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Small heat shock proteins (sHSPs) help prevent the irreversible aggregation of denatured proteins that occurs in response to organismal stress. In this study, we identified two intron-free genes encoding sHSPs from Frankliniella occidentalis; these were designated FoHSP11.6 and FoHSP28.0 and belonged to an atypical and typical sHSP family, respectively. Both FoHSPs were transcribed in all developmental stages of F. occidentalis with the highest expression levels in pupae and adults and greater expression in males than females. Although the FoHSPs had different temperature-induced expression profiles, they were generally induced by both low and high temperatures and reached maximal expression levels after 0.5-1 h of temperature stress. The FoHSPs expression levels in pupae were induced by drought and high humidity, and higher expression levels were correlated with lower survival rates. The thermotolerance of F. occidentalis decreased when theFoHSPs were silenced by RNA interference. Our results show that FoHSP11.6 and FoHSP28.0 are involved in the response to temperature and drought and may also function in growth and development of F. occidentalis.

The leafminer Liriomyza trifolii is an important insect pest of ornamental and vegetable crops worldwide. Cyromazine is an effective, commonly-used insecticide that functions as a growth regulator, but its effect on L. trifolii has not been previously reported. In this study, transcriptome analysis was undertaken in L. trifolii exposed to cyromazine. Clusters of orthologous groups analysis indicated that a large number of differentially expressed genes responding to cyromazine were categorized as "lipid transport and metabolism", "post-translational modification, protein turnover, chaperones", and "cell wall/membrane/envelope biogenesis". Gene ontology analysis indicated that pathways associated with insect hormones, growth and development, and cuticle synthesis were significantly enriched. In general, the transcriptome results showed that the genes related to insect hormones were significantly expressed after treatment with cyromazine. Furthermore, the combined exposure of L. trifolii to cyromazine and the hormone analogues 20-hydroxyecdysone (20E) or juvenile hormone (JH) indicated that hormone analogues can change the expression pattern of hormone-related genes (20EP and JHEH) and pupal length. The combined application of cyromazine with 20E improved the survival rate of L. trifolii, whereas the combination of JH and cyromazine reduced survival. The results of this study help elucidate the mechanistic basis for cyromazine toxicity and provide a foundation for understanding cyromazine resistance.

The primary function of heat shock transcription factor (HSF) in the heat shock response is to activate the transcription of genes encoding heat shock proteins (HSPs). The phloem-feeding insect Bemisia tabaci (Gennadius) is an important pest of cotton, vegetables and ornamentals that transmits several plant viruses and causes enormous agricultural losses. In this study, the gene encoding HSF (Bthsf1) was characterized in MED B. tabaci. The full-length cDNA encoded a protein of 652 amino acids with an isoelectric point of 5.55. The BtHSF1 deduced amino acid sequence showed strong similarity to HSF in other insects. Expression analyses using quantitative real-time PCR indicated that Bthsf1 was significantly up-regulated in B. tabaci adults and pupae during thermal stress. Although Bthsf1 was induced by both hot and cold stress, the amplitude of expression was greater in the former. Bthsf1 had distinct, significant differences in expression pattern during different duration of high but not low temperature stress. Oral ingestion of dsBthsf1 repressed the expression of Bthsf1 and four heat shock proteins (Bthsp90, Bthsp70-3, Bthsp20 and Bthsp19.5) in MED B. tabaci during hot and cold stress. In conclusion, our results show that Bthsf1 is differentially expressed during high and low temperature stress and regulates the transcription of multiple hsps in MED B. tabaci.

The primary function of heat shock transcription factor (HSF) in the heat shock response is to activate the transcription of genes encoding heat shock proteins (HSPs). The phloem-feeding insect Bemisia tabaci (Gennadius) is an important pest of cotton, vegetables and ornamentals that transmits several plant viruses and causes enormous agricultural losses. In this study, the gene encoding HSF (Bthsf1) was characterized in MED B. tabaci. The full-length cDNA encoded a protein of 652 amino acids with an isoelectric point of 5.55. The BtHSF1 deduced amino acid sequence showed strong similarity to HSF in other insects. Expression analyses using quantitative real-time PCR indicated that Bthsf1 was significantly up-regulated in B. tabaci adults and pupae during thermal stress. Although Bthsf1 was induced by both hot and cold stress, the amplitude of expression was greater in the former. Bthsf1 had distinct, significant differences in expression pattern during different duration of high but not low temperature stress. Oral ingestion of dsBthsf1 repressed the expression of Bthsf1 and four heat shock proteins (Bthsp90, Bthsp70-3, Bthsp20 and Bthsp19.5) in MED B. tabaci during hot and cold stress. In conclusion, our results show that Bthsf1 is differentially expressed during high and low temperature stress and regulates the transcription of multiple hsps in MED B. tabaci.

The heat shock protein 70 family (HSP70) is among the most varied HSP family with respect to structure and function. The phloem-feeding insect Bemisia tabaci (Gennadius) is an important pest of cotton, vegetables and ornamentals that transmits several plant viruses and causes enormous agricultural losses. In this study, two new HSP70 genes (Bthsp70-2 and Bthsp70-3) were isolated from the MED cryptic species B. tabaci, an important phloem-feeding pest of vegetables and ornamentals. Bthsp70-2 and Bthsp70-3 encoded proteins comprised of 652 and 676 amino acids, and the deduced proteins were closely related to other HSP70s in Hemiptera. Expression analyses using real-time quantitative PCR indicated that Bthsp70-2 and Bthsp70-3 were induced in B. tabaci pupae and adults during high and low thermal stress. Bthsp70-2 and Bthsp70-3 exhibited similar, but not identical, expression patterns when exposed to different durations of high temperature stress. Oral ingestion of dsBthsp70 reduced the expression level of Bthsp70-2 and Bthsp70-3 in B. tabaci and increased the mortality of B. tabaci during heat shock. In conclusion, Bthsp70-2 and Bthsp70-3 exhibit different expression patterns during thermal stress, thus expanding the roles of HSPs in B. tabaci.

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 ℃ 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.

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.

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.