Jin-Jun Wang

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae).

BackgroundQuantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel).ResultsTwo different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs.ConclusionsIn this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.

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.

SEASONAL ABUNDANCE OF THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI (HOMOPTERA: PSYLLIDAE) IN SOUTHERN FLORIDA

Abstract Seasonal abundance of the Asian citrus psyllid, Diaphorina citri Kuwayama, was studied weekly in two orange jasmine [Murraya paniculata (L.) Jack] plots in southern Florida from October 1998 to October 1999. Psyllid populations occur throughout the season on orange jasmine in southern Florida. Population peaks were observed in October, November, and December in 1998, and May and August in 1999. Psyllid population levels were positively related to the availability of new shoot flushes which were in turn related to the weekly minimum temperature and rainfall. Natural enemies were not key factors in regulating populations during the study period. The populations of adult psyllids were also studied weekly on potted orange jasmine and grapefruit (Citrus paradisi Macfadyen) plants from June 1999 to July 2000. The population levels of psyllid on both host plants were not significantly different and general population trends on the two hosts were similar over time. Continuous shoot flushes produced by orange jasmine could play an important role in maintaining high populations of this insect when new shoot flushes were not available in the commercial citrus groves.

Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress.

Relatively low or high temperatures are responsible for a variety of physiological stress responses in insects and mites. Induced thermal stress was recently associated with increased reactive oxygen species (ROS) generation, which caused oxidative damage. In this study, we examined the time-related effect of the relatively low (0, 5, 10, and 15 °C) or high (32, 35, 38, and 41 °C) temperatures on the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), and glutathione-S-transferase (GST), and the total antioxidant capacity (TEAC) of the citrus red mite, Panonychus citri (McGregor). The malondialdehyde (MDA) concentration, as a marker of lipid peroxidation in organisms, was also measured in the citrus red mite under thermal stress conditions. Results showed that SOD and GST activities were significantly increased and play an important role in the process of antioxidant response to thermal stress. Lipid peroxidation levels increased significantly (P<0.001) and changed in a time-dependent manner. CAT and POX activity, as well as TEAC, did not vary significantly and play a minor role to remove the ROS generation. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play an important role in reducing oxidative damage in the citrus red mite.

Analysis of Transcriptome Differences between Resistant and Susceptible Strains of the Citrus Red Mite Panonychus citri (Acari: Tetranychidae)

Background The citrus red mite is a worldwide citrus pest and a common sensitizing allergen of asthma and rhinitis. It has developed strong resistance to many registered acaricides, However, the molecular mechanisms of resistance remain unknown. we therefore used next generation sequencing technology to investigate the global transcriptomes between resistant strains and susceptible strains. Results We obtained 34,159, 30,466 and 32,217 unigenes by assembling the SS reads, RS reads and SS&RS reads respectively. There are total 17,581 annotated unigenes from SS&RS reads by BLAST searching databases of nr, the Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) with an E-value ≤ 1e-5, in which 7,075 unigenes were annotated in the COG database, 12, 712 unigenes were found in the KEGG database and 3,812 unigenes were assigned to Gene ontology (GO). Moreover, 2,701 unigenes were judged to be the differentially expressed genes (DEGs) based on the uniquely mapped reads. There are 219 pathways in all annotated unigenes and 198 pathways in DEGs that mapped to the KEGG database. We identified 211 metabolism genes and target genes related to general insecticide resistance such as P450 and Cytochrome b, and further compared their differences between RS and SS. Meanwhile, we identified 105 and 194 genes related to growth and reproduction, respectively, based on the mode of action of Hexythiazox. After further analyses, we found variation in sequences but not in gene expression related to mite growth and reproduction between different strains. Conclusion To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in phytophagous mite resistance. This study identified differential unigenes related to general pesticide resistance and organism growth and reproduction in P. citri. The assembled, annotated transcriptomes provide a valuable genomic resource for further understanding the molecular basis of resistance mechanisms.

