Absence of juvenile hormone signalling regulates the dynamic expression profiles of nutritional metabolism genes during diapause preparation in the cabbage beetle Colaphellus bowringi

Temperate insects have evolved diapause, a period of programmed developmental arrest during specific life stages, to survive unfavourable conditions. During the diapause preparation phase (DPP), diapause‐destined individuals generally store large amounts of fat by regulating nutrition distribution for the energy requirement during diapause maintenance and postdiapause development. Although nutritional patterns during the DPP have been investigated at physiological and biochemical levels in many insects, it remains largely unknown how nutritional metabolism is regulated during the DPP at molecular levels. We used RNA sequencing to compare gene expression profiles of adult female cabbage beetles Colaphellus bowringi during the preoviposition phase (POP) and the DPP. Most differentially expressed genes were involved in specific metabolic pathways during the DPP. Genes related to lipid and carbohydrate metabolic pathways were clearly highly expressed during the DPP, whereas genes related to protein metabolic pathways were highly expressed during the POP. Hormone challenge and RNA interference experiments revealed that juvenile hormone via its nuclear receptor methoprene‐tolerant mediated the expression of genes associated with nutritional metabolism during the DPP. This work not only sheds light on the mechanisms of diapause preparation, but also provides new insights into the molecular basis of environmental plasticity in insects.     

Cytochrome P450 CYP4DE1 and CYP6CW3v2 contribute to ethiprole resistance in Laodelphax striatellus (Fallén)

Laodelphax striatellus Fallén (Hemiptera: Delphacidae), a destructive pest of rice, has developed high resistance to multiple insecticides, threatening the success of pest management programmes. The present study investigated ethiprole resistance mechanisms in a field population that is highly resistant to ethiprole. That population was used to establish a laboratory population that was subjected to further selection to produce a resistant strain. Target genes were cloned and compared between the resistant and the susceptible strains, the role of detoxification enzymes was examined, and the relative expression levels of 71 detoxification enzyme genes were tested using quantitative real time (RT)‐PCR. The laboratory selection enhanced the resistance from 107‐fold to 180‐fold. The Rdl‐type target site mutation seldom occurred in the resistant strain and is unlikely to represent the major mechanism underlying the observed resistance. Of the three important detoxification enzymes, only P450 monooxygenase was found to be associated with ethiprole resistance. Moreover, two genes, CYP4DE1 and CYP6CW3v2, were found to be overexpressed in the resistant strain. Furthermore, gene‐silencing via a double‐stranded RNA feeding test was carried out, and the results showed that the mRNA levels of CYP4DE1 and CYP6CW3v2 were reduced in the resistant strain, whereas ethiprole susceptibility was increased. These results suggest that CYP4DE1 and CYP6CW3v2 play an important role in ethiprole resistance in L. striatellus.     

Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin‐resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene‐silencing experiments via double‐stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively.     

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.     

Comparison of orthologous cytochrome P450 genes relative expression patterns in the bark beetles Dendroctonus rhizophagus and Dendroctonus valens (Curculionidae: Scolytinae) during host colonization

Bark beetles of the genus Dendroctonus are important components of coniferous forests. During host colonization, they must overcome the chemical defences of their host trees, which are metabolized by cytochrome P450 (CYP or P450) enzymes to compounds that are readily excreted. In this study, we report the relative expression (quantitative real‐time PCR) of four orthologous cytochrome P450 genes (CYP6BW5, CYP6DG1, CYP6DJ2 and CYP9Z20) in Dendroctonus rhizophagus and Dendroctonus valens forced to attack host trees at 8 and 24 h following forced attack and in four stages during natural colonization [solitary females boring the bark (T1); both male and female members of couples before oviposition (T2); both male and female members of couples during oviposition (T3), and solitary females inside the gallery containing eggs (T4)]. For both species gene expression was different compared with that observed in insects exposed to single monoterpenes in the laboratory, and the expression patterns were significantly different amongst species, sex, gut region and exposure time or natural colonization stage. The induction of genes (CYP6BW5v1, CYP6DJ2v1 and CYP9Z20v1 from D. rhizophagus, as well as CYP6DG1v3 from D. valens) correlated with colonization stage as well as with the increase in oxygenated monoterpenes in the gut of both species throughout the colonization of the host. Our results point to different functions of these orthologous genes in both species.     

Identification of an E‐box DNA binding protein,activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis‐activating neuropeptide in Helicoverpa armigera

Activated protein 4 (AP‐4), an E‐box DNA‐binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har‐AP‐4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro‐translated Har‐AP‐4 can bind specifically to the E‐box motif on the promoter of the diapause hormone and pheromone biosynthesis‐activating neuropeptide (DH‐PBAN). Har‐AP‐4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har‐AP‐4 can significantly activate the promoter of the DH‐PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har‐AP‐4, which binds to the promoter of DH‐PBAN, may play a role in regulating pupal development in H. armigera.     

Exome‐wide association of deltamethrin resistance in Aedes aegypti from Mexico

Aedes aegypti is the major vector of a number of arboviruses that cause disease in humans. Without vaccines or pharmaceuticals, pyrethroid insecticides remain the major tool for public health protection. Pyrethroid resistance is now widespread. Replacement substitutions in the voltage‐gated sodium channel (vgsc) that reduce the stability of pyrethroid binding account for most of the resistance, but metabolic mechanisms also inactivate pyrethroids. High‐throughput sequencing and the A. aegypti L5 annotated physical map has allowed interrogation of the exome for genes and single‐nucleotide polymorphisms associated with pyrethroid resistance. We exposed females of A. aegypti from Mexico to a deltamethrin discriminating dose to designate them as resistant (active after 1 h) or susceptible (knocked down with no recovery after 4 h). The vgsc on chromosome 3 had the highest association, followed by genes proximal to vgsc. We identified potential detoxification genes located singly (eg HPX8C) or within clusters in chromosome 2 [three esterase clusters, two of cytochrome P450 monooxygenases (CYP)] and chromosome 3 (one cluster of 16 CYP325 and seven CYP9 genes). Deltamethrin resistance in A. aegypti is associated with mutations in the vgsc gene and a large assortment of genes.     

