Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Comparative Analysis of Transcriptome Responses to Cold Stress in Galeruca daurica (Coleoptera: Chrysomelidae).

Galeruca daurica (Joannis) has become a new insect pest in the Inner Mongolia grasslands since 2009, and its larvae and eggs have strong cold tolerance. To get a deeper insight into its molecular mechanisms of cold stress responses, we performed de novo transcriptome assembly for G. daurica by RNA-Seq and compared the transcriptomes of its larvae exposed to five different temperature treatments (-10, -5, 0, 5, and 25°C for 1 h and then recovered at 25°C for 1 h), respectively. Compared with the control (25°C), the numbers of differentially expressed genes (DEGs) decreased from 1,821 to 882, with the temperature declining from 5 to -10°C. Moreover, we obtained 323 coregulated DEGs under different low temperatures. Under four low temperatures (-10, -5, 0, and 5°C), a large number of genes were commonly upregulated during recovery from cold stresses, including those related to cuticle protein, followed by cytochrome P450, clock protein, fatty acid synthase, and fatty acyl-CoA reductase; meanwhile, lots of genes encoding cuticle protein, RNA replication protein, RNA-directed DNA polymerase, and glucose dehydrogenase were commonly downregulated. Our findings provide important clues for further investigations of key genes and molecular mechanisms involved in the adaptation of G. daurica to harsh environments.

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis.

Galeruca daurica (Joannis) is a new outbreak pest in the Inner Mongolia grasslands in northern China. Heat shock protein 10 and 60 (Hsp10 and Hsp60) genes of G. daurica, designated as GdHsp10 and GdHsp60, were cloned by rapid amplification of cDNA ends techniques. Sequence analysis showed that GdHsp10 and GdHsp60 encoded polypeptides of 104 and 573 amino acids, respectively. Sequence alignment and phylogenetic analysis clearly revealed that the amino acids of GdHsp10 and GdHsp60 had high homology and were clustered with other Hsp10 and Hsp60 genes in insects which are highly relative with G. daurica based on morphologic taxonomy. The mRNA expression analysis by real-time PCR revealed that GdHsp10 and GdHsp60 were expressed at all development stages and in all tissues examined, but expressed highest in eggs and in adults' abdomen; both heat and cold stresses could induce mRNA expression of GdHsp10 and GdHsp60 in the 2nd instar larvae; the two Hsp genes were expressed from high to low with the extension of treatment time in G. daurica eggs exposed to freezing point. Overall, our study provides useful information to understand temperature stress responses of Hsp60 and Hsp10 in G. daurica, and provides a basis to further study functions of Hsp60/Hsp10 relative to thermotolerance and cold hardiness mechanism.

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis).

Quantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔC t method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

Identification of odorant-binding protein genes in Galeruca daurica (Coleoptera: Chrysomelidae) and analysis of their expression profiles.

Odorant-binding proteins (OBPs) play a fundamental role in insect olfaction. In recent years, Galeruca daurica (Joannis) (Coleoptera: Chrysomelidae) has become one of the most important insect pests in the Inner Mongolian grasslands of China. This pest only feeds on the species of Allium plants, implying the central role of olfaction in its search for specific host plants. However, the olfaction-related proteins have not been investigated in this beetle. In this study, we identified 29 putative OBP genes, namely GdauOBP1-29, from the transcriptome database of G. daurica assembled in our laboratory by using RNA-Seq. All 29 genes had the full-length open reading frames except GdauOBP29, encoding proteins in length from 119 to 202 amino acids with their predicted molecular weights from 12 to 22 kDa with isoelectric points from 3.88 to 8.84. Predicted signal peptides consisting of 15-22 amino acid residues were found in all except GdauOBP6, GdauOBP13 and GdauOBP29. The amino acid sequence identity between the 29 OBPs ranged 8.33-71.83%. GdauOBP1-12 belongs to the Classic OBPs, while the others belong with the Minus-C OBPs. Phylogenetic analysis indicated that GdauOBPs are the closest to CbowOBPs from Colaphellus bowringi. RT-PCR and qRT-PCR analyses showed that all GdauOBPs were expressed in adult antennae, 11 of which with significant differences in their expression levels between males and females. Most GdauOBPs were also expressed in adult heads (without antennae), thoraxes, abdomens, legs and wings. Moreover, the expression levels of the GdauOBPs varied during the different development stages of G. daurica with most GdauOBPs expressed highly in the adult antennae but scarcely in eggs and pupae. These results provide insights for further research on the molecular mechanisms of chemical communications in G. daurica.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica.

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

MicroRNA miR-285 modulates the metamorphosis in Galeruca daurica by targeting Br-C.

