-controlled protein" gene is sensitive to photoperiod and significantly decreased during the pre-diapause phase. Genes encoding two small heat shock proteins, hsp21.4 and hsp27.2, were significantly expressed on August 15 as compared to three other sampling times in August 2018. Eight DEGs were randomly chosen and evaluated by real-time quantitative PCR (RT-qPCR) to validate the accuracy of the transcriptome data. The expression of six DEGs (gene-evm_000752, gene-evm_006486, gene-evm_008626, gene-evm_002485, gene-evm_011981 and Chilo_suppressalis_newGene_18103) showed significant same patterns of differential expression in both the RT-qPCR and RNA-Seq analyses. This study increases our understanding of the complex physiological and molecular mechanisms involved in C. suppressalis at the pre-diapause phase.

Characterization and functional analysis of Cshsp19.0 encoding a small heat shock protein in Chilo suppressalis (Walker).

Small heat shock proteins (sHSPs) function as ATP-independent chaperones that preserve cellular proteostasis under stressful conditions. In this study, Cshsp19.0, which encodes a new small heat shock protein, was isolated and characterized from Chilo suppressalis (Walker) to better understand the contribution of sHSPs to insect development and stress tolerance. The full-length Cshsp19.0 cDNA was 697 bp and encoded a 19.0 kDa protein with an isoelectric point of 5.95. Phylogenetic analysis and amino acid alignments indicated that Cshsp19.0 is a member of the sHSP family. Cshsp19.0 was expressed at maximal levels in foreguts and showed the least amount of expression in fat bodies. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsp19.0 was most highly expressed in 1st instar larvae. Furthermore, Cshsp19.0 was upregulated when insects were exposed to heat and cold stress for a 2-h period. There were significant differences in the male and female pupae in response to humidity; Cshsp19.0 expression increased in male pupae as RH increased, whereas the inverse pattern was observed in female pupae. Larvae exhibited a lower rate of survival when Cshsp19.0 was silenced by a nanomaterial-promoted RNAi method. The results confirm that Cshsp19.0 functions to increase environmental stress tolerance and regulates physiological activities in C. suppressalis.

Identification and physiological function of CsPrip, a new aquaporin in Chilo suppressalis.

Aquaporin (AQP) transport solutes across cell membranes in both unicellular and multicellular organisms. In this study, the aquaporin CsPrip was identified in Chilo suppressalis, an important pest of rice. CsPrip was comprised of two variants, CsPrip_v1 and CsPrip_v2; the former variant was <103 bp was shorter than the latter, although both exhibited the same open reading frame (ORF). Transmembrane topology and protein structure analyses showed that CsPrip retained the conserved features of water-selective insect AQPs, including six transmembrane domains, two conserved hydrophobic asparagine-proline-alanine motifs and the aromatic/arginine constriction region. Expression in Xenopus oocytes revealed that CsPrip preferentially transported water and urea instead of trehalose and glycerol. The CsPrip transcript was expressed in multiple organs and tissues of C. suppressalis larvae and was most abundant in the hindgut and Malpighian tubules. CsPrip transcription was highest in male adults and was relatively stable throughout development. CsPrip expression in larvae was significantly altered by thermal stress, and relative humidity levels impacted CsPrip transcription in 3rd and 5th instar larvae. This study confirms that the aquaporin CsPrip performs multiple critical functions in maintaining water equilibrium in C. suppressalis.

Temperature affects the tolerance of Liriomyza trifolii to insecticide abamectin.

The leafminer fly, Liriomyza trifolii, is an invasive pest of horticultural and vegetable crops that possesses a robust competitive ability when compared to congeneric species, especially with respect to temperature and insecticide tolerance. Abamectin, which is commonly used to control L. trifolii in the field, was selected as the target insecticide in this study. Our objective was to study the effect of abamectin and high temperature stress on L. trifolii mortality and the expression of genes encoding cytochrome P450 (CYP450s) and heat shock proteins (Hsps) by quantitative real-time reverse transcriptase PCR (qRT-PCR). When L. trifolii was exposed to abamectin followed by exposure to 40 °C (LC50 +HT40), mortality showed a significant increase, whereas exposure to 40 â„ƒ followed by abamectin (HT40+LC50) reduced mortality relative to abamectin or HT40 alone. Expression of three CYP450s in the CYP4 family was highest in the HT40+LC50 treatment, followed by the LC50+HT40 treatment. The expression levels of CYP18A1 (CYP18 family) were not significantly different among treatments, and CYP301A1 (CYP301 family) was only sensitive to temperature (HT40). The expression of five sHsps showed similar expression patterns and were highly responsive to the LC50+HT40 treatment, followed by the HT40 and HT40+LC50 treatments. Based on CYP450s and Hsps expression levels, our findings that suggest that L. trifolii exhibits adaptive cross-tolerance to high temperature and abamectin. This study provides a framework for selecting the most effective application time for abamectin with respect to controlling L. trifolii, which will ultimately reduce the overuse of pesticides.

Comparison of morphology, development and expression patterns of hsf and hsp11.0 of Cotesia chilonis under normal and high temperature.

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.

Molecular Characterization of Heat-Induced HSP11.0 and Master-Regulator HSF from Cotesia chilonis and Their Consistent Response to Heat Stress.

