Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

HSP superfamily of genes in the malaria vector Anopheles sinensis: diversity, phylogenetics and association with pyrethroid resistance.

BACKGROUND: Heat shock proteins (HSPs) are molecular chaperones that are involved in many normal cellular processes and various kinds of environmental stress. There is still no report regarding the diversity and phylogenetics research of HSP superfamily of genes at whole genome level in insects, and the HSP gene association with pyrethroid resistance is also not well known. The present study investigated the diversity, classification, scaffold location, characteristics, and phylogenetics of the superfamily of genes in Anopheles sinensis genome, and the HSP genes associated with pyrethroid resistance. METHODS: The present study identified the HSP genes in the An. sinensis genome, analysed their characteristics, and deduced phylogenetic relationships of all HSPs in An. sinensis, Anopheles gambiae, Culex quinquefasciatus and Aedes aegypti by bioinformatic methods. Importantly, the present study screened the HSPs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and looked over the HSP gene expression pattern for the first time in An. sinensis on the time-scale post insecticide treatment with RT-qPCR. RESULTS: There are 72 HSP genes in An. sinensis genome, and they are classified into five families and 11 subfamilies based on their molecular weight, homology and phylogenetics. Both RNA-seq and qPCR analysis revealed that the expression of AsHSP90AB, AsHSP70-2 and AsHSP21.7 are significantly upregulated in at least one field pyrethroid-resistant population. Eleven genes are significantly upregulated in different period after pyrethroid exposure. The HSP90, sHSP and HSP70 families are proposed to be involved in pyrethroid stress response based in expression analyses of three field pyrethroid-resistant populations, and expression pattern on the time scale post insecticide treatment. The AsHSP90AB gene is proposed to be the essential HSP gene for pyrethroid stress response in An. sinensis. CONCLUSIONS: This study provides the information frame for HSP superfamily of genes, and lays an important basis for the better understanding and further research of HSP function in insect adaptability to diverse environments.

UDP-glycosyltransferase genes and their association and mutations associated with pyrethroid resistance in Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: UDP-glycosyltransferase (UGT) is an important biotransformation superfamily of enzymes. They catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules, and function in several physiological processes, including detoxification, olfaction, cuticle formation, pigmentation. The diversity, classification, scaffold location, characteristics, phylogenetics, and evolution of the superfamily of genes at whole genome level, and their association and mutations associated with pyrethroid resistance are still little known. METHODS: The present study identified UGT genes in Anopheles sinensis genome, classified UGT genes in An. sinensis, Anopheles gambiae, Aedes aegypti and Drosophila melanogaster genomes, and analysed the scaffold location, characteristics, phylogenetics, and evolution of An. sinensis UGT genes using bioinformatics methods. The present study also identified the UGTs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and the mutations associated with pyrethroid resistance with genome re-sequencing in An. sinensis. RESULTS: There are 30 putative UGTs in An. sinensis genome, which are classified into 12 families (UGT301, UGT302, UGT306, UGT308, UGT309, UGT310, UGT313, UGT314, UGT315, UGT36, UGT49, UGT50) and further into 23 sub-families. The UGT308 is significantly expanded in gene number compared with other families. A total of 119 UGTs from An. sinensis, An. gambiae, Aedes aegypti and Drosophila melanogaster genomes are classified into 19 families, of which seven are specific for three mosquito species and seven are specific for Drosophila melanogaster. The UGT308 and UGT302 are proposed to main families involved in pyrethroid resistance. The AsUGT308D3 is proposed to be the essential UGT gene for the participation in biotransformation in pyrethroid detoxification process, which is possibly regulated by eight SNPs in its 3' flanking region. The UGT302A3 is also associated with pyrethroid resistance, and four amino acid mutations in its coding sequences might enhance its catalytic activity and further result in higher insecticide resistance. CONCLUSIONS: This study provides the diversity, phylogenetics and evolution of UGT genes, and potential UGT members and mutations involved in pyrethroid resistance in An. sinensis, and lays an important basis for the better understanding and further research on UGT function in defense against insecticide stress.