BACKGROUND: Galeruca daurica has become a new pest on the Inner Mongolia grasslands since an abrupt outbreak in 2009 caused serious damage. As a pupa indicator during insect metamorphosis, the early response gene of the ecdysone signaling pathway, Broad-Complex (Br-C), plays a vital role in the growth and development of insects. MicroRNAs (miRNAs) are small non-coding RNAs which mediate various biological activities, but it is unknown whether and how Br-C is regulated by miRNAs. RESULTS: Temporal expression profiles revealed that miR-285 and Br-C basically displayed an opposite trend during larval-adult development, and Br-C was sharply up-regulated on the last day of final-instar larvae while miR-285 was significantly down-regulated. Both dual-luciferase reporter assay and miRNA-mRNA interaction assay indicated that miR-285 interacts with the coding sequence of Br-C and represses its expression. Not only overexpression but also downexpression of miR-285 led to the failure of larval to pupal to adult metamorphosis. In addition, both overexpression of miR-285 and silence of Br-C inhibited the expression of Br-C and other ecdysone signaling pathway genes, including E74, E75, ECR, FTZ-F1, and HR3. On the contrary, suppressing miR-285 obtained opposite results. Further experiments showed that 20-hydroxyecdysone down-regulated miR-285 and up-regulated Br-C and above-mentioned genes, whereas juvenile hormone alalogue (JHA) resulted in opposite effects. CONCLUSION: Our results reveal that miR-285 is involved in mediating the metamorphosis in G. daurica by targeting Br-C in the ecdysone signaling pathway. miR-285 and its target Br-C could be as a potential target for G. daurica management. © 2024 Society of Chemical Industry.

MicroRNA miR-2765-3p regulates reproductive diapause by targeting FoxO in Galeruca daurica.

The forkhead box O (FoxO), as a conserved transcription factor, plays an indispensable role in regulating insect diapause. However, how FoxO is regulated to control diapause in insects remains unknown. In this study, we discovered functional binding sites for miR-2765-3p in the 3' untranslated region of FoxO in Galeruca daurica. The luciferase reporter assay showed that miR-2765-3p targeted FoxO and suppressed its expression. The expression profiles of miR-2765-3p and FoxO displayed opposite patterns during the female developmental process. Overexpression of miR-2765-3p by the injection of the miR-2765-3p agomir into adult females reduced FoxO expression, leading to the suppression of lipid accumulation, promotion of ovarian development, and inhibition of reproductive diapause. This is similar to the phenotype that results from the depletion of FoxO by injecting dsFoxO into adult females. In addition, the repression of miR-2765-3p by injecting the miR-2765-3p antagomir increased the FoxO transcript level, leading to the stimulation of lipid accumulation, depression of ovarian development, and induction of reproductive diapause. A hormone injection assay showed that the juvenile hormone (JH) agonist (methoprene) upregulated miR-2765-3p and downregulated FoxO. Notably, injecting methoprene rescued ovarian development defects associated with miR-2765-3p inhibition. These findings indicate that the JH/miR-2765-3p/FoxO axis plays a vital role in the regulation of reproductive diapause in G. daurica.

Comparative Transcriptome Analysis of Galeruca daurica Reveals Cold Tolerance Mechanisms.

Galeruca daurica (Joannis) is a pest species with serious outbreaks in the Inner Mongolian grasslands in recent years, and its larvae and eggs are extremely cold-tolerant. To gain a deeper understanding of the molecular mechanism of its cold-tolerant stress response, we performed de novo transcriptome assembly of G. daurica via RNA-Seq and compared the differentially expressed genes (DEGs) of first- and second-instar larvae grown and developed indoors and outdoors, respectively. The results show that cold tolerance in G. daurica is associated with changes in gene expression mainly involved in the glycolysis/gluconeogenesis pathway, the fatty acid biosynthesis pathway and the production of heat shock proteins (HSPs). Compared with the control group (indoor), the genes associated with gluconeogenesis, fatty acid biosynthesis and HSP production were up-regulated in the larvae grown and developed outdoors. While the changes in these genes were related to the physiological metabolism and growth of insects, it was hypothesized that the proteins encoded by these genes play an important role in cold tolerance in insects. In addition, we also investigated the expression of genes related to the metabolic pathway of HSPs, and the results show that the HSP-related genes were significantly up-regulated in the larvae of G. daurica grown and developed outdoors compared with the indoor control group. Finally, we chose to induce significant expression differences in the Hsp70 gene (Hsp70A1, Hsp70-2 and Hsp70-3) via RNAi to further illustrate the role of heat stress proteins in cold tolerance on G. daurica larvae. The results show that separate and mixed injections of dsHSP70A1, dsHsp70-2 and dsHsp70-3 significantly reduced expression levels of the target genes in G. daurica larvae. The super-cooling point (SCP) and the body fluid freezing point (FP) of the test larvae were determined after RNAi using the thermocouple method, and it was found that silencing the Hsp70 genes significantly increased the SCP and FP of G. daurica larvae, which validated the role of heat shock proteins in the cold resistance of G. daurica larvae. Our findings provide an important theoretical basis for further excavating the key genes and proteins in response to extremely cold environments and analyzing the molecular mechanism of cold adaptation in insects in harsh environments.

