CCE Gene Research Articles

Identification and Functional Characterization of Carboxylesterase Genes Involved in Spirodiclofen Resistance in Panonychus citri (McGregor).

Overexpression of carboxyl/cholinesterase (CCE) genes has been reported to be associated with many cases of pesticide resistance in arthropods. However, it has been rarely documented that CCE genes participate in spirodiclofen resistance in Panonychus citri. In previous research, we found that spirodiclofen resistance is related to increased P450 and CCE enzyme activities in P. citri. In this study, we identified two CCE genes, PcCCE3 and PcCCE5, which were significantly upregulated in spirodiclofen-resistant strain and after exposure to spirodiclofen. RNA interference of PcCCE3 and PcCCE5 increased the spirodiclofen susceptibility in P. citri. In vitro metabolism indicated that PcCCE3 and PcCCE5 could interact with spirodiclofen, but metabolites were detected only in the PcCCE3 treatment. Our results indicated that PcCCE3 participates in spirodiclofen resistance through direct metabolism, and PcCCE5 may be involved in the spirodiclofen resistance by passive binding and sequestration, which provides new insights into spirodiclofen resistance in P. citri.

Overexpression of a nuclear receptor HR96 contributes to spirodiclofen susceptibility in Panonychus citri (McGregor)

The citrus red mite, Panonychus citri, is one of the most notorious and devastating citrus pests around the world that has developed resistance to multiple chemical acaricides. In previous research, we found that spirodiclofen-resistant is related to overexpression of P450, CCE, and ABC transporter genes in P. citri. However, the regulatory mechanisms of these detoxification genes are still elusive. This study identified all hormone receptor 96 genes of P. citri. 8 PcHR96 genes contained highly conserved domains. The expression profiles showed that PcHR96h was significantly upregulated in spirodiclofen resistant strain and after exposure to spirodiclofen. RNA interference of PcHR96h decreased expression of detoxification genes and increased spirodiclofen susceptibility in P. citri. Furthermore, molecular docking, heterologous expression, and drug affinity responsive target stability demonstrated that PcHR96h can interact with spirodiclofen in vitro. Our research results indicate that PcHR96h plays an important role in regulating spirodiclofen susceptibility and provides theoretical support for the resistance management of P. citri.

Transcriptome analysis revealed that multiple genes were related to the cyflumetofen resistance of Tetranychus cinnabarinus (Boisduval)

Metabolic resistance is one of the main causes of acaricide resistance. Many previous studies focused on the function of specific genes in insecticides/acaricides resistance. However, during the development of resistance, the overall dynamic of expression levels of detoxification enzyme genes in mites is still unclear. Tetranychus cinnabarinus, a major agricultural pest, which is notorious for developing resistance to acaricides rapidly. In this study, a field susceptible strain (YS) was continuously selected for 16, 25 and 32 generations, and developed to low resistance (7.83-fold, L), medium resistance (17.23-fold, M) and high resistance (86.05-fold, H), respectively. Transcriptome sequencing was performed in YS, L, M and H strains. Overall, compared with YS strain, the number of differential expression genes increased slightly with the development of cyflumetofen-resistance. As for detoxification genes, the median of fold change of up-regulated P450、CCE and GST genes was higher than those of all up-regulated genes in three resistance level, but only the number and the median of fold change of up-regulated P450 genes was increased slightly with the development of resistance. In addition, synergism experiments also proved that P450 and GST genes were the major contributors to the metabolic resistance of cyflumetofen of T. cinnabarinus. These results showed that the resistance of T. cinnabarinus to cyflumetofen was related to many resistant genes, among which P450 genes could play crucial roles in cyflumefen resistance.

Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control

BackgroundAedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. chikungunya). Development of insecticide resistance in the vector is a major concern as its control mainly relies on the use of biocides. Data on the species’ resistance status are essential for efficient and sustainable control. To date the insecticide resistance status of Ae. albopictus populations from Greece against major insecticides used in vector control remains largely unknown.MethodsWe investigated the insecticide resistance status of 19 Ae. albopictus populations from 11 regions of Greece. Bioassays were performed against diflubenzuron (DFB), Bacillus thuringiensis var. israelensis (Bti), deltamethrin and malathion. Known insecticide resistance loci were molecularly analysed, i.e. voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance; presence and frequency of carboxylesterases 3 (CCEae3a) and 6 (CCEae6a) gene amplification associated with organophosphate (OP) resistance and; chitin synthase-1 (CHS-1) for the possible presence of DFB resistance mutations.ResultsBioassays showed full susceptibility to DFB, Bti and deltamethrin, but resistance against the OP malathion (range of mortality: 55.30–91.40%). VGSC analysis revealed a widespread distribution of the mutations F1534C (in all populations, with allelic frequencies between 6.6–68.3%), and I1532T (in 6 populations; allelic frequencies below 22.70%), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations (overall frequency: 33%). Co-presence of the F1534C mutation and CCEae3a amplification was reported in 39 of the 156 samples analysed by both assays. No mutations at the CHS-1 I1043 locus were detected.ConclusionsThe results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece; (ii) possible incipient pyrethroid resistance due to the presence of kdr mutations; and (iii) possible reduced efficacy of OPs, in a scenario of re-introducing them for vector control. The study highlights the need for systematic resistance monitoring for developing and implementing appropriate evidence-based control programmes.

Downregulation of carboxylesterase contributes to cyflumetofen resistance in Tetranychus cinnabarinus (Boisduval).

Increased expression or point mutations of carboxyl/cholinesterases (CCEs) have been involved in many cases of insecticide and acaricide resistance. However, it has been only rarely documented that downregulation of CCE genes is associated with resistance, although many insecticides and acaricides need hydrolytic activation in vivo. Previously, expression analysis of a laboratory-selected cyflumetofen-resistant strain of Tetranychus cinnabarinus indicated that resistance was associated with increased expression of a CCE gene of TcCCE04, but also the downregulation of two CCE genes, TcCCE12 and TcCCE23. Synergism experiments revealed the importance of ester hydrolysis in cyflumetofen toxicity, because treatment with S,S,S-tributylphosphorotrithioate (DEF) caused strong inhibition of cyflumetofen hydrolysis, in both the susceptible and resistant strains. Moreover, silencing expression of TcCCE12 and TcCCE23 via RNAi further decreased the susceptibility of mites to cyflumetofen significantly, suggesting that downregulated CCE genes could be involved in cyflumetofen resistance. In addition, it was shown that recombinant TcCCE12 protein could hydrolyze cyflumetofen effectively. Decreased esterase activity via downregulation of specific CCE genes most likely contributes to cyflumetofen resistance by decreased activation of cyflumetofen to its active metabolite. Mixtures of cyflumetofen and esterase-inhibition acaricides (e.g. organophosphates or carbamates) should be avoided in field applications. © 2019 Society of Chemical Industry.

Latrophilin mediates insecticides susceptibility and fecundity through two carboxylesterases, esterase4 and esterase6, in Tribolium castaneum.

Latrophilin (LPH) is known as an adhesion G-protein-coupled receptor which involved in multiple physiological processes in organisms. Previous studies showed that lph not only involved the susceptibility to anticholinesterase insecticides but also affected fecundity in Tribolium castaneum. However, its regulatory mechanisms in these biological processes are still not clear. Here, we identified two potential downstream carboxylesterase (cce) genes of Tclph, esterase4 and esterase6, and further characterized their interactions with Tclph. After treatment of T. castaneum larvae with carbofuran or dichlorvos insecticides, the transcript levels of Tcest4 and Tcest6 were significantly induced from 12 to 72 h. RNAi against Tcest4 or Tcest6 led to the higher mortality compared with the controls after the insecticides treatment, suggesting that these two genes play a vital role in detoxification of insecticides in T. castaneum. Furthermore, with insecticides exposure to Tclph knockdown beetles, the expression of Tcest4 was upregulated but Tcest6 was downregulated, indicating that beetles existed a compensatory response against the insecticides. Additionally, RNAi of Tcest6 resulted in 43% reductions in female egg laying and completely inhibited egg hatching, which showed the similar phenotype as that of Tclph knockdown. These results indicated that Tclph affected fecundity by positively regulating Tcest6 expression. Our findings will provide a new insight into the molecular mechanisms of Tclph involved in physiological functions in T. castaneum.

