Hong-Bo Jiang

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.

Functional Phylogenetics Reveals Contributions of Pleiotropic Peptide Action to Ligand-Receptor Coevolution

The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of functions, a phenomenon known as pleiotropy. Here we examine whether pleiotropic actions of peptide genes might influence ligand-receptor coevolution. Four test groups of neuropeptides characterized by conserved C-terminal amino acid sequence motifs and their cognate receptors were examined in the red flour beetle (Tribolium castaneum): 1) cardioacceleratory peptide 2b (CAPA); CAPAr, 2) pyrokinin/diapause hormone (PK1/DH); PKr-A, -B, 3) pyrokinin/pheromone biosynthesis activating hormone (PK2/PBAN); PKr-C, and 4) ecdysis triggering hormone (ETH); ETHr-b. Ligand-receptor specificities were established through heterologous expression of receptors in cell-based assays for 9 endogenous ligands. Based on ligand-receptor specificity analysis, we found positive pleiotropism exhibited by ETH on ETHR-b and CAPAr, whereas PK1/DH and CAPA are more highly selective for their respective authentic receptors than would be predicted by phylogenetic analysis. Disparities between evolutionary trees deduced from receptor sequences vs. functional ligand-receptor specificities lead to the conclusion that pleiotropy exhibited by peptide genes influences ligand-receptor coevolution.

Molecular characterization of two novel deltamethrin-inducible P450 genes from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

Two novel P450 genes, CYP6CE1 and CYP6CE2 (GenBank accession number: EF421245 and EF421246), were cloned and characterized from psocid, Liposcelis bostrychophila. CYP6CE1 and CYP6CE2 contain open reading frames of 1,581 and 1,563 nucleotides that encode 527 and 521 amino acid residues, respectively. The putative proteins of CYP6CE1 and CYP6CE2 show predicted molecular weights of 60.76 and 59.83 kDa with a theoretical pI of 8.58 and 8.78, respectively. CYP6CE1 and CYP6CE2 share 74% identity with each other, and the deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Both CYP6CE1 and CYP6CE2 share the closest identities with Hodotermopsis sjoestedti CYP6AM1 at 48% among the published sequences. Phylogenetic analysis showed a closer relationship of CYP6CE1 and CYP6CE2 with CYP6 members of other insects than with those from other families. Quantitative real-time RT-PCR showed that both CYP6CE1 and CYP6CE2 are expressed at all developmental stages tested. Interestingly, CYP6CE2 transcripts decreased from the highest in 1st nymph to the lowest in adults, which seemed to suggest developmental regulation. However, neither CYP6CE1 nor CYP6CE2 were stage specific. The CYP6CE1 and CYP6CE2 transcripts in adults increased significantly after deltamethrin exposure. Recombinant protein expression studies are needed to determine the real functions of these proteins.

Molecular Cloning and Sequence Analysis of a Novel P450 Gene Encoding CYP345D3 from the Red Flour Beetle, Tribolium castaneum

Abstract A novel cDNA clone encoding a cytochrome P450 gene has been isolated from the insecticide-susceptible strain of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The nucleotide sequence of the clone, designated CYP345D3, was determined. The cDNA is 1554 bp in length and contains an open reading frame from base pairs 32 to 1513, encoding a protein of 493 amino acid residues and a predicted molecular weight of 57466 Daltons. The putative protein contains the classic heme-binding sequence motif FxxGxxxCxG (residues 430–439) conserved among all P450 enzymes as well as other characteristic motifs of the cytochrome P450s. Comparison of the deduced amino acid sequence with other CYP members shows that CYP345D3 shares 91% identity with the previously published sequence of CYP345D1 from the T. castaneum genome project and the nucleotide sequence identity between them is less than 80%. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated close phylogenetic relationship of CYP345D3 with CYP6 of other insects than those from mammals and amore distant relationship to P450 from other families. CYP345D3 was submitted to GenBank, accession number EU008544.

Molecular characterization of three heat shock protein 70 genes and their expression profiles under thermal stress in the citrus red mite

Three heat shock protein 70 family transcripts, named PcHsp70-1, PcHsp70-2 and PcHsp70-3, were isolated from the citrus red mite, Panonychus citri. PcHsp70-1, PcHsp70-2, and PcHsp70-3 contained an open reading frame of 1977, 1968, and 2028 nucleotides that encoded 658, 655 and 675 amino acid residues, respectively. Comparison of deduced amino acid sequences of PcHsp70-1 and PcHsp70-2 showed 86.34% identity, while the amino acid sequence of PcHsp70-3 was only 57.39 and 58.75% identical to that of PcHsp70-1 and PcHsp70-2, respectively. Sequences and phylogenetic analyses suggested that PcHsp70-1 and PcHsp70-2 were cytosolic Hsps, whereas PcHsp70-3 was located in ER (endoplasmic reticulum). To accurately validate mRNA expression profiles of the three Hsp70s under thermal stress conditions, seven housekeeping genes were evaluated. Alpha-tubulin and RpII were selected as optimal endogenous references for cold shock and heat shock conditions, respectively. Real-time quantitative RT-PCR revealed that only the mRNA expression of PcHsp70-2 was up-regulated under heat shocks, and all of the three Hsp70s were constitutively expressed under cold shocks. The results suggest that the three Hsp70s were more critical to coping with heat than cold shocks.