Transcriptome-wide identification of microRNAs in response to 20-hydroxyecdysone in Galeruca daurica.

Both 20-hydroxyecdysone (20E) and miRNAs have multiple functions in the regulation of various physiological processes in insects. However, little is known about the interaction between 20E and miRNAs. In this study, six small RNA libraries were constructed from the adult Galeruca daurica treated with 20E and dimethyl sulfoxide (DMSO), respectively. Using small RNA sequencing, a total of 183 miRNAs, including 140 known and 43 novel miRNAs, were identified. Compared with the control (DMSO), 52 miRNAs (21 up-regulated and 31 down-regulated) were significantly differentially expressed after 20E treatment. The KEGG and GO analysis of the predicted genes targeted by 20E-responsive miRNAs indicate that 20E may influence the metabolic change during reproductive diapause in G. daurica via regulating miRNAs.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Detection of ryanodine receptor G4911E and I4754M mutation sites and analysis of binding modes of diamide insecticides with RyR on Galeruca daurica (Coleoptera: Chrysomelidae).

In recent years, the leaf beetle Galeruca daurica has broken out in the northern grasslands of Inner Mongolia, its management still mainly depends on chemical control using traditional insecticides or with novel action. The study was aim to identify mutation locus associated with resistance to diamide insecticides in field population of G. daurica, to provide a reference for rational selection of insecticides and to avoid the rapid resistance development to diamide insecticides. We cloned the full length of the ryanodine receptor gene of G. daurica (GdRyR), constructed 3D model and transmembrane regions by homologous modeling based on deduced amino acid sequence. Two potential mutation loci (Gly4911Glu and Ile4754Met) and allelic mutation frequencies were detected in individuals of G. daurica. In addition, their binding patterns to two diamide insecticides (chlorantraniliprole, cyantraniliprole) were analyzed separately using a molecular docking method. The full-length cDNA sequence of GdRyR (GenBank accession number: OP828593) was obtained by splicing and assembling, which is 15,399 bp in length and encodes 5,133 amino acids. The amino acid similarity of GdRyR with that of other Coleopteran insects were 86.70%-91.33%, which possessed the typical structural characteristics. An individual resistance allelic mutation frequency test on fifty field leaf beetles has identified 12% and 32% heterozygous individuals at two potential mutation loci Gly4911Glu and Ile4754Met, respectively. The affinity of the I4754M mutant model of GdRyR for chlorantraniliprole and cyantraniliprole was not significantly different from that of the wild type, and all had non-covalent interactions such as hydrogen bonding, hydrophobic interactions and π-cation interactions. However, the G4911E mutant model showed reduced affinity and reduced mode of action with two diamide insecticides, thus affecting the binding stability of the ryanodine receptor to the diamide insecticides. In conclusion, the G4911E mutation in GdRyR may be a potential mechanism for the development of resistance to diamide insecticides on G. daurica and should be a key concern for resistance risk assessment and reasonable applications of diamide insecticides for control in future. Moreover, this study could provide a reference for ryanodine receptor structure-based insecticides design.

Regulation of Juvenile Hormone on Summer Diapause of Geleruca daurica and Its Pathway Analysis.

Juvenile hormone (JH) signaling plays an important role in regulation of reproductive diapause in insects. However, we have little understanding of the effect of JH on gene expression at the transcriptome level in diapause. Galeruca daurica is a new pest in the Inner Mongolia grasslands with obligatory summer diapause in the adult stage. Topical application of a JH analog methoprene at the pre-diapause stage delayed the adults entering diapause and inhibited lipid accumulation whereas it did not during diapause. Using Illumina sequencing technology and bioinformatics tools, 54 and 138 differentially expressed genes (DEGs) were detected at 1 and 2 d after treatment, respectively. The KEGG analysis showed that the DEGs were mainly enriched in the metabolism pathways. qRT-PCR analysis indicated that methoprene promoted the expression of genes encoding vitellogenin, fork head transcription factor and Krüppel homolog 1, whereas suppressed the expression of genes encoding juvenile hormone-binding protein, juvenile hormone esterase, juvenile hormone acid methyltransferase, juvenile hormone epoxide hydrolase and fatty acid synthase 2. These results indicate that JH signaling plays an important role in regulating reproductive diapause of G. daurica.

