Knockdown of the ABCG23 Gene Disrupts the Development and Lipid Accumulation of Panonychus citri (Acari/Tetranychidae).

Panonychus citri is a worldwide citrus pest that is currently controlled through the use of insecticides. However, alternative strategies are required to manage P. citri. Recent studies suggest that the ATP-binding cassette (ABC) transporter G subfamily plays a crucial role in transporting cuticular lipids, which are essential for the insect's barrier function against microbial penetration. Therefore, investigating the potential of the ABC transporter G subfamily as a control measure for P. citri could be a promising approach. Based on the genome database, the gene was cloned, and the transcriptional response of ABCG23 for the different developmental stages of P. citri and under spirobudiclofen stress was investigated. Our results showed that the expression level of ABCG23 was significantly lower in adult females exposed to treatment compared to the control and was higher in females than males. The knockdown of ABCG23 using RNAi led to a decrease in the survival rate, fecundity, and TG contents of P. citri. Additionally, a lethal phenotype was characterized by body wrinkling and darkening. These results indicate that ABCG23 may be involved in cuticular lipid transportation and have adverse effects on the development and reproduction of P. citri, providing insight into the discovery of new targets for pest management based on the insect cuticle's penetration barrier function.

Fatty Acid Elongase Gene PcELO7 is Essential for Lipid Accumulation and Fecundity of Panonychus citri (Acari: Tetranychidae).

RNA interference (RNAi) has been proposed as a promising strategy for sustainable and ecofriendly pest control. The insect cuticle lipids were deposited on the body surface and functioned as a defense against chemical xenobiotics. They consisted of aliphatic compounds, including free fatty acids (FFAs). However, elongase of very long chain fatty acids (ELOs) is essential for FFA biosynthesis; the function of ELO is still unknown in many arthropods, including Panonychus citri (P. citri). In this study, three ELOs were cloned. Developmental-specific mRNA expression results revealed that three PcELOs were highly expressed in egg and adult females. Whereas PcELO7 was dominantly expressed in adult females. Under spirobudiclofen stress, ELOs mRNA expression had different changes, and PcELO7 was down-regulated. The silencing of PcELO7 resulted in a dramatic reduction of oviposition and hatchability. Significant reduction of FFA contents was also examined within PcELO7-repressed P. citri. In addition, we found that PcELO7 mRNA levels were related to fecundity and could affect triacylglycerol (TG) contents. The findings demonstrated that the introduction of dsPcELO7 via oral feeding induced the RNA interference-mediated silencing of a special target gene and could result in mortality and reproduction. In conclusion, PcELO7 is a special RNAi target for P. citri control, and its lethal mechanism might be disturbing lipids biosynthesis.

Sublethal Effects of Emamectin Benzoate on Development and Reproduction and RNAi of the Vitellogenin Gene in Spodoptera frugiperda.

Spodoptera frugiperda, one of the most destructive corn pests in the world, invaded China in December 2018. In this study, sublethal concentrations (LC10 and LC30) of emamectin benzoate (EB) were used to treat pesticide-free treatment (PFT) and EB treatment (ET) of S. frugiperda. In PFT, compared with the control (CK), the pupal weight, hatching rate, and pupation rate of LC10 and LC30 groups were significantly reduced. The fecundity and the expression of vitellogenin gene (SfVg) were decreased after LC30 treatment, while the LC10 treatment groups showed no significant difference from the control group. In ET, compared to CK, the fecundity was increased by 11.14 and 18.8%. The expression of SfVg was upregulated by 2.6 times after LC30 treatment. Moreover, RNAi-mediated SfVg knockdown resulted in a nearly 70% reduction in oviposition. The result provided a theoretical basis for optimizing the application of EB and Vg-dsRNA in the control of S. frugiperda.

Regulation of three subtypes of SOD gene in Aleuroglyphus ovatus (Acari:Acaridae) under lead stress.

Superoxide dismutase (SOD) is an important enzyme that acts as the first line of protection in the mite antioxidant defense system, involved in eliminating reactive oxygen species (ROS) under harsh environmental conditions. Nevertheless, the SOD gene family was yet to be reported in stored grain pest mite (Aleuroglyphus ovatus). In this study, A. ovatus was used to evaluate the response of SOD gene during lead stress. A. ovatus were separately exposed to different concentration lead (12.5, 25, 50, and 100 mg/kg), which induce the dynamic trend of SOD enzyme activity initially increased and then reduced with an increase in lead concentration, whereas they were still substantially higher than the control group. Moreover, after lead stress, it was found that all of the three SOD genes showed enhanced relative messenger RNA expression at high concentrations and decreased relative expression at low concentrations, which indicated that lead stress induces the expression of AoSODs. The present work implies that AoSODs play an important role in resisting oxidative damage caused by lead stress.

