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.

Electroporation-mediated nucleic acid delivery during non-embryonic stages for gene-function analysis in Anopheles sinensis.

The delivery of exogenous nucleic acids to eggs or non-embryonic individuals by microinjection is a vital reverse genetics technique used to determine gene function in mosquitoes. However, DNA delivery to eggs is complex and time-consuming, and conventional, non-embryonic-injection techniques may result in unobvious phenotypes caused by insufficient absorption of nucleic acid fragments by cells at target body parts or tissues. In this study, we developed a set of electroporation-mediated non-embryonic microinjections for the delivery of exogenous nucleic acids in Anopheles sinensis. Gene silencing using this method led to down-regulation of target gene expression (AsCPR128) by 77% in targeted body parts, compared with only 10% in non-targeted body parts, thus increasing the defect-phenotype rate in the target area by 5.3-fold, compared with non-shock injected controls. Electroporation-mediated somatic transgenesis resulted in stable phenotypic characteristics of the reporter gene at the shocked body parts during the pupal-adult stages in about 69% of individuals. Furthermore, injecting plasmid DNA near the ovaries of female mosquitoes after a blood meal followed by electric shock produced three germline G1 transgenic lines, with a transformation rate of about 11.1% (calculated from ovulatory G0 females). Among the positive G1 lines, 42%, 40%, and 31% of individuals emitted red fluorescence in the larval stage. When the red fluorescent larvae developed into adults, green fluorescence was emitted from the ovaries of the females upon feeding. These results suggest that electroporation-mediated non-embryonic microinjection can be an efficient, rapid, and simple technique for analyzing gene function in non-model mosquitoes or other small insects.

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.

Comparative genomics of chemosensory protein genes (CSPs) in twenty-two mosquito species (Diptera: Culicidae): Identification, characterization, and evolution.

Chemosensory proteins (CSP) are soluble carrier proteins that may function in odorant reception in insects. CSPs have not been thoroughly studied at whole-genome level, despite the availability of insect genomes. Here, we identified/reidentified 283 CSP genes in the genomes of 22 mosquitoes. All 283 CSP genes possess a highly conserved OS-D domain. We comprehensively analyzed these CSP genes and determined their conserved domains, structure, genomic distribution, phylogeny, and evolutionary patterns. We found an average of seven CSP genes in each of 19 Anopheles genomes, 27 CSP genes in Cx. quinquefasciatus, 43 in Ae. aegypti, and 83 in Ae. albopictus. The Anopheles CSP genes had a simple genomic organization with a relatively consistent gene distribution, while most of the Culicinae CSP genes were distributed in clusters on the scaffolds. Our phylogenetic analysis clustered the CSPs into two major groups: CSP1-8 and CSE1-3. The CSP1-8 groups were all monophyletic with good bootstrap support. The CSE1-3 groups were an expansion of the CSP family of genes specific to the three Culicinae species. The Ka/Ks ratios indicated that the CSP genes had been subject to purifying selection with relatively slow evolution. Our results provide a comprehensive framework for the study of the CSP gene family in these 22 mosquito species, laying a foundation for future work on CSP function in the detection of chemical cues in the surrounding environment.

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 identification and characterization of odorant-binding protein (OBP) genes in the malaria vector Anopheles sinensis (Diptera: Culicidae).

Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins (OBPs) may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBP, and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motif's conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes (OBP23-28, OBP63-64) might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis.

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.

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.

Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi.

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.