Small heat shock proteins (sHSPs) are members of the heat shock protein (HSP) family that play an important role in temperature stress, and heat shock factors (HSFs) are transcriptional activators that regulate HSP expression. Cotesia chilonis, the major endoparasitoid of Chilo suppressalis, modulates the C. suppressalis population in the field. In this study, we cloned and characterized two genes from C.chilonis: the heat-induced HSP11.0 gene (Cchsp11.0) that consisted of a 306-bp ORF, and the master regulator HSF (Cchsf) containing an 1875-bp ORF. CcHSP11.0 contained a chaperonin cpn10 signature motif that is conserved in other hymenopteran insects. CcHSF is a typical HSF and contains a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal trans-activation domain. Neither Cchsp11.0 or Cchsf contain introns. Real-time quantitative PCR revealed that Cchsp11.0 and Cchsf were highly induced at 36 °C and 6 °C after a 2-h exposure. Overall, the induction of Cchsf was lower than Cchsp11.0 at low temperatures, whereas the opposite was true at high temperatures. In conclusion, both Cchsp11.0 and Cchsf are sensitive to high and low temperature stress, and the expression pattern of the two genes were positively correlated during temperature stress.

High temperature stress induces expression of CYP450 genes and contributes to insecticide tolerance in Liriomyza trifolii.

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.

Comparative transcriptome analysis of three invasive leafminer flies provides insights into interspecific competition.

Liriomyza spp. (Diptera: Agromyzidae) represent a group of economically-significant highly polyphagous pests of plants grown in field and greenhouse conditions. Liriomyza spp. share similar biological and morphological characteristics, and complex interspecific interactions have been documented among these species in various geographical regions. Where the displacement of one of these species by the other has been studied, no unique mechanisms have been identified as causing it. The impact of competitive factors (such as, insecticide tolerance, thermotolerance, and adaptability to cropping systems) may be unique to specific geographic regions of Liriomyza spp., but more research is needed to confirm these hypotheses. In this study, RNA-seq was used to determine the transcriptomes of three closely-related leafminers, e.g. L. sativae, L. trifolii, and L. huidobrensis. Over 20 Gb of clean reads were generated and assembled into unique transcriptomes, and 38,747 unigenes were annotated in different databases. In pairwise comparisons, L. trifolii and L. sativae had more up-regulated genes than L. huidobrensis. With respect to common differentially-expressed genes (Co-DEGs), the three leafminers exhibited distinct groups of highly-expressed gene clusters. When genes related to competitive factors were compared, expression patterns in L. trifolii and L. sativae were more closely related to each other than to L. huidobrensis. The data suggest that DEGs involved in competitive factors may play a key role in competition and displacement of leafminers. The divergent genes identified in this study will be valuable in revealing possible mechanisms of invasion, displacement and interspecific competition in Liriomyza spp.

Transcriptome analysis of Liriomyza trifolii (Diptera: Agromyzidae) in response to temperature stress.

The leafminer Liriomyza trifolii is an important insect pest of ornamental and vegetable crops worldwide. Temperature is a critical environmental factor that impacts both the distribution and interspecific competition of Liriomyza spp. In this study, we compared the transcriptomes of L. trifolii exposed to ambient (25 °C), hot (43 °C), and cold (-7 °C) temperatures. RNA-seq revealed 100,041 assembled unigenes, and 50,546 of these were annotated in L. trifolii transcriptome libraries. A total of 207 and 2904 differentially expressed genes (DEGs) were identified in response to hot and cold stress, respectively. Functional classification indicated that "cellular process", "single organism processes" and "metabolic processes" pathways were significantly enriched, along with "binding activity" and "catalytic activity". With respect to clusters of orthologous genes (COG) classification, DEGs were assigned to "post-translational modification, protein turnover, chaperones", "carbohydrate transport and metabolism" and "lipid transport and metabolism" categories. Subsequent annotation and enrichment analyses indicated that genes encoding heat shock proteins (HSPs) and cuticular proteins were significantly up-regulated during heat and cold stress, respectively. This study expands our knowledge of gene expression in L. trifolii during temperature stress and provides a basis for further studies aimed at understanding the mechanism of thermotolerance in this important invasive leafminer fly.

Characterization of an inducible HSP70 gene in Chilo suppressalis and expression in response to environmental and biological 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.

Selection of valid reference genes for quantitative real-time PCR in Cotesia chilonis (Hymenoptera: Braconidae) exposed to different temperatures.

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 ß-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.

Cloning of a new HSP70 gene from western flowerthrips, Frankliniella occidentalis, and expression patterns during thermal stress.

Frankliniella occidentalis (Pergande) is an invasive pest that endangers a wide variety of horticultural and agronomic crops. HSP70 is the most important member of the heat shock protein (HSP) family and plays an important role in insect thermal tolerance. In this study, a new gene encoding HSP70 from F. occidentalis, Fohsp706, was selected from the F. occidentalis transcriptome exposed to thermal stress (40 °C) and cloned by RT-PCR and RACE. Further characterization indicated that Fohsp706 localizes to the cytoplasm and does not contain introns. Quantitative real-time reverse transcriptase PCR indicated that Fohsp706 expression was significantly up-regulated by thermal stress; furthermore, there were significant differences in Fohsp706 expression in adults and second instar nymphs after heat stress. Our results indicated that Fohsp706 contributes to thermotolerance in F. occidentalis and provides another example of how this pest adapts to unfavorable environmental conditions.