Chronic lead exposure prolongs the immature stages of brown-legged grain mite, Aleuroglyphus ovatus, in a long-term population study.

An important aspect of environmental pollution, lead contamination is a widespread problem in several ecosystems. The present study aimed to evaluate the potential effects of low concentration lead stress on the development and reproduction of Aleuroglyphus ovatus. They were fed with artificial diet containing four different concentrations of lead (12.5, 25, 50, and 100 mg/kg). The results showed that there were both accelerating effect of lead (at low concentrations), as well as retarding effects (at high concentrations) on the development of the mite, and lead stress significantly prolonged the immature stages of A. ovatus and this inhibitory effect was greater with greater lead concentrations. The immature stages in the L group were shorter than those in the S group. In the S and L groups, the oviposition periods were significantly longer in the treatments with lower lead concentrations than in the control, while they were significantly shorter in those treatments of higher lead concentrations. Age-specific survival rate (lx) started to decline earlier in the S group, whereas there were no differences between the L group and CK. Age-specific fecundity rate (mx) peaked earlier in the S group than in CK, while mx peaked later in L1 and L2 than in CK. The rm value and net reproduction rate (R0) of treated A. ovatus decreased with increasing lead concentrations. Lower lead concentrations could promote population expansion while higher concentrations could inhibit population size. These results confirmed the developmental effect of lead stress on A. ovatus, highlighting that heavy metal contamination has negative effects on organisms in their natural environment.

Stress response and tolerance mechanisms of spirobudiclofen exposure based on multiomics in Panonychus citri (Acari: Tetranychidae).

The toxicity of insecticides used in the field decreases gradually to sublethal concentrations over time. Therefore, it is necessary to study sublethal effects of pesticides for controlling population explosion. Panonychus citri is a global pest which control is based on insecticides. This study explores the stress responses of spirobudiclofen on the P. citri. Spirobudiclofen significantly inhibited survival and reproduction of P. citri, and the effects aggravated as concentration increased. The transcriptomes and metabolomes of spirobudiclofen-treated and control were compared to characterize spirobudiclofen molecular mechanism. Transcriptomics indicated stress induced by spirobudiclofen stimulated immune defense, antioxidative system, cuticle formation, and lipid metabolism, as deduced from RNA-seq analysis. Meanwhile, our study found that tolerance metabolism in P. citri was regulated by promoting the metabolism of glycerophospholipids, glycine, serine, and threonine. The results of this study can provide a basis for exploring the adaptation strategies of P. citri to spirobudiclofen stress.

The metabolism and detoxification effects of lead exposure on Aleurolyphus ovatus (Acari: Acaridae) via transcriptome analysis.

Aleurolyphus ovatus Troupeau is one of the most predominant species of the Acaridae family worldwide. Recent reports have demonstrated that the accumulation of lead in stored grains and dietary items exceeds the required standards. However, the molecular mechanism of heavy metal stress on mites has not been reported. To understand the mechanism underlying the heavy metal response of A. ovatus, comparative transcriptome analysis was performed in this study using an Illumina high throughput mRNA sequencing (RNA-seq) platform. A. ovatus was fed on artificial diets containing two different concentrations of lead, namely, a low concentration of 12.5 mg/kg (LAO) and a high concentration of 100 mg/kg (HAO), while the mites in the control (NAO) group were not exposed to lead. A total of 44,362 unigenes, with an average length of 1547 bp, were identified. Of these, 996 unigenes were successfully annotated in seven functional databases. The number of differentially expressed genes (DEGs) in A. ovatus under different lead concentrations was compared. In NAO versus LAO group, including 310 up-regulated and 1580 down-regulated DEGs. In NAO versus HAO group, including 3928 up-regulated and 1761 down-regulated DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that detoxification enzyme genes were significantly expressed in pathways, such as cytochrome P450 foreign body metabolism, glutathione metabolism and drug metabolism-cytochrome pathway. The results of gene annotation and quantitative real-time PCR showed that high concentration of lead significantly stimulated the expression of metabolic detoxification enzyme genes such as glutathione S transferase (GST) and superoxide dismutase (SOD), while low concentration inhibited their expression. This study will provide a basis for the molecular mechanism of A. ovatus in response to heavy metal lead stimulation in stored grain.

