Wen-Jia Yang

Transcriptome Analysis of the Oriental Fruit Fly (Bactrocera dorsalis)

Background The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most economically important pests in the world, causing serious damage to fruit production. However, lack of genetic information on this organism is an obstacle to understanding the mechanisms behind its development and its ability to resist insecticides. Analysis of the B. dorsalis transcriptome and its expression profile data is essential to extending the genetic information resources on this species, providing a shortcut that will support studies on B. dorsalis. Methodology/Principal Findings We performed de novo assembly of a transcriptome using short read sequencing technology (Illumina). The results generated 484,628 contigs, 70,640 scaffolds, and 49,804 unigenes. Of those unigenes, 27,455 (55.13%) matched known proteins in the NCBI database, as determined by BLAST search. Clusters of orthologous groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to better understand the functions of these unigenes. Genes related to insecticide resistance were analyzed in additional detail. Digital gene expression (DGE) libraries showed differences in gene expression profiles at different developmental stages (eggs, third-instar larvae, pupae, and adults). To confirm the DGE results, the expression profiles of six randomly selected genes were analyzed. Conclusion/Significance This transcriptome greatly improves our genetic understanding of B. dorsalis and makes a huge number of gene sequences available for further study, including both genes of known importance and genes of unknown function. The DGE data provide comprehensive insight into gene expression profiles at different developmental stages. This facilitates the study of the role of each gene in the developmental process and in insecticide resistance.

Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis.

Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5′- and 3′-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E.

De novo cloning and annotation of genes associated with immunity, detoxification and energy metabolism from the fat body of the oriental fruit fly, Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis, is a destructive pest in tropical and subtropical areas. In this study, we performed transcriptome-wide analysis of the fat body of B. dorsalis and obtained more than 59 million sequencing reads, which were assembled into 27,787 unigenes with an average length of 591 bp. Among them, 17,442 (62.8%) unigenes matched known proteins in the NCBI database. The assembled sequences were further annotated with gene ontology, cluster of orthologous group terms, and Kyoto encyclopedia of genes and genomes. In depth analysis was performed to identify genes putatively involved in immunity, detoxification, and energy metabolism. Many new genes were identified including serpins, peptidoglycan recognition proteins and defensins, which were potentially linked to immune defense. Many detoxification genes were identified, including cytochrome P450s, glutathione S-transferases and ATP-binding cassette (ABC) transporters. Many new transcripts possibly involved in energy metabolism, including fatty acid desaturases, lipases, alpha amylases, and trehalose-6-phosphate synthases, were identified. Moreover, we randomly selected some genes to examine their expression patterns in different tissues by quantitative real-time PCR, which indicated that some genes exhibited fat body-specific expression in B. dorsalis. The identification of a numerous transcripts in the fat body of B. dorsalis laid the foundation for future studies on the functions of these genes.

Transcriptome profiling of the testis reveals genes involved in spermatogenesis and marker discovery in the oriental fruit fly, Bactrocera dorsalis

The testis is a highly specialized tissue that plays a vital role in ensuring fertility by producing spermatozoa, which are transferred to the female during mating. Spermatogenesis is a complex process, resulting in the production of mature sperm, and involves significant structural and biochemical changes in the seminiferous epithelium of the adult testis. The identification of genes involved in spermatogenesis of Bactrocera dorsalis (Hendel) is critical for a better understanding of its reproductive development. In this study, we constructed a cDNA library of testes from male B. dorsalis adults at different ages, and performed de novo transcriptome sequencing to produce a comprehensive transcript data set, using Illumina sequencing technology. The analysis yielded 52 016 732 clean reads, including a total of 4.65 Gb of nucleotides. These reads were assembled into 47 677 contigs (average 443 bp) and then clustered into 30 516 unigenes (average 756 bp). Based on BLAST hits with known proteins in different databases, 20 921 unigenes were annotated with a cut‐off E‐value of 10−5. The transcriptome sequences were further annotated using the Clusters of Orthologous Groups, Gene Orthology and the Kyoto Encyclopedia of Genes and Genomes databases. Functional genes involved in spermatogenesis were analysed, including cell cycle proteins, metalloproteins, actin, and ubiquitin and antihyperthermia proteins. Several testis‐specific genes were also identified. The transcripts database will help us to understand the molecular mechanisms underlying spermatogenesis in B. dorsalis. Furthermore, 2913 simple sequence repeats and 151 431 single nucleotide polymorphisms were identified, which will be useful for investigating the genetic diversity of B. dorsalis in the future.

