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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.

Simple Summary Small heat shock proteins (sHSPs) are members of the heat shock protein (HSP) family that play an important role in heat stress, and heat shock factors (HSFs) are transcriptional activators that mainly regulate the expression of HSPs. Cotesia chilonis, the major endoparasitoid of Chilo suppressalis, widely distributes in China and other Asian regions. Previous studies have shown that C. chilonis has a certain thermal tolerance. Here, heat-induced HSP11.0 and master-regulator HSF were cloned and characterized from C. chilonis. The transcription patterns of them in response to different temperatures and time course after temperature treatment were analyzed. This study is the first report on the analysis on hsf gene of C. chilonis. The results of expression patterns will provide new insights into thermoregulation of C. chilonis in response to climate change. Abstract 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.

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.

Small heat shock proteins (sHSPs) comprise numerous proteins with diverse structure and function. As molecular chaperones, they play essential roles in various biological processes, especially under thermal stresses. In this study, we identified three sHSP-encoding genes, LtHSP19.5, LtHSP20.8 and LtHSP21.7b from Liriomyza trifolii, an important insect pest of ornamental and vegetable crops worldwide. Putative proteins encoded by these genes all contain a conserved &alpha, crystallin domain that is typical of the sHSP family. Their expression patterns during temperature stresses and at different insect development stages were studied by reverse-transcription quantitative PCR (RT-qPCR). In addition, the expression patterns were compared with those of LtHSP21.3 and LtHSP21.7, two previously published sHSPs. When pupae were exposed to temperatures ranging from -20 to 45 &deg, C for 1 h, all LtsHSPs were strongly induced by either heat or cold stresses, but the magnitude was lower under the low temperature range than high temperatures. Developmentally regulated differential expression was also detected, with pupae and prepupae featuring the highest expression of sHSPs. Results suggest that LtsHSPs play a role in the development of the invasive leaf miner fly and may facilitate insect adaptation to climate change.

Liriomyza chinensis is a serious pest of onions in many countries, especially in East Asia. We sequenced the complete mitochondrial genome of this species and compared it with five other Agromyzidae species. The L. chinensis mitogenome is a double-stranded 16,175 bp circular molecule with an A + T content of 78.3%. It contains 37 genes and a control region as do the sequenced Liriomyza species. The mitogenomes of L. chinensis and other Agromyzidae species showed a clear bias in nucleotide composition with a positive AT-skew. Most PCGs used standard ATN as start codons, and TAN as termination codons. The tRNAs exhibited the typical clover-leaf structure, except for tRNASer(AGN) and the two rRNA genes are conserved with those of other Agromyzids. The L. chinensis mitogenome control region included several conserved regions, including a poly-T, two (TA)n and one poly-A stretch, which are considered important replication and transcription. The 13 PCGs were used to study the phylogeny of L. chinensis and five related Agromyzids. Analysis by maximum likelihood, Bayesian inference and genetic distance suggest congruent phylogenetic relationships in Liriomyza spp. and provide a useful supplement to taxonomic classification by morphology.

Heat shock protein 70 (HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length cDNAs of HSP70s gene (Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of cDNA. Real-time PCR was used to analyze the HSP70 expression patterns. The cDNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids (aa) with a calculated molecular weight of 75 and 54 kDa, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported FoHSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.

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.

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.

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.

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.

Aquaporins (AQPs), which are members of the major intrinsic protein (MIP) family, play an important role in the transport of water and other small, uncharged solutes across membranes. In this study, we identified gene encoding two aquaporin 12-like (AQP12L) proteins, CsAqp12L_v1 and CsAqp12L_v2, from Chilo suppressalis, a serious rice pest in Asia. Phylogenetic analysis indicated that CsAQP12L_V1 and CsAQP12L_V2 were grouped in a well-supported cluster that included other members of Lepidoptera. The two proteins are almost identical, except that CsAQP12L_V1 lacks 34 amino acids that are present in CsAQP12L_V2 at site 217. The qRT-PCR indicated that both CsAqp12L and CsAqp12L_v2 were expressed in heads, epidermis, foregut, midgut, and hindguts, with the highest level of expression in hindguts, heads, and epidermis. Expression of CsAqp12L and CsAqp12L_v2 was detected in all life stages and both sexes and was highest in first instar larvae and lowest in eggs. Expression of CsAqp12L and CsAqp12L_v2 was not significantly altered by exposure to brief changes in temperature. There were no significant differences in the third instar larvae, male and female pupae, and female adults in response to adverse humidity. However, the mRNA level of CsAqp12L in the fifth instar larvae and CsAqp12L_v2 in male adults was induced significantly by low humidity, respectively. Moreover, Xenopus oocytes injected with cRNAs of CsAQP12L_V1 and CsAQP12L_V2 showed no significant changes in permeability to water, glycerol, trehalose, or urea. The two CsAQP12L variants likely localize to an intracellular location in C. suppressalis and may respond to novel stimuli.

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.

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.

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.

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 polyphagous agromyzid fly, Liriomyza trifolii, is a significant and important insect pest of ornamental and vegetable crops worldwide. The adaptation of insects to different environments is facilitated by heat shock proteins (HSPs), which play an important role in acclimation to thermal stress. In this study, we cloned and characterized five HSP-encoding genes of L. trifolii (Lthsp20, Lthsp40, Lthsp60, Lthsp70, and Lthsp90) and monitored their expression under different thermal stresses using real-time quantitative PCR. Pupae of L. trifolii were exposed to 19 different temperatures ranging from -20 to 45°C. The results revealed that Lthsp20, Lthsp40, Lthsp70 and Lthsp90 were significantly upregulated in response to both heat and cold stress, while Lthsp60 was induced only by heat temperatures. The temperatures of the onset (Ton) and maximal (Tmax) expression of the five Lthsps were also determined and compared with published Ton and Tmax values of homologous genes in L. sativae and L. huidobrensis. Although L. trifolii occurs primarily in southern China, it has cold tolerance comparable with the other two Liriomyza species. Based on the heat shock proteins expression patterns, L. trifolii has the capacity to tolerate extreme temperatures and the potential to disseminate to northern regions of China.

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.