The loss of Halloween gene function seriously affects the development and reproduction of Diaphorina citri (Hemiptera: Liviidae) and increases its susceptibility to pesticides.

The citrus industry has suffered severe losses as a result of Huanglongbing spread by Diaphorina citri. Controlling the population of D. citri is the key to preventing and controlling the spread of Huanglongbing. Ecdysteroids are key hormones that regulate insect development and reproduction. Therefore, the Halloween gene family involved in the ecdysone synthesis of D. citri is an ideal target for controlling the population growth of this insect. In this study, we successfully cloned four Halloween genes expressed during D. citri development. Silencing of one of the four genes resulted in a significant decrease in 20E titers in nymphs and significant decreases in the developmental, survival and emergence rates. Inhibiting Halloween gene expression in adults impeded the growth of the female ovary, diminished yolk formation, lowered vitellogenin transcription levels, and hence impaired female fecundity. This showed that Halloween genes were required for D. citri development and reproduction. DcCYP315A1 and DcCYP314A1 were highly expressed when D. citri was exposed to thiamethoxam and cypermethrin, and silencing these two genes made D. citri more sensitive to these two pesticides. Inhibition of DcCYP315A1 and DcCYP314A1 expression not only significantly delayed the development and reproduction of D. citri but also increased its susceptibility to pesticides. Therefore, these two genes are more suitable as potential target genes for controlling D. citri.

Age-Stage, Two-Sex Life Table and Functional Response of Amblyseius orientalis (Acari: Phytoseiidae) Feeding on Different Nutrient Sources.

Amblyseius orientalis Ehara is a predatory mite that belongs to the family Phytoseiidae. It is mainly found in Jiangxi, Shanghai, Guangdong, and other areas of China. Although A. orientalis is a dominant predatory mite species in China and is also important for agriculture and biological control, not many studies have investigated it. Thus, research on A. orientalis is necessary. However, its application in biological control is hindered by the absence of techniques for the mass rearing of A. orientalis in captivity. We conducted experiments to determine the growth, development, reproduction, and functional response of A. orientalis in this study by indoor single-head rearing at 25 ± 1 °C, 65 ± 5% relative humidity, and a photoperiod of a 16 h:8 h light/dark cycle under laboratory conditions. The results of the age stage, two-sex life table showed that the individuals in the pollen + yeast and pollen + yeast + sucrose groups had significantly higher oviposition period, fecundity, net reproductive rate (R0), and gross reproduction rate (GRR) than those in the pollen group. The results of the function response showed that the pollen + yeast + sucrose group was the most favorable for captive breeding of A. orientalis and had the best predatory ability along with rejuvenation and recovery ability. The results of the study provided a theoretical basis for indoor rearing, propagation, and utilization of A. orientalis.

Characterization of Chitin Synthase A cDNA from Diaphorina citri (Hemiptera: Liviidae) and Its Response to Diflubenzuron.

Diaphorina citri Kuwayama is the vector of HLB and one of the most common pests in citrus orchards in southern China. One of the most significant genes in D. citri's growth and development is the chitin synthase gene. In this study, the CHS gene (DcCHSA) of D. citri was cloned and analyzed by bioinformatics. According to RT-qPCR findings, DcCHSA was expressed at many growth processes of D. citri, with the greatest influence in the fifth-instar nymph. The molting failure rate and mortality of D. citri rose as DFB concentration increased in this research, as did the expression level of DcCHSA. Feeding on DcCHSA caused a large drop in target gene expression, affected nymph molting, caused failure or even death in freshly eclosion adults, increased mortality, and reduced the molting success rate over time. These findings showed that DcCHSA was involved in nymph to adult development and may aid in the identification of molecular targets for D. citri regulation. It provided new ideas for further control of D. citri.

Knockdown of hexokinase in Diaphorina citri Kuwayama (Hemiptera: Liviidae) by RNAi inhibits chitin synthesis and leads to abnormal phenotypes.