Identification of carboxylesterase genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera: Culicidae).

Carboxylesterases (CCEs) are one of three large detoxification enzyme families. Some CCEs are active on synthetic insecticides with ester structures. Anopheles sinensis is an important malaria vector in eastern Asia. This study identified and characterized the CCE genes in the A. sinensis genome and determined CCE genes associated with pyrethroid resistance using RNA sequencing (RNA-seq) and quantitative reverse transcription - polymerase chain reaction (qRT-PCR), in A. sinensis from Anhui, Chongqing, and Yunnan in China. Fifty-seven putative CCEs were identified and placed into three classes, 12 subfamilies and 14 clades through phylogenetic and homology analyses. Exon sizes ranged from 31 to 4317 bp, with 49 CCEs having two to five exons and eight having six to 11 exons. A total of 183 introns were recognized with sizes ranging from 31 to 4317 bp. The 57 CCEs were located on 14 scaffolds, with 70% located on four scaffolds. The alpha-esterase subfamily was significantly expanded compared with that of Anopheles gambiae. In a pyrethroid-resistant strain, RNA-seq detected five upregulated CCE genes and qRT-PCR detected 12 upregulated CCE genes. The α-esterase 10 (AsAe10) and acetylcholinesterase 1 (AsAce1) genes were the main CCE genes associated with pyrethroid resistance. This information will be useful for further study of the CCE gene family and pyrethroid resistance mechanisms mediated by CCEs. © 2017 Society of Chemical Industry.

Rhodnius prolixus supergene families of enzymes potentially associated with insecticide resistance

Chagas disease or American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi. Once known as an endemic health problem of poor rural populations in Latin American countries, it has now spread worldwide. The parasite is transmitted by triatomine bugs, of which Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) is one of the vectors and a model organism. This species occurs mainly in Central and South American countries where the disease is endemic. Disease prevention focuses on vector control programs that, in general, rely intensely on insecticide use. However, the massive use of chemical insecticides can lead to resistance. One of the major mechanisms is known as metabolic resistance that is associated with an increase in the expression or activity of detoxification genes. Three of the enzyme families that are involved in this process – carboxylesterases (CCE), glutathione s-transferases (GST) and cytochrome P450s (CYP) – are analyzed in the R. prolixus genome. A similar set of detoxification genes to those of the Hemipteran Acyrthosiphon pisum but smaller than in most dipteran species was found in R. prolixus genome. All major CCE classes (43 genes found) are present but the pheromone/hormone processing class had fewer genes than usual. One main expansion was detected on the detoxification/dietary class. The phosphotriesterase family, recently associated with insecticide resistance, was also represented with one gene. One microsomal GST gene was found and the cytosolic GST gene count (14 genes) is extremely low when compared to the other hemipteran species with sequenced genomes. However, this is similar to Apis mellifera, a species known for its deficit in detoxification genes. In R. prolixus 88 CYP genes were found, with representatives in the four clans (CYP2, CYP3, CYP4 and mitochondrial) usually found in insects. R. prolixus seems to have smaller species-specific expansions of CYP genes than mosquitoes and beetles, among others. The number of R. prolixus CYP genes is similar to the hemipteran Ac. pisum, although with a bigger expansion in CYP3 and CYP4 clans, along with several gene fragments, mostly in CYP4 clan. Eleven founding members of new families were detected, consisting of ten genes in the CYP3 clan and 1 gene in the CYP4 clan. Members of these clans were proposed to have important detoxification roles in insects. The identification of CCE, GST and CYP genes is of utmost importance for directing detoxification studies on triatomines that can help insecticide management strategies in control programs.

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus.

BackgroundThe control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated.Principal findingsTemephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance.SignificanceThe identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.