A Role of Corazonin Receptor in Larval-Pupal Transition and Pupariation in the Oriental Fruit Fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Corazonin (Crz) is a neuropeptide hormone, but also a neuropeptide modulator that is internally released within the CNS, and it has a widespread distribution in insects with diverse physiological functions. Here, we identified and cloned the cDNAs of Bactrocera dorsalis that encode Crz and its receptor CrzR. Mature BdCrz has 11 residues with a unique Ser11 substitution (instead of the typical Asn) and a His in the evolutionary variable position 7. The BdCrzR cDNA encodes a putative protein of 608 amino acids with 7 putative transmembrane domains, typical for the structure of G-protein-coupled receptors. When expressed in Chinese hamster ovary (CHO) cells, the BdCrzR exhibited a high sensitivity and selectivity for Crz (EC50 ≈ 52.5 nM). With qPCR, the developmental stage and tissue-specific expression profiles in B. dorsalis demonstrated that both BdCrz and BdCrzR were highly expressed in the larval stage, and BdCrzR peaked in 2-day-old 3rd-instar larvae, suggesting that the BdCrzR may play an important role in the larval-pupal transition behavior. Immunochemical localization confirmed the production of Crz in the central nervous system (CNS), specifically by a group of three neurons in the dorso-lateral protocerebrum and eight pairs of lateral neurons in the ventral nerve cord. qPCR analysis located the BdCrzR in both the CNS and epitracheal gland, containing the Inka cells. Importantly, dsRNA-BdCrzR-mediated gene-silencing caused a delay in larval-pupal transition and pupariation, and this phenomenon agreed with a delayed expression of tyrosine hydroxylase and dopa-decarboxylase genes. We speculate that CrzR-silencing blocked dopamine synthesis, resulting in the inhibition of pupariation and cuticular melanization. Finally, injection of Crz in head-ligated larvae could rescue the effects. These findings provide a new insight into the roles of Crz signaling pathway components in B. dorsalis and support an important role of CrzR in larval-pupal transition and pupariation behavior.

Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lbalpha1 and Lbalpha8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full-length cDNAs of Lbalpha1 (GenBank accession number: EU871527) and Lbalpha8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608 bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lbalpha1 had the closest phylogenetic relationship to Agamalpha1 from Anopheles gambiae and Amelalpha1 from Apis mellifera, and Lbalpha8 shares the highest identity with Agamalpha8 from An. gambiae and Amelalpha8 from A. mellifera. Quantitative real-time PCR analysis showed that Lbalpha1 was expressed 2.03-6.54-fold higher than Lbalpha8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lbalpha1 and Lbalpha8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lbalpha1, whereas there was a descending expression pattern for Lbalpha8 in the 1st through the 4th nymphal stadia.

The short neuropeptide F modulates olfactory sensitivity of Bactrocera dorsalis upon starvation

The insect short neuropeptide F (sNPF) family has been shown to modulate diverse physiological processes, such as feeding, appetitive olfactory behavior, locomotion, sleep homeostasis and hormone release. In this study, we identified the sNPF (BdsNPF) and its receptor (BdsNPFR) in an important agricultural pest, the oriental fruit fly Bactrocera dorsalis (Hendel). Afterwards, the receptor cDNA was functionally expressed in Chinese hamster ovary cell lines. Activation of BdsNPFR by sNPF peptides caused an increase in intracellular calcium ions, with a 50% effective concentration values at the nanomolar level. As indicated by qPCR, the BdsNPF and BdsNPFR transcripts were mainly detected in the central nervous system and antennae, and they showed significantly starvation-induced expression patterns. Furthermore, we found that the starved flies had an increased electroantennogram response compared to the normally fed flies. However, this enhanced olfactory sensitivity was reversed when we decreased the expression of BdsNPF by double-stranded RNA injection in adults. We concluded that sNPF plays an important role in modulating the olfactory sensitivity of B. dorsalis upon starvation. Our results will facilitate the understanding of the regulation of early olfactory processing in B. dorsalis.

The Tribolium castaneum cell line TcA: a new tool kit for cell biology

The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

Differential expression of genes in the alate and apterous morphs of the brown citrus aphid, Toxoptera citricida

Winged and wingless morphs in insects represent a trade-off between dispersal ability and reproduction. We studied key genes associated with apterous and alate morphs in Toxoptera citricida (Kirkaldy) using RNAseq, digital gene expression (DGE) profiling, and RNA interference. The de novo assembly of the transcriptome was obtained through Illumina short-read sequencing technology. A total of 44,199 unigenes were generated and 27,640 were annotated. The transcriptomic differences between alate and apterous adults indicated that 279 unigenes were highly expressed in alate adults, whereas 5,470 were expressed at low levels. Expression patterns of the top 10 highly expressed genes in alate adults agreed with wing bud development trends. Silencing of the lipid synthesis and degradation gene (3-ketoacyl-CoA thiolase, mitochondrial-like) and glycogen genes (Phosphoenolpyruvate carboxykinase [GTP]-like and Glycogen phosphorylase-like isoform 2) resulted in underdeveloped wings. This suggests that both lipid and glycogen metabolism provide energy for aphid wing development. The large number of sequences and expression data produced from the transcriptome and DGE sequencing, respectively, increases our understanding of wing development mechanisms.