BACKGROUND: Silencing specific genes in pests using RNA interference (RNAi) technology is a promising new pest-control strategy. The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important citrus pest because it transmits Candidatus Liberibacter asiaticus, which causes huanglongbing. Chitin is essential for insect development, and enzymes in this pathway are attractive targets for pest control. RESULTS: The hexokinase gene DcHK was characterized from D. citri to impair proper growth and chitin synthesis through RNAi. The transcription of DcHK was more highly developed in third-instar nymphs, adults and the Malpighian tube. The RNAi needed for D. citri is dose-dependent, with 600 ng µl-1 dsDcHK sufficient to knockdown endogenous DcHK expression. The messenger RNA (mRNA) level was lowest at 36 h after dosing, and there were significant effects on the relative levels of mRNA in the chitin synthesis pathway (DcTre, DcG6PI, DcGNAT, DcGFAT, DcPGM, DcUAP and DcCHS), leading to mortality, reduced body weight and abnormal or lethal phenotypes. CONCLUSION: RNAi can be triggered by orally delivered double-stranded RNA in D. citri. These results can provide support for HK genes as a new potential target for citrus psyllid control. © 2022 Society of Chemical Industry.

Complete mitochondrial genome of Idea leuconoe (Lepidoptera: Danaidae) and related phylogenetic analyses.

In the present study, we first sequenced and determined the complete mitochondrial genome (mitogenome) of the giant Danaidae butterfly, Idea leuconoe (Lepidoptera: Danaidae). The mitogenome was a typical closed, circular, double-stranded DNA molecule of 15,278 bp length (GenBank accession number: KR815449), similar to the metazoan mitogenomes containing 37 genes and one A + T-rich region. All the protein-coding genes (PCGs) were initiated with a typical ATN codon. Seven genes (COII, ATP6, COIII, nad4, nad4L, cytb, and nad1) adopted the standard ATG start codon, but the remaining six genes were initiated with ATA. All the 13 PCGs harbored complete termination codons (TAA). The overlap nucleotides ATGATAA were conserved for the ATP8/ATP6 gene. The largest intergenic spacer was located between trnGln and nad2, a common finding in Lepidoptera butterflies. All the transfer RNA genes in the I. leuconoe mitogenome could be folded into typical clover-leaf secondary structures, except for trnSer (AGN) that lacked a dihydrouridine arm. The control region with 94.8% A + T content was 444 bp in length and located between rrnS and trnMet. Finally, the phylogenetic relationships obtained using the maximum likelihood and Bayesian methods confirmed two well-supported phylogenetic trees of Danaidae, Papilionidae, and Nymphalidae from the order Lepidoptera, which were consistent with the traditional morphological classification. Results provided additional information for butterfly phylogenetic analysis and insights into the evolution of genomes.

The Primary Complete Mitochondrial Genome of the Lappet Moth Brahmophthalma hearseyi (Lepidoptera: Brahmaeidae) and Related Phylogenetic Analysis.

Background: Brahmophthalma hearseyi (Lepidoptera: Brahmaeidae) is widely distributed across China. Its larvae damage the leaves of many plants such as those belonging to the Oleaceae family, causing significant economic losses and seriously affecting the survival and reproduction of Cervus nippon; however, genetic data for this species are scarce. Methods: The complete mitochondrial genome (mitogenome) of B. hearseyi was sequenced using long-PCR and primer-walking methods. Phylogenetic analysis that was based on 13 PCGs and two rRNAs was carried out using the neighbor-joining and Bayesian interference methods. Results: The mitogenome is a typical circular molecule that is made up of 15,442 bp, which includes 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and an A + T-rich region (456 bp). All of the PCGs, except for COX1 and COX2, start with ATN codons. COX2 and ND5 use the incomplete termination codon T, and 11 other PCGs use the typical stop codon TAA. All tRNA genes, except for trnS1 and trnS2, display a typical cloverleaf structure; trnS1 lacks the "DHU" arm, whereas trnS2 exhibits two mismatched base pairs in the anticodon stem. Phylogenetic analysis showed that B. hearseyi is clustered into Brahmaeidae, and the phylogenetic relationships are (Brahmaeidae + Lasiocampidae) + (Bombycidae + (Sphingidae + Saturniidae)). Conclusions: This study provides the first mitogenomic resources for the Brahmaeidae.

Sublethal effects of bifenazate on biological traits and enzymatic properties in the Panonychus citri (Acari: Tetranychidae).