Dynamics and regulation of glycolysis‐tricarboxylic acid metabolism in the midgut of Spodoptera litura during metamorphosis

Significant changes usually take place in the internal metabolism of insects during metamorphosis. The glycolysis‐tricarboxylic acid (glycolysis‐TCA) pathway is important for energy metabolism. To elucidate its dynamics, the mRNA levels of genes involved in this pathway were examined in the midgut of Spodoptera litura during metamorphosis, and the pyruvate content was quantified. The expression patterns of these genes in response to starvation were examined, and the interaction between protein phosphatase 1 (PP1) and phosphofructokinase (PFK) was studied. The results revealed that the expression or activities of most glycolytic enzymes was down‐regulated in prepupae and then recovered in some degree in pupae, and all TCA‐related genes were remarkably suppressed in both the prepupae and pupae. Pyruvate was enriched in the pupal midgut. Taken together, these results suggest that insects decrease both glycolysis and TCA in prepupae to save energy and then up‐regulate glycolysis but down‐regulate TCA in pupae to increase the supply of intermediates for construction of new organs. The expression of all these genes were down‐regulated by starvation, indicating that non‐feeding during metamorphosis may be a regulator of glycolysis‐TCA pathway in the midgut. Importantly, interaction between PP1 and PFK was identified and is suggested to be involved in the regulation of glycolysis.     

Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2

Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L. striatellus imidacloprid resistant strain (Imid‐R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L. striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co‐expressed with D. melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good‐quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P‐nitroanisole to p‐nitrophenol with a maximum velocity (V_max) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'‐phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (K_cat) = 0.34 pmol/min/pmol P450, michaelis constant (K_m) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L. striatellus.     

Lack of association between schizophrenia and polymorphisms in dopamine metabolism and transport genes

We investigated the relationship between several functional polymorphisms in genes coding for dopamine metabolism and transport enzymes (MAO‐A VNTR; MAO‐A 941T>G; DAT VNTR; DAT ‐67A/T; CYP2D6*3; CYP2D6*4; CYP2D6*5; CYP2D6*6) and the frequency of schizophrenia. Participants in the study were 242 subjects diagnosed with schizophrenia and related disorders and 290 hospital‐based controls. Genomic DNA was isolated from whole blood and genotyped by several methods. However, there was no association between schizophrenia and the alleles, genotypes or diplotypes that were studied or their interactions. Polymorphisms in genes coding for dopamine metabolism and transport enzymes did not predispose to or protect from schizophrenia and related disorders.     

The effect of transcatheter arterial chemoembolization on CYP1A2 activity in patients with hepatocellular carcinoma

Aim: CYP1A2, a constitutive enzyme expressed in the liver, is among the phase I enzymes responsible for polycyclic aromatic hydrocarbons metabolism. Phenacetin O‐de‐ethylation is a marker for CYP1A2 activity. This study investiagtes the metabolism of phenacetin in patients with hepatocellular carcinoma (HCC). Methods: The phenacetin test was performed in 56 normal subjects and 92 HCC patients. The test was repeated in HCC patients after treatment with transcatheter arterial chemoembolization (TACE). The recovery of phenacetin’s urinary metabolites was studies in 12 normal subjects and 14 patients with HCC. Results: Compared with normal controls, the recovery of phenacetin O‐de‐ethylated metabolites decreased by 42·5% (P < 0·01) in patients with HCC and diminished further after TACE (P < 0·05). The ratio of plasma total paracetamol to phenacetin was much lower than in normal controls (P < 0·01) and was reduced by 40·7% more after TACE (P < 0·05). Conclusions: The metabolism of phenacetin is impaired in patient with HCC. TACE damages the activity of CYP1A2. The phenacetin test can be used to predict effect of TACE on liver function in HCC patients.     

A cluster of CYP6 gene family associated with the major quantitative trait locus is responsible for the pyrethroid resistance in Culex pipiens pallen

The emergence and rapid spread of insecticide resistance in several mosquito species has become a significant obstacle in management of mosquito‐borne diseases, including deltamethrin resistance in Culex pipiens pallens. Previous study identified a major deltamethrin resistance quantitative trait locus (DR‐6) that alone explained 62% of the genetic variance. In this study, the marker L4B1.102 and L4B1.175 associated with the DR‐6 were characterized. We searched for potential candidate genes in the flank region of two markers in the genome sequence and showed that a cluster of CYP6 cytochrome P450 genes (CYP6BB4, CYP6BB3, CYP6CC2, CYP6P14, CYP6BZ2, CYP6AA9, CYP6AA8, CYP6AA7) was in the vicinity of DR‐6. Significant differences in the expression of these P450s in the larval and adult stages were identified in the resistant strains compared with the susceptible strain. For CYP6AA9 and CYP6BB4, the correlation analysis showed a highly positive correlation between relative gene expression quantification and the resistance level in different strains. Knockdown of CYP6BB4 increased the sensitivity of mosquitoes to deltamethrin. We identified that the deltamethrin resistance was in a cluster of CYP6 genes in C. pipiens pallens, and CYP6BB4 may play a significant role in the development of deltamethrin resistance.