Functional analysis of five trypsin-like protease genes in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Insect midgut proteases catalyze the release of free amino acids from dietary proteins and are essential for insect normal development. To date, digestive proteases as potential candidates have made great progress in pest control. To clarify the function of trypsin-like protease genes in the digestive system of Bactrocera dorsalis, a serious pest of a wide range of tropical and subtropical fruit and vegetable crops, five trypsin genes (BdTry1, BdTry2, BdTry3, BdTry4 and BdTry5) were identified from transcriptome dataset, and the effects of feeding condition on their expression levels were examined subsequently. RNA interference (RNAi) was applied to further explore their function on the growth of B. dorsalis. The results showed that all the BdTrys in starving midgut expressed at a minimal level but up-regulated upon feeding (except BdTry3). Besides, RNAi by feeding dsRNAs to larvae proved to be an effective method to cause gene silencing and the mixed dsRNAs of the five BdTrys slowed larvae growth of B. dorsalis. The current data suggest that trypsin genes are actively involved in digestion process of B. dorsalis larvae and thereafter play crucial roles in their development.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction.

In insects, the accessory gland proteins (Acps) secreted by male accessory glands (MAGs) account for the majority of seminal fluids proteins. Mixed with sperm, they are transferred to the female at mating and so impact reproduction. In this project, we identified 2,927 proteins in the MAG secretions of the oriental fruit fly Bactrocera dorsalis, an important agricultural pest worldwide, using LC-MS analysis, and all sequences containing open reading frames were analyzed using signalP. In total, 90 Acps were identified. About one third (26) of these 90 Acps had a specific functional description, while the other two thirds (64) had no functional description including dozens of new classes of proteins. Hence, several of these novel Acps were abundant in the MAG secretions, and we confirmed their MAG-specific expression by qPCR. Finally and interestingly, one of these novel proteins was functionally predicted as juvenile hormone-binding protein, suggesting the impact of Acps with reproductive events in the female. Our results will aid in the development of an experimental method to identify Acps in insects, and in turn this information with new Acps in B. dorsalis will pave the way of further exploration their function in reproduction and potential development as new insecticide targets.

Comparative proteomic analysis of Bactrocera dorsalis (Hendel) in response to thermal stress.

Temperature is one of the most important environmental variables affecting growth, reproduction and distribution of insects. The rise of comparative proteomics provides a powerful tool to explore the response in proteins to thermal stress. As an important worldwide pest, the oriental fruit fly Bactrocera dorsalis causes severe economic losses to crops. To understand the response of B. dorsalis to thermal stress, we performed a comparative proteome analysis of this insect after exposure to extreme low and high temperatures using two-dimensional electrophoresis. Among the separated proteins, 51 diverse protein spots were present differently in response to extreme temperatures. Using tandem mass spectrometry sequencing analysis 39 proteins were successfully identified, which included 13 oxidoreductases, 10 binding proteins, 5 transferases, and 2 each of lyases, isomerases, ligases, and developmental proteins. Subsequently, the expression of these protein transcripts was studied by RT-qPCR to validate the proteomic results. In conclusion, this study provides a first look into the thermal stress response of B. dorsalis at the protein level, and thus it paves the way for further functional studies in the physiological mechanism related to thermal stress.

Multiple glutathione S-transferase genes: identification and expression in oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel), is widely distributed in Asia-Pacific regions, where it is a serious pest of a wide range of tropical and subtropical fruit and vegetable crops. In this study, 17 cDNA encoding glutathione S-transferases (GSTs) in B. dorsalis were sequenced and characterised. RESULTS: Phylogenetic analysis revealed that 16 GSTs belonged to five different cytosolic classes, including four in delta, eight in epsilon, two in omega, one in theta, and one in zeta. The remaining GST (BdGSTu1) was unclassified. RT-qPCR assay showed that the relative expression levels of five GST genes were significantly higher in larval stages than in adulthood. Tissue-specific expression analysis found that BdGSTe3, BdGSTe9 and BdGSTd5 were expressed highly in the midgut, BdGSTe4, BdGSTe6, BdGSTd6 and BdGSTz2 were higher in the fat body, and six GSTs were higher in Malpighian tubules. RT-qPCR confirmed that the expressions of nine GST genes were increased by malathion exposure at various times and doses, while BdGSTe4, BdGSTe9 and BdGSTt1 were increased by ß-cypermethrin exposure. CONCLUSION: The increases in GST gene expression levels after malathion and ß-cypermethrin exposure in B. dorsalis might increase the ability of this species to detoxify other insecticides and xenobiotics.

Involvement of superoxide dismutase in oxidative stress in the oriental fruit fly, Bactrocera dorsalis: molecular cloning and expression profiles.