Molecular Characterization of the cDNA Encoding Ecdysone Receptor Isoform B1 and Its Expression in the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

ABSTRACT The 20-hydroxyecdysone (20E) plays a critical role in a series of biological processes, via the ecdysone receptor/USP heterodimeric complex in arthropod. In order to clarify the regulatory mechanism of 20E, we characterized a full-length cDNA encoding a putative ecdysone receptor isoform B1 and named it as BdEcR-B1 in the oriental fruit fly, Bactrocera dorsalis. The BdEcR-B1 gene was 3,111 bp long, with an open reading frame of 2,304 bp, which encoded 767 amino acids with a predicated molecular weight of 83.3 kDa and an isoelectric point of 6.74. Alignment analysis revealed that the deduced protein sequence had 80% identity to EcR-B1 isoforms of various dipteran species, indicating that this gene was highly conserved during the evolution of the Diptera. Phylogenetic analysis suggested that BdEcR-B1 was orthologous to the EcR-B1 proteins identified in other insect species. Quantitative real-time PCR showed that BdEcR-B1 was expressed at all tested developmental stages, and the expression of BdEcR-B1 reached a significantly higher level just prior to the larval-pupa molt stage and in 4-d old pupa than those in other stages. Moreover, the BdEcR-B1 gene much more strongly expressed in gut and Malpighian tubule than those in the trachea and fat body, which suggests that this gene may be involved in tissue-specific function during larval development.

Transcriptional Regulation of a Chitinase Gene by 20-Hydroxyecdysone and Starvation in the Oriental Fruit Fly, Bactrocera dorsalis

Insect chitinases are hydrolytic enzymes that are required for the degradation of glycosidic bonds of chitin. In this study, we identified and characterized a full-length cDNA of the chitinase gene (BdCht2) in the oriental fruit fly, Bactrocera dorsalis. The cDNA contains an open reading frame (ORF) of 1449 bp that encodes 483 amino acid residues and 126- and 296-bp non-coding regions at the 5′- and 3′-ends, respectively. The BdCht2 genome has four exons and three introns. The predicted molecular mass of the deduced BdCht2 is approximately 54.3 kDa, with an isoelectric point of 5.97. The 977 bp 5′ flanking region was identified and the transcription factor binding sites were predicted. Bioinformatic analyses showed that the deduced amino acid sequence of BdCht2 had 34%–66% identity to that of chitinases identified in other insect species. Quantitative real-time PCR (qPCR) analyses indicated that BdCht2 was mainly expressed during the larval-pupal and pupal-adult transitions. The tissue-specific expression showed that the highest expression was in the integument, followed by the fat body and other tissues. Moreover, the expression of BdCht2 was upregulated significantly upon 20-hydroxyecdysone (20E) at different dose injections after 8 h compared to that of the control. Starvation also increased the expression of BdCht2 in the third-instar larvae and was suppressed again by re-feeding the insects. These results suggest that BdCht2 plays an important role in the molting process of B. dorsalis larvae and can be regulated by 20E.

Exposure to diflubenzuron results in an up-regulation of a chitin synthase 1 gene in citrus red mite, Panonychus citri (Acari: Tetranychidae).

Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.

Two Chitin Biosynthesis Pathway Genes in Bactrocera dorsalis (Diptera: Tephritidae): Molecular Characteristics, Expression Patterns, and Roles in Larval—Pupal Transition

ABSTRACT Glucose-6-phosphate isomerase (G6PI) and UDP-N-acetylglucosamine pyrophosphorylase (UAP), two key components in the chitin biosynthesis pathway, are critical for insect growth and metamorphosis. In this study, we identified the genes BdG6PI and BdUAP from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frames (ORFs) of BdG6PI (1,491 bp) and BdUAP (1,677 bp) encoded 496 and 558 amino acid residues, respectively. Multiple sequence alignments showed that BdG6PI and BdUAP had high amino acid sequence identity with other insect homologues. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated that BdG6PI was mainly expressed in the early stages of third-instar larvae and adults, while significantly higher expression of BdUAP was observed in adults. Both transcripts were expressed highly in the Malpighian tubules, but only slightly in the tracheae. The expression of both BdG6PI and BdUAP was significantly up-regulated by 20-hydroxyecdysone exposure and down-regulated by starvation. Moreover, injection of double-stranded RNAs of BdG6PI and BdUAP into third-instar larvae significantly reduced the corresponding gene expressions. Additionally, silencing of BdUAP resulted in 65% death and abnormal phenotypes of larvae, while silencing of BdG6PI had a slight effect on insect molting. These findings provide some data on the roles of BdG6PI and BdUAP in B. dorsalis and demonstrate the potential role for BdUAP in larval—pupal transition.

The Essential Role of Vitellogenin Receptor in Ovary Development and Vitellogenin Uptake in Bactrocera dorsalis (Hendel)

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.