Panonychus citri, a major citrus pest. In pest management, bifenazate is a novel acaricide with high biological activity against red mites, such as Tetranychus urticae Koch. However, in the field, pests are frequently exposed to sublethal or lethal concentrations of pesticides. At present, its sublethal effects on P. citri have not been reported. Therefore, in order to investigate sublethal effect of bifenazate on biological traits and enzymatic properties of P. citri. The newly emerged females were treated with two concentrations of bifenazate: LC10 and LC30, the development and fecundity were observed. The results showed that female adult duration, fecundity, oviposition days, longevity were decrease compared with control, but pre-oviposition period was longer, net reproductive rate (R0), mean generation (T) were decreased, intrinsic rate of increase (rm), finite rate (λ) were decreased in LC30, however, doubling time was increased. Enzymatic tests showed that CAT, POD, CarE activities were higher in treatments than control. The SOD and GST activities were lower in LC30 than control and LC10, the CYP450 activity was decreased with the increasing concentrations. This study demonstrated that low lethal concentrations of bifenazate adversely affected life table parameters, enzymatic properties in P. citri. Therefore, bifenazate has the potential to control this pest.

Characteristic and expression of Hsp70 and Hsp90 genes from Tyrophagus putrescentiae and their response to thermal stress.

The development of insects is critically affected by temperature, which therefore plays an important role in the control of stored grain pests. Extreme temperature stress conditions lead to biological responses in mites, such as the synthesis of heat shock proteins. Tyrophagus putrescentiae (Tp) is a pest mite in stored grain that has negative effects on both economy and health. Since T. putrescentiae population dynamics are strongly influenced by temperature, in the present study we have cloned the cDNA of HSP70 and HSP90 (referred to as TpHSP70-1, TpHSP70-2 and TpHSP90) and determined their expression by fluorescence real time quantitative PCR. TpHSP70 and TpHSP90 showed high homology with similar genes in other species and the open reading frames of TpHSP70-1, TpHSP70-2 and TpHSP90 encoded proteins of 665, 661 and 718 amino acid residues, respectively. Under thermal stress, expression of TpHsp70-1 and TpHsp90 was up-regulated at higher temperatures, suggesting their role in the defense against thermal stress.

Molecular Characterization of Chitin Synthase Gene in Tetranychus cinnabarinus (Boisduval) and Its Response to Sublethal Concentrations of an Insecticide.

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is one of the most important acarine pest species. At present, its control remains primarily dependent on using various chemical insecticides/acaricides in agricultural crops worldwide. To clarify the mechanism whereby T. cinnabarinus responds to insecticide exposure, we identified the chitin synthase 1 gene (TcCHS1) and then explored the gene expression levels of TcCHS1 at different developmental stages of T. cinnabarinus. We also investigated the effects of sublethal concentrations of diflubenzuron on the toxicities and survivals of T. cinnabarinus eggs and larvae as well as TcCHS1 expression levels. The full-length cDNA sequence contains an open reading frame (ORF) of 4881 nucleotides that encoded for a 1474 amino acid residues protein. The predicted TcCHS1 protein had a molecular mass of 168.35 kDa and an isoelectric point of 6.26, and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The results of phylogenetic analyses demonstrated that the putative CHS1 amino acid sequence of T. cinnabarinus revealed high similarities with chitin synthases in other insects and mites. Additionally, at the molecular level, transcriptional analysis by real-time quantitative PCR in different developmental stages of T. cinnabarinus revealed that TcCHS1 mRNA was expressed in all stages, and highest in eggs and female adults, but lowest in deutonymphs. Furthermore, the results of toxicity bioassays indicated that diflubenzuron treatment resulted in high mortality rates in eggs and larvae of T. cinnabarinus. The mRNA expression levels of TcCHS1 from the eggs and larvae of T. cinnabarinus were up-regulated in response to sublethal concentrations of diflubenzuron exposures. Together, all these results demonstrate that diflubenzuron has ovicidal and larvicidal effects and TcCHS1 may play an important role in the growth and development of T. cinnabarinus and may disrupt the chitin biosynthesis, thereby controlling T. cinnabarinus populations.

Knockdown of the Trehalose-6-Phosphate Synthase Gene Using RNA Interference Inhibits Synthesis of Trehalose and Increases Lethality Rate in Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae).