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae).

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

Glutathione S-transferase (GST) genes and their function associated with pyrethroid resistance in the malaria vector Anopheles sinensis.

BACKGROUND: Glutathione S-transferases (GSTs), a multifunctional protein family, are involved in insecticide resistance. However, a systematic analysis of GSTs in Anopheles sinensis, an important vector for malaria transmission, is lacking. In this study, we investigated the diversity and characteristics of GST genes, and analyzed their expression patterns and functions associated with insecticide resistance in this species. RESULTS: We identified 32 putative cytosolic and three putative microsomal GST genes in the An. sinensis genome. Transcriptome analysis showed that GSTs were highly expressed in larvae, and mainly expressed in the antennae, midgut and Malpighian tubules of adults. In addition, we found that GSTd2 and GSTe2 were significantly upregulated in four An. sinensis pyrethroid-resistant field populations. Furthermore, silencing of GSTd2 and GSTe2 significantly increased the susceptibility of An. sinensis to deltamethrin, and recombinant GSTd2 and GSTe2 exhibited high enzymatic activity in the metabolism of 1-chloro-2,4-dinitrobenzene and dichlorodiphenyltrichloroethane (DDT). CONCLUSION: These results showed that GSTs are involved in the development of insecticide resistance in An. sinensis through transcriptional overexpression and enzymatic metabolization, facilitating our understanding of insecticide resistance in insects. © 2022 Society of Chemical Industry.

Mitochondrial genes associated with pyrethroid resistance revealed by mitochondrial genome and transcriptome analyses in the malaria vector Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Insecticides are still the main method of mosquito control, but mosquito resistance presents a large obstacle. The function of mitochondrial genes in the evolution of insecticide resistance is still poorly understood. Pyrethroid is the most commonly used insecticide, and Anopheles sinensis is an important malaria vector in China and Southeast Asia. In this study, we investigated the mitochondrial genes associated with pyrethroid resistance through their genetic and expression variation based on analyses of transcriptomes and 36 individuals with resequencing in three geographical populations in China. RESULTS: The nucleotide diversity (Pi) in 18 resistant individuals was much lower than that in 18 susceptible individuals, which suggests that some sites experienced purifying selection subject to pyrethroid stress. Ka/Ks and amino acid analyses showed that ND4 experienced positive selection and had 23 amino acid mutations due to pyrethroid stress. These mutations might change the ND4 structure and function and thus alter the efficiency of the respiratory chain. ND5 was significantly upregulated, and ATP8 was significantly downregulated in these three pyrethroid resistant populations, which suggests that these two genes function in the production and maintenance of pyrethroid resistance. There are differences in mitochondrial genes involved in pyrethroid resistance among these three populations. CONCLUSION: This is the first study to reveal the association of mitochondrial genes in the evolution of insecticide resistance through amino acid mutation and expression patterns and can help us further understand insecticide resistance mechanisms. © 2019 Society of Chemical Industry.

The complete mt genomes of Lutzia halifaxia, Lt. fuscanus and Culex pallidothorax (Diptera: Culicidae) and comparative analysis of 16 Culex and Lutzia mt genome sequences.