The Multipartite Mitochondrial Genome of Liposcelis bostrychophila: Insights into the Evolution of Mitochondrial Genomes in Bilateral Animals

Booklice (order Psocoptera) in the genus Liposcelis are major pests to stored grains worldwide and are closely related to parasitic lice (order Phthiraptera). We sequenced the mitochondrial (mt) genome of Liposcelis bostrychophila and found that the typical single mt chromosome of bilateral animals has fragmented into and been replaced by two medium-sized chromosomes in this booklouse; each of these chromosomes has about half of the genes of the typical mt chromosome of bilateral animals. These mt chromosomes are 8,530 bp (mt chromosome I) and 7,933 bp (mt chromosome II) in size. Intriguingly, mt chromosome I is twice as abundant as chromosome II. It appears that the selection pressure for compact mt genomes in bilateral animals favors small mt chromosomes when small mt chromosomes co-exist with the typical large mt chromosomes. Thus, small mt chromosomes may have selective advantages over large mt chromosomes in bilateral animals. Phylogenetic analyses of mt genome sequences of Psocodea (i.e. Psocoptera plus Phthiraptera) indicate that: 1) the order Psocoptera (booklice and barklice) is paraphyletic; and 2) the order Phthiraptera (the parasitic lice) is monophyletic. Within parasitic lice, however, the suborder Ischnocera is paraphyletic; this differs from the traditional view that each suborder of parasitic lice is monophyletic.

Development and Reproduction of the Psocid Liposcelis bostrychophila (Psocoptera: Liposcelididae) as a Function of Temperature

Abstract The development, survival, and reproduction of the psocid Liposcelis bostrychophila Badonnel were evaluated at 8 constant temperatures (17.5, 20, 22.5, 25, 27.5, 30, 32.5, and 35°C). The population reared at 17.5°C failed to develop or reproduce. Between 20 and 35°C, developmental period from egg to adult varied from 41.9 d at 20°C to 18.1 d at 32°C. Based on a nonlinear model, the lower-temperature developmental thresholds from egg, 1st through 4th stadia, and combined immature stages were estimated at 14.8, 17.4, 14.6, 15.5, 17.4, and 15.5°C, respectively; the upper temperature thresholds ranged from 36.4 to 38.8°C. The survival from egg to adult was 82.9% at 27.5°C; 37.4 and 50.7% at 20 and 35°C, respectively. After emergence the adult had a preoviposition period that ranged from 17.9 d at 20°C to 3.3 d at 27.5°C. L. bostrychophila produced the most eggs (74.7) at 27.5°C and the fewest (51.9) at 20°C. The population reared at 30°C had the highest intrinsic rate of increase (0.0946), net reproductive rate (59.59), the shortest population doubling time (7.3 d), and shorter mean generation time (43.2 d) compared with the populations reared at 6 other constant temperatures. The populations reared at 27.5–32.5°C had type I survivorship pattern; whereas populations reared at other temperatures had type III survivorship curve as determined by a Weibull frequency distribution. The optimal range of temperature for L. bostrychophila population growth was 25–32.5°C.

The complete mitochondrial genome of the citrus red mite Panonychus citri (Acari: Tetranychidae): high genome rearrangement and extremely truncated tRNAs