BACKGROUND: Bactrocera dorsalis, one of the most economically important fruit fly pests in East Asia, is well adapted to various environmental conditions. Pesticides, pathogens and other stresses can cause oxidative damage in most organisms. The superoxide dismutase (SOD) family contains some of the most important enzymes in the antioxidant protection system of the fruit fly and other organisms. RESULTS: Four full-length cDNA sequences encoding one MnSOD (BdSOD2-1) and three Cu-ZnSODs (BdSOD1-1, BdSOD1-2 and BdSOD1-3) were cloned. The expression profiles of these four genes under different stresses showed them to be involved in response to detrimental conditions including heavy metals, pesticides, extreme temperatures and lipopolysaccharide (LPS) stresses. More specifically, the expression levels of these genes were found to be depressed in the presence of copper, zinc and manganese. The expression of all four SOD genes increased upon exposure to lead, cadmium, low temperature (0 °C) and LPS stresses. Only BdSOD1-3 transcription increased significantly at high temperature (40 °C) exposure. The expressions levels of BdSOD1-2 and BdSOD1-3 increased significantly in the presence of ß-cypermethrin and malathion, but only the expression of BdSOD2-1 increased in the presence of avermectin treatment. CONCLUSION: These different expression profiles suggest that the four BdSODs play different roles and respond to different oxidative stresses in B. dorsalis. Some BdSODs undergo specific reaction in the response to specific oxidative stresses.

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.

Purification and biochemical characterization of glutathione S-transferases from four field populations of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

Glutathione S-transferases (GSTs) are a group of detoxification enzymes that catalyze the nucleophilic addition of glutathione to a wide variety of endogenous and xenobiotic compounds. In this study, GSTs were purified from four field populations of Bactrocera dorsalis with different insecticide susceptibilities by glutathione-agarose affinity chromatography. The populations were collected from Dongguan (DG) and Guangzhou (GZ) of the Guangdong Province, Haikou of the Hainan province (HN), and Kunming of the Yunnan province (YN), China. Differences in GST characteristics among the four populations were studied using purified enzyme samples through comparative SDS-PAGE, kinetic, and inhibition experiments. The specific activities of the purified enzymes were similar, but the purification yield of the GZ population (31.54%) was the lowest. SDS-PAGE analysis showed only one band at approximately 23 kDa for these four populations. Kinetic analyses showed that the affinities of the purified GSTs from the GZ and YN populations for 1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those of GSTs from the other two populations, whereas the HN population had the highest catalytic capability in terms of V(max) value. The optimum temperature for CDNB conjugation was 37 °C and the optimum pH was 7.5 in all four populations. Inhibition kinetics showed that ethacrynic acid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfalein, and ß-cypermethrin had excellent inhibitory effects on GSTs in the four populations of B. dorsalis, but the low inhibitory effects of malathion and avermectin did not differ between populations. These results suggest that GSTs may have a role in detoxification of ß-cypermethrin in B. dorsalis.

Influence of continuous high temperature conditions on Wolbachia infection frequency and the fitness of Liposcelis tricolor (Psocoptera: Liposcelididae).

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in arthropods, and their appeal to researchers' interest is growing. In this study, the influence of continuous high temperature conditions on Wolbachia infection frequency and the fitness of Liposcelis tricolor (Psocoptera: Liposcelididae) was studied in a laboratory. The results showed that the Wolbachia infection frequency was gradually decreased when L. tricolor was reared at 33 degrees C; after six generations of treatment, no Wolbachia wsp gene product was detected, suggesting that the Wolbachia infection was completely eliminated. The combined immature development periods and survival rates of L. tricolor did not vary significantly among six generations; however, the female longevities and fecundities dramatically declined from F(1) through F(6), resulting in decreasing values of population intrinsic rate of increase (r(m)). Using r(m) values, the fitness for F(2), F(3), F(4), F(5), and F(6) relative to F(1) was calculated as 0.995, 0.953, 0.811, 0.700, and 0.552, respectively. We realize that it is not possible to distinguish between the effects of high temperature may have on the metabolic processes of the psocids and the effects of temperature-induced reduction in bacterial infection frequencies. Our study confirms that the combined use of antibiotics and heat treatment is a good control measure for psocids.

Influence of wolbachia infection on the fitness of the stored-product pest Liposcelis tricolor (Psocoptera: Liposcelididae).

Wolbachia are maternally inherited intracellular bacteria (Rickettsiaceae) that infect a wide range of arthropods and nematodes and that are associated with various reproductive abnormalities in their hosts. In this study, the effects of removal of Wolbachia infection on development, survival, and reproduction of Liposcelis tricolor Badonnel (Psocoptera: Liposcelididae) were investigated in laboratory. The Wolbachia-free strain was obtained by the removal of Wolbachia infection by using 1% rifampicin treatment on the Wolbachia-infected strain (control) for 4 wk, and no Wolbachia gene product was detected in this strain throughout the experiment. The results showed that the removal of Wolbachia infection had negative effects on the fitness of L. tricolor. Compared with the control strain, the Wolbachia-free strain (both in the first [F1] and second [F2] generation) had prolonged developmental times, reduced survivorship of immature stages, and reduced fecundity and longevity, resulting in much smaller rm values. Using rm values, the fitness for Wolbachia-free F1 and F2 relative to the control were calculated as 0.45 and 0.27, respectively. The results of this study further confirmed our previous conclusion that Wolbachia infection have positive effects on fecundity and fertility of L. tricolor, and for optimal reproduction of L. tricolor, Wolbachia must be present in psocids.