Identification of Carboxylesterase Genes Implicated in Temephos Resistance in the Dengue Vector Aedes aegypti

BackgroundThailand is currently experiencing one of its worst dengue outbreaks in decades. As in most countries where this disease is endemic, dengue control in Thailand is largely reliant on the use of insecticides targeting both immature and adult stages of the Aedes mosquito, with the organophosphate insecticide, temephos, being the insecticide of choice for attacking the mosquito larvae. Resistance to temephos was first detected in Aedes aegypti larvae in Thailand approximately 25 years ago but the mechanism responsible for this resistance has not been determined.Principal FindingsBioassays on Ae. aegypti larvae from Thailand detected temephos resistance ratios ranging from 3.5 fold in Chiang Mai to nearly 10 fold in Nakhon Sawan (NS) province. Synergist and biochemical assays suggested a role for increased carboxylesterase (CCE) activities in conferring temephos resistance in the NS population and microarray analysis revealed that the CCE gene, CCEae3a, was upregulated more than 60 fold in the NS population compared to the susceptible population. Upregulation of CCEae3a was shown to be partially due to gene duplication. Another CCE gene, CCEae6a, was also highly regulated in both comparisons. Sequencing and in silico structure prediction of CCEae3a showed that several amino acid polymorphisms in the NS population may also play a role in the increased resistance phenotype.SignificanceCarboxylesterases have previously been implicated in conferring temephos resistance in Ae aegypti but the specific member(s) of this family responsible for this phenotype have not been identified. The identification of a strong candidate is an important step in the development of new molecular diagnostic tools for management of temephos resistant populations and thus improved control of dengue.

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786bp, containing a 1641bp open reading frame encoding 546 amino acids, a 38bp 5′-untranslated region (5′-UTR), and a 107bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.

Transcriptomic and phylogenetic analysis of Culex pipiens quinquefasciatus for three detoxification gene families

BackgroundThe genomes of three major mosquito vectors of human diseases, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), and carboxyl/cholinesterases (CCE). However, unlike An. gambiae and Ae. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE) technique.ResultsA total of 302 detoxification genes were found in C. p. quinquefasciatus, including 71 CCE, 196 P450, and 35 cytosolic GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of α-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. For the five DGE libraries, 3.5-3.8 million raw tags were generated and mapped to 13314 reference genes. Among 302 detoxification genes, 225 (75%) were detected for expression in at least one DGE library. One fourth of the CCE and P450 genes were detected uniquely in one stage, indicating potential developmentally regulated expression. A total of 1511 genes showed different expression levels between a parathion-resistant and a susceptible strain. Fifteen detoxification genes, including 2 CCEs, 6 GSTs, and 7 P450s, were expressed at higher levels in the resistant strain.ConclusionsThe results of the present study provide new insights into the functions and evolution of three detoxification gene families in mosquitoes and comprehensive transcriptomic resources for C. p. quinquefasciatus, which will facilitate the elucidation of molecular mechanisms underlying the different biological characteristics of the three major mosquito vectors.

Molecular characterization and functional analysis of novel carboxyl/cholinesterases with GQSAG motif in the silkworm Bombyx mori

We have previously cloned and characterized BmJHE, a juvenile hormone (JH)-selective esterase (JHE) that is important for JH titer regulation in the silkworm Bombyx mori. Here, we sought to determine whether multiple genes might function as JH-specific esterase in this species. We searched for putative carboxyl/cholinesterase (CCE) genes having GQSAG, a highly conserved motif in JHE, by the use of silkworm genomic database. Five novel CCE genes ( Bmcce-1–5) were identified and their cDNA sequences and intron–exon structures were determined. We investigated the developmental expression patterns of these CCE genes by real-time quantitative PCR analysis and found that their expression patterns varied among developmental stages and organs. Of the proteins produced by the five genes, only BmCCE-5 had the ability to degrade JH; however, this protein might not function as a JH-specific esterase in vivo as it had a high K m value for JH. On the other hand, BmCCE-5 degraded general esterase substrates efficiently. Since Bmcce-5 was strongly expressed in Malpighian tubules and the gut, it might function in digestion or xenobiotic metabolism. Our results suggest that of the CCEs containing a GQSAG motif only BmJHE can function as a JH-specific degradation enzyme in the silkworm.