BackgroundThe family Tetranychidae (Chelicerata: Acari) includes ~1200 species, many of which are of agronomic importance. To date, mitochondrial genomes of only two Tetranychidae species have been sequenced, and it has been found that these two mitochondrial genomes are characterized by many unusual features in genome organization and structure such as gene order and nucleotide frequency. The scarcity of available sequence data has greatly impeded evolutionary studies in Acari (mites and ticks). Information on Tetranychidae mitochondrial genomes is quite important for phylogenetic evaluation and population genetics, as well as the molecular evolution of functional genes such as acaricide-resistance genes. In this study, we sequenced the complete mitochondrial genome of Panonychus citri (Family Tetranychidae), a worldwide citrus pest, and provide a comparison to other Acari.ResultsThe mitochondrial genome of P. citri is a typical circular molecule of 13,077 bp, and contains the complete set of 37 genes that are usually found in metazoans. This is the smallest mitochondrial genome within all sequenced Acari and other Chelicerata, primarily due to the significant size reduction of protein coding genes (PCGs), a large rRNA gene, and the A + T-rich region. The mitochondrial gene order for P. citri is the same as those for P. ulmi and Tetranychus urticae, but distinctly different from other Acari by a series of gene translocations and/or inversions. The majority of the P. citri mitochondrial genome has a high A + T content (85.28%), which is also reflected by AT-rich codons being used more frequently, but exhibits a positive GC-skew (0.03). The Acari mitochondrial nad1 exhibits a faster amino acid substitution rate than other genes, and the variation of nucleotide substitution patterns of PCGs is significantly correlated with the G + C content. Most tRNA genes of P. citri are extremely truncated and atypical (44-65, 54.1 ± 4.1 bp), lacking either the T- or D-arm, as found in P. ulmi, T. urticae, and other Acariform mites.ConclusionsThe P. citri mitochondrial gene order is markedly different from those of other chelicerates, but is conserved within the family Tetranychidae indicating that high rearrangements have occurred after Tetranychidae diverged from other Acari. Comparative analyses suggest that the genome size, gene order, gene content, codon usage, and base composition are strongly variable among Acari mitochondrial genomes. While extremely small and unusual tRNA genes seem to be common for Acariform mites, further experimental evidence is needed.

Toxic effects of six plant oils alone and in combination with controlled atmosphere on Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Abstract Six plant essential oils alone as repellent and fumigant, and in combination with the controlled atmosphere against Liposcelis bostrychophila Badonnel were assessed in the laboratory. These essential oils were extracted from the leaves of six source plants: Citrus tangerina Tanaka, Citrus aurantium L., Citrus bergamia Risso et Poiteau, Pinus sylvestris L., Cupressus funebris Endl., and Eucalyptus citriodora Hook. The repellency test indicated that L. bostrychophila adults were repelled by filter paper strips treated with six essential oils. Of these essential oils, the C. funebris oil was most effective followed by that of P. sylvestris, C. tangerina, C. bergamia, and E. citriodora. The average repellency of the C. aurantium oil against L. bostrychophila adults was significantly lower than other five test oils by day 14. These essential oils had a high level of toxicity in the fumigation assay against L. bostrychophila adults at both 10 and 20 ppm. When combined with two controlled atmosphere treatments (12% CO2 + 9% O2, and 10% CO2 + 5% O2, balanced N2), the toxicity of plant oils was enhanced significantly.

Toxicities and Synergistic Effects of Several Insecticides Against the Oriental Fruit Fly (Diptera: Tephritidae)

ABSTRACT The oriental fruit fly, Bactrocera dorsalis (Hendel), is a serious insect pest that causes large losses to orchards globally. In this study, we conducted experiments to investigate the susceptibility of two populations (Kunming of Yunnan and Dongguan of Guangdong province) of B. dorsalis to nine insecticides. Bioassay results demonstrated that fipronil was the most effective insecticide, followed by phoxim, abamectin, triazophos, &bgr;-cypermethrin, chlorpyrifos, deltamethrin, malathion, and imidacloprid against the Kunming of Yunnan province population, with LD50 values that ranged from 1.55 to 187.48 ng/ fly. For the Dongguan of Guangdong province population, fipronil was also most toxic, followed by triazophos, phoxim, chlorpyrifos, abamectin, deltamethrin, β-cypermethrin, malathion, and imidacloprid, with LD50 values from 2.07 to 439.11 ng/fly. The addition of synergists triphenyl phosphate, piperonyl butoxide, and diethyl maleate yielded different levels of synergistic effects on different insecticides against each population. However, the synergistic effects on the nine insecticides against the two populations are different. The treatment of a sublethal dose (LD20) of &lgr;-cypermethrin together with three synergists could induce increased specific activity of carboxylesterases at the beginning of exposure, followed by a decline within 24 h. The specific activity of carboxylesterases was higher in the fat body, midgut, and Malpighian tubules, suggesting these are important tissues for detoxification. Overall, the data developed in this study provide useful information for designing an insecticide management strategy for controlling this insect in the field.