BACKGROUND: Despite the medical importance of the genus Culex, the mitochondrial genome (mt genome) characteristics of Culex spp. are not well understood. The phylogeny of the genus and particularly the generic status of the genus Lutzia and the subgenus Culiciomyia remain unclear. METHODS: The present study sequenced and analyzed the complete mt genomes of Lutzia halifaxia, Lutzia fuscanus and Cx. (Culiciomyia) pallidothorax and assessed the general characteristics and phylogenetics of all known 16 mt genome sequences for species in the genera Culex and Lutzia. RESULTS: The complete mt genomes of Lt. halifaxia, Lt. fuscanus and Cx. pallidothorax are 15,744, 15,803 and 15,578 bp long, respectively, including 13 PCGs, 22 tRNAs, two tRNAs and a control region (CR). Length variations in the Culex and Lutzia mt genomes involved mainly the CR, and gene arrangements are the same as in other mosquitoes. We identified four types of repeat units in the CR sequences, and the poly-T stretch exists in all of these mt genomes. The repeat units of CR are conserved to different extent and provide information on their evolution. Phylogenetic analyses demonstrated that the Coronator and Sitiens groups are each monophyletic, whereas the monophyletic status of the Pipiens Group was not supported; Cx. pallidothorax is more closely related to the Sitiens and Pipiens groups; and both phylogenetics analysis and repeat unit features in CR show that Lutzia is a characteristic monophyletic entity, which should be an independent genus. CONCLUSIONS: To our knowledge, this is the first comprehensive review of the mt genome sequences and taxonomic discussion based on the mt genomes of Culex spp. and Lutzia spp. The research provides general information on the mt genome of these two genera, and the phylogenetic and taxonomic status of Lutzia and Culiciomyia.

Genome-wide identification, characterization and classification of ionotropic glutamate receptor genes (iGluRs) in the malaria vector Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Ionotropic glutamate receptors (iGluRs) are conserved ligand-gated ion channel receptors, and ionotropic receptors (IRs) were revealed as a new family of iGluRs. Their subdivision was unsettled, and their characteristics are little known. Anopheles sinensis is a major malaria vector in eastern Asia, and its genome was recently well sequenced and annotated. METHODS: We identified iGluR genes in the An. sinensis genome, analyzed their characteristics including gene structure, genome distribution, domains and specific sites by bioinformatic methods, and deduced phylogenetic relationships of all iGluRs in An. sinensis, Anopheles gambiae and Drosophila melanogaster. Based on the characteristics and phylogenetics, we generated the classification of iGluRs, and comparatively analyzed the intron number and selective pressure of three iGluRs subdivisions, iGluR group, Antenna IR and Divergent IR subfamily. RESULTS: A total of 56 iGluR genes were identified and named in the whole-genome of An. sinensis. These genes were located on 18 scaffolds, and 31 of them (29 being IRs) are distributed into 10 clusters that are suggested to form mainly from recent gene duplication. These iGluRs can be divided into four groups: NMDA, non-NMDA, Antenna IR and Divergent IR based on feature comparison and phylogenetic analysis. IR8a and IR25a were suggested to be monophyletic, named as Putative in the study, and moved from the Antenna subfamily in the IR family to the non-NMDA group as a sister of traditional non-NMDA. The generated iGluRs of genes (including NMDA and regenerated non-NMDA) are relatively conserved, and have a more complicated gene structure, smaller ω values and some specific functional sites. The iGluR genes in An. sinensis, An. gambiae and D. melanogaster have amino-terminal domain (ATD), ligand binding domain (LBD) and Lig_Chan domains, except for IR8a that only has the LBD and Lig_Chan domains. However, the new concept IR family of genes (including regenerated Antenna IR, and Divergent IR), especially for Divergent IR are more variable, have a simpler gene structure (intron loss phenomenon) and larger ω values, and lack specific functional sites. These IR genes have no other domains except for Antenna IRs that only have the Lig_Chan domain. CONCLUSIONS: This study provides a comprehensive information framework for iGluR genes in An. sinensis, and generated the classification of iGluRs by feature and bioinformatics analyses. The work lays the foundation for further functional study of these genes.

Genome-wide and expression-profiling analyses suggest the main cytochrome P450 genes related to pyrethroid resistance in the malaria vector, Anopheles sinensis (Diptera Culicidae).