Diaphorina citri Kuwayama is the vector of citrus "huanglongbing", a citrus disease which poses a significant threat to the global citrus industry. Trehalose-6-phosphate synthase (TPS) plays an important role in the regulation of trehalose levels of insects, while its functions in D. citri are unclear. In this study, full-length cDNA sequences of the TPS gene from D. citri (DcTPS) were cloned and its expression patterns at various developmental stages were investigated. The results indicated that DcTPS mRNA was expressed at each developmental stage and the highest DcTPS expression was found in the fifth-instar nymphs of D. citri. Additionally, mortality and deformity of D. citri were observed after 24 and 48 h by feeding with three different dsRNA concentrations (20, 100 and 500 ng/µL). The results indicated that DcTPS expression was declined, and mortality and malformation in nymphs were increased via feeding with dsDcTPS. Moreover, the enzyme and trehalose content were decreased, while the content of glucose was significantly higher than that of untreated (control) individuals. This suggests that DcTPS might be vital for the growth and development of D. citri and further studies of the genes should be related to molting and metabolism for controlling D. citri.

Diaphorina citri (Hemiptera: Psylloidea) in China: Two Invasion Routes and Three Transmission Paths.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is one of the most common pests impacting citrus orchards in southern China. Samples of D. citri were collected in southern China in order to systematically explore the genetic architecture of the species. Mitochondrial cytochrome b (Cytb) and cytochrome coxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) which allowed highlighting low haplotype and nucleotide diversities among the population. Two clades could be observed in the haplotype network. Moreover, Bayesian and maximum parsimony phylogenetic trees were constructed based on the sequences of Cytb and COI. Here, we report on the significant genetic variation of the species when comparing southwestern China with other regions of southern China (southern and southeastern). This analysis also suggested that the genetic structure of D. citri in China originates may from long-term climate fluctuations concomitant with recent disturbances resulting from human activity. Combined with previous data, the present work indicates that D. citri potentially entered China through two distinct invasion routes and spread within the country via three transmission paths.

Effect of temperature on development and reproduction of the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychiae), fed on cassava leaves.

The effect of five constant temperatures (16, 20, 24, 28 and 32 °C) on the development, survival and reproduction of Tetranychus cinnabarinus (Boisduval) [= Tetranychus urticae Koch (red form)] fed on cassava leaves was examined in the laboratory at 85% relative humidity. Development time of various immature stages decreased with increasing temperature, with total egg-to-adult development time varying from 27.7 to 6.7 days. The lower thermal threshold for development was 10.8 °C and the thermal constant from egg to adult was 142.4 degree-days. Pre- and post-oviposition period and female longevity all decreased as temperature increased. The longest oviposition period was observed at 20 °C with 20.4 days. Under different temperatures, mated females laid, on average, 1.0, 2.9, 4.7, 4.7 and 4.9 eggs per day, respectively. The maximum fecundity (81.5 eggs per female) was at 28 °C and the intrinsic rate of increase (r m ) was highest (0.25) at 32 °C. The results of this study indicate that T. cinnabarinus population could increase rapidly when cassava leaves serve as a food source. At the appropriate temperature T. cinnabarinus could seriously threaten growth of cassava.

Differential expression patterns of two delta-9-acyl-CoA desaturases in Thitarodes pui (Lepidoptera: Hepialidae) during different seasons and cold exposure.

Thitarodes pui larvae have a limited distribution in the Tibetan Plateau and are the host of a parasitic fungus, Ophiocordyceps sinensis. Low temperature is a main environmental stress. However, understanding of T. pui cold adaptation mechanisms is insufficient. Delta-9-acyl-CoA desaturase (D9D) is closely correlated with cold adaptation for many organisms. To further understand the cold adaptation processes in T. pui larvae, two D9Ds, TpdesatA and TpdesatB were sequenced, and expression patterns were investigated during different seasons and cold exposure (under 0°C) in the laboratory. The full lengths of two cDNAs are 1,290 bp and 1,603 bp, and the ORFs encode a polypeptide of 348 and 359 amino acids, respectively. Four transmembrane domains, three conserved histidine residues and five hydrophobic regions exist in these two sequences. The expression level of TpdesatA is up-regulated in the long-term cold exposure and negatively correlated with temperature in seasonal patterns. TpdesatB responds to cold temperature in short-term cold exposure and positively corresponds temporarily in seasonal expression. Two D9Ds may have different substrate specificities, TpdesatA tends to use C16:0 and C18:0 as substrate while TpdesatB prefers C18:0. In conclusion, TpdesatA may play a very important role in T. pui cold tolerance and TpdesatB regulates function in short-term cold exposure and content change of fatty acids in the body.