Identification and functional analysis of microRNAs in the regulation of summer diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. Although the regulatory roles of miRNAs in various physiological processes throughout insect development have been investigated, it is almost unknown about the roles of miRNAs involved in regulation of diapause in insects. We constructed nine small RNA libraries from Galeruca daurica adults at different diapause stages: pre-diapause (PD), diapause (D), and post-diapause (TD). Using Illumina sequencing, a total of 95.06 million valid reads was obtained, and 222 miRNAs, including 135 conserved and 87 novel miRNAs, were identified from G. daurica. The expression profiles of these miRNAs were analyzed across different diapause stages. The 30 and 13 miRNAs were differentially expressed in the D/PD and TD/D comparisons, respectively. The KEGG and GO analysis of the predicted target genes suggested the essential roles of miRNAs in the regulation of summer diapause in G. daurica, especially via the juvenile hormone, ribosome, MAPK signaling, and Ca2+ signaling pathways. Our research results indicate that miRNAs may be involved in the regulation of summer diapause in G. daurica, and these results also provide an important new small RNA genomics resource for further studies on insect diapause.

Functional Characterization of Olfactory Proteins Involved in Chemoreception of Galeruca daurica.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1, GdauOBP6, GdauOBP10, GdauOBP15, GdauCSP4, and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5, RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants.

Proteomic analysis of adult Galeruca daurica (Coleoptera: Chrysomelidae) at different stages during summer diapause.

Galeruca daurica is a new pest causing great losses in the Inner Mongolian grasslands of China. The adults enter obligatory diapause during summer. However, the molecular mechanism of summer diapause remains unknown. We used iTRAQ to conduct proteomic analysis of adult G. daurica at the pre-diapause (PD), diapause (D) and post-diapause (TD) stages during summer diapause. A total of 139 and 118 differentially expressed proteins (DEPs) were detected in D/PD and TD/D comparisons, respectively. Besides a large number of DEPs involved in metabolic process, stress response, cytoskeletal reorganization, and phagosome pathway, many new proteins related to diapause were found in this study, such as encapsulation-relating proteins, odorant binding proteins, chemosensory proteins and ribosomal proteins. KEGG analysis revealed that the phagosome pathway was the only common significantly enriched pathway in both D/PD and TD/D. In addition, juvenile hormone regulation and Ca2+ signaling may play an important role in the regulation of summer diapause in G. daurica. Our proteomic analysis provides a new insight into the mechanism of obligatory summer diapause, and lays a foundation for future molecular level studies.

Effects of Temperature on the Termination of Egg Diapause and Post-Diapause Embryonic Development of Galeruca daurica (Coleoptera: Chrysomelidae).

Galeruca daurica (Joannis) is a new pest on the grasslands of Inner Mongolia, China. It is univoltine and overwinters in the egg stage. Larvae and adults feed on the foliage of Allium plants. To assess the requirements to terminate egg diapause and subsequent effects on post-diapause development rate, eggs were held at different temperature regimes. Exposure to low temperatures was required to terminate egg diapause. Prolonged exposure (2 mo vs 1 mo) to 5°C and outside ambient conditions (mean temperature: 10.5°C; range: -7.1-21.6°C) enhanced the termination of egg diapause. Prolonged exposure also reduced the time to egg hatch; e.g., eggs held for 2 mo versus 1 mo at 5°C developed more quickly when subsequently placed at warmer temperatures. Egg hatch was observed at 17, 21, 25, and 29°C, but not at 15°C. Regression analysis identified 16.2°C as the minimum temperature for post-diapause development. The temperature requirement to complete embryonic development (from diapause termination to egg hatch) was calculated to be 103.1 to 140.9 degree-days.

The complete mitochondrial genome of Galeruca daurica (Joannis) (Coleoptera: Chrysomelidae).

Galeruca daurica (Joannis) (Coleoptera: Chrysomelidae) is one of important pests in the Inner Mongolia grasslands. The complete mitochondrial genome was sequenced. The genome is 16 615 bp long, with an AT content of 78.1%, containing 37 typical animal mitochondrial genes and an AT-rich region. All 13 PCGs share the start codon ATN, and the usual termination codons (TAA and TAG) are found from 13 protein-coding genes, except for COI, COII, and ND4 (T). All the 22 typical animal tRNA genes are found in G. daurica mt-genome, and most of the tRNAs could be folded into the classic cloverleaf secondary structure except for tRNA-Ser (AGN), which lacks the dihydrouracil (DHU) stem. The sizes of the large ribosomal RNA genes are 1276 bp long and small ribosomal RNA genes are 747 bp long. The AT content of the AT-rich region is 79.0%. Phylogenetic analysis supports that the coleopteran insects from the same family cluster in the same group, and Chrysomelidae and Tenebrionidae are basal to the Cerambycidae. Galeruca daurica has a closest relationship with Diabrotica barberi and Diabrotica virgifera.