BACKGROUND: Anopheles sinensis is one of the major malaria vectors. However, pyrethroid resistance in An. sinensis is threatening malaria control. Cytochrome P450-mediated detoxification is an important pyrethroid resistance mechanism that has been unexplored in An. sinensis. In this study, we performed a comprehensive analysis of the An. sinensis P450 gene superfamily with special attention to their role in pyrethroid resistance using bioinformatics and molecular approaches. RESULTS: Our data revealed the presence of 112 individual P450 genes in An. sinensis, which were classified into four major clans (mitochondrial, CYP2, CYP3 and CYP4), 18 families and 50 subfamilies. Sixty-seven genes formed nine gene clusters, and genes within the same cluster and the same gene family had a similar gene structure. Phylogenetic analysis showed that most of An. sinensis P450s (82/112) had very close 1: 1 orthology with Anopheles gambiae P450s. Five genes (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 and AsCYP306A1) were significantly upregulated in three pyrethroid-resistant populations in both RNA-seq and RT-qPCR analyses, suggesting that they could be the most important P450 genes involved in pyrethroid resistance in An. sinensis. CONCLUSION: Our study provides insight on the diversity of An. sinensis P450 superfamily and basis for further elucidating pyrethroid resistance mechanism in this mosquito species. © 2018 Society of Chemical Industry.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Suppression of Laccase 2 severely impairs cuticle tanning and pathogen resistance during the pupal metamorphosis of Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. METHODS: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. RESULTS: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. CONCLUSIONS: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies.

Insight into the possible mechanism of the summer diapause of Delia antiqua (Diptera: Anthomyiidae) through digital gene expression analysis.

The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause (SD), high-throughput RNA-Seq data were generated from non-diapause (ND) and SD (initial, maintenance and quiescence phase) pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization of transcriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

The complete mitochondrial genome of Anopheles minimus (Diptera: Culicidae) and the phylogenetics of known Anopheles mitogenomes.

Anopheles minimus is an important vector of human malaria in southern China and Southeast Asia. The phylogenetics of mosquitoes has not been well resolved, and the mitochondrial genome (mtgenome) has proven to be an important marker in the study of evolutionary biology. In this study, the complete mtgenome of An. minimus was sequenced for the first time. It is 15 395 bp long and encodes 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and a non-coding region. The gene organization is consistent with those of known Anopheles mtgenomes. The mtgenome performs a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew. All 13 PCGs prefer to use the codon UUA (Leu), ATN as initiation codon but cytochrome-oxidase subunit 1 (COI) and ND5, with TCG and GTG, and TAA as termination codon, but COI, COII, COIII and ND4, all with the incomplete T. tRNAs have the typical clover-leaf structure, but tRNA(Ser(AGN)) is consistent with known Anopheles mtgenomes. The control region includes a conserved T-stretch and a (TA)n stretch, and has the highest A+T content at 93.1%. The phylogenetics of An. minimus with 18 other Anopheles species was constructed by maximum likelihood and Bayesian inference, based on concatenated PCG sequences. The subgenera, Cellia and Anopheles, and Nyssorhynchus and Kerteszia have mutually close relationships, respectively. The Punctulatus group and Leucosphyrus group of Neomyzomyia Series, and the Albitarsis group of Albitarsis Series were suggested to be monophyletic. The monophyletic status of the subgenera, Cellia, Anopheles, Nyssorhynchus and Kerteszia need to be further elucidated.

The Complete Mitochondrial Genome of Delia antiqua and Its Implications in Dipteran Phylogenetics.

Delia antiqua is a major underground agricultural pest widely distributed in Asia, Europe and North America. In this study, we sequenced and annotated the complete mitochondrial genome of this species, which is the first report of complete mitochondrial genome in the family Anthomyiidae. This genome is a double-stranded circular molecule with a length of 16,141 bp and an A+T content of 78.5%. It contains 37 genes (13 protein-coding genes, 22 tRNAs and 2 rRNAs) and a non-coding A+T rich region or control region. The mitochondrial genome of Delia antiqua presents a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew. All of the 13 protein-coding genes use ATN as an initiation codon except for the COI gene that starts with ATCA. Most protein-coding genes have complete termination codons but COII and ND5 that have the incomplete termination codon T. This bias is reflected in both codon usage and amino acid composition. The protein-coding genes in the D. antiqua mitochondrial genome prefer to use the codon UUA (Leu). All of the tRNAs have the typical clover-leaf structure, except for tRNASer(AGN) that does not contain the dihydrouridine (DHU) arm like in many other insects. There are 7 mismatches with U-U in the tRNAs. The location and structure of the two rRNAs are conservative and stable when compared with other insects. The control region between 12S rRNA and tRNAIle has the highest A+T content of 93.7% in the D. antiqua mitochondrial genome. The control region includes three kinds of special regions, two highly conserved poly-T stretches, a (TA)n stretch and several G(A)nT structures considered important elements related to replication and transcription. The nucleotide sequences of 13 protein-coding genes are used to construct the phylogenetics of 26 representative Dipteran species. Both maximum likelihood and Bayesian inference analyses suggest a closer relationship of D. antiqua in Anthomyiidae with Calliphoridae, Calliphoridae is a paraphyly, and both Oestroidea and Muscoidea are polyphyletic.

Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua (Diptera: Anthomyiidae).

Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09µg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36µg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions.

Identification and characterization of a gene encoding a UBX domain-containing protein in the migratory locust, Locusta migratoria manilensis.

Ubiquitin regulatory X (UBX) domain-containing proteins are believed to function as cofactors for p97/CDC48, an adenosine triphosphatase shown to be involved in multiple cellular processes. In the present study, a full-length complementary DNA (cDNA) of UBX domain-containing gene, termed LmUBX1, was cloned from Locusta migratoria manilensis and characterized, using random amplification of cDNA ends polymerase chain reaction (RACE PCR), sequence analysis and quantitative real-time PCR. LmUBX1, 1 600 bp in length, is predicted to encode a 446-amino acid protein with a predicted molecular weight of 51.18 kDa that contains a central PUB domain and a carboxy-terminal UBX domain. Homology analysis revealed that LmUBX1 has higher similarity to the known UBX domain-containing proteins from insects than from other species. Moreover, based on sequence characteristics and phylogenetic relationships, it is suggested that LmUBX1 can be classified into the UBXD1 subfamily. Expression analysis founded that LmUBX1 exhibited significant expression variations at different developmental stages and in different tissues, suggesting that the expression of LmUBX1 was highly regulated. Interestingly, its messenger RNA transcript was more abundant in ovary and testis than in other tissues examined, suggesting that it may have more important roles in the reproductive system. In addition, LmUBX1 was differentially expressed in gregarious and solitary locusts and was significantly up-regulated in third and fifth instars of gregarious locusts, implying that LmUBX1 was also likely involved in the phase polyphenisms in L. migratoria manilensis. To our knowledge, this is the first report of cloning of a full-length cDNA of UBX domain-containing gene from L. migratoria manilensis.

Differential gene expression between summer and winter diapause pupae of the onion maggot Delia antiqua, detected by suppressive subtractive hybridization.

To gain a better understanding of the molecular mechanisms regulating pupal diapause of the onion maggot Delia antiqua, PCR-based suppressive subtractive hybridization was performed to identify genes involved in summer and/or winter diapause. A total of 209 unique sequences were obtained including 89 in forward library for winter diapausing pupae and 120 in the reverse library for summer diapausing pupae. 76.4% (68/89) and 68.3% (82/120) unique sequences had significant hits to non-redundant proteins database. Gene functional annotation showed these non-redundant sequences are involved in stress response and innate immunity, metabolism and energy, information processing and regulation, binding, food storage, morphogenesis and development, cell skeleton and cycle, protein synthesis and folding. Approximately 28.2% (59/209) transcripts showed no significant similarity to any other sequence in the public databases, probably representing unique genes of the onion maggot. Semi-quantitative RT-PCR revealed that the relative expression levels of 18 genes were comparable between summer and winter diapause. This study elucidates the temporal expression of diapause-related genes in onion maggot, also provides new insights into the differences in the physiological changes in summer and winter pupae. Functional characterization of some candidate genes will further enhance the understanding of the generating, maintaining, and breaking mechanism of diapause.