Dan-Dan Wei

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Liposcelis bostrychophila and found that the typical single mt chromosome of bilateral animals has fragmented into and been replaced by two medium-sized chromosomes in this booklouse; each of these chromosomes has about half of the genes of the typical mt chromosome of bilateral animals.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.

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.

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.

De novo assembly, gene annotation, and marker discovery in stored-product pest Liposcelis entomophila (Enderlein) using transcriptome sequences.

Background As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. Methodology/Principal Findings We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. Conclusions/Significance We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future.

Overexpression of two α‐esterase genes mediates metabolic resistance to malathion in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). However, the underlying molecular mechanism of the esterase‐mediated resistance remains largely unknown in this species. Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed α‐esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B. dorsalis. Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides. The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B. dorsalis. Both genes were significantly up‐regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain. However, no changes in either the coding sequence or gene copy number were observed between the two strains. Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion. Furthermore, RNA interference‐mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults. In conclusion, these results expand our molecular understanding of the important role of α‐esterases during the development of resistance to organophosphorous insecticides in B. dorsalis.

Sequence analysis of the ribosomal internal transcribed spacers region in psocids (Psocoptera: Liposcelididae) for phylogenetic inference and species discrimination.

ABSTRACT Psocids (Psocoptera: Liposcelididae: Liposcelis spp.) are major pests of stored grain and commonly occur on a wide range of stored products. Increasingly, the genus of Liposcelis has gained recognition of their importance due to their feeding on stored grains, contaminating food, and agricultural commodities as well as transmitting harmful microorganisms, including fungi and bacteria. Psocids have close morphological similarities and often commix occur at the same ecosystems. Therefore, a first step necessary to further implement population studies is the accurate identification of species, based on molecular methods. In this study, we determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS) 1-5.8S-ITS2 region in 100 individuals of six Liposcelis species (including Liposcelis bostrychophila Badonnel, Liposcelis entomophila (Enderlein), Liposcelis decolor (Pearman), Liposcelis tricolor Badonnel, Liposcelis pacta Pearman, and Liposcelis yunnaniensis Li & Li) from 16 locations of China. We evaluated the suitability of this marker for phylogenetic inference study in the Liposcelis species. We also developed a molecular identification method for six Liposcelis species based on ITS2 sequence. Results demonstrate that ITS1-5.8S-ITS2 sequences are a useful tool for the population genetic study and phylogeny estimation of Liposcelis species. The results of this study indicate that the ITS2 sequences can be a reliable tool for species discrimination of the six species of psocids tested here. In addition, the multiplex method described proved reliable when tested across different geographical populations.

Reference Gene Validation for Quantitative PCR Under Various Biotic and Abiotic Stress Conditions in Toxoptera citricida (Hemiptera, Aphidiae).

ABSTRACT The regulation of mRNA expression level is critical for gene expression studies. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly used to investigate mRNA expression level of genes under various experimental conditions. An important factor that determines the optimal quantification of qRT-PCR data is the choice of the reference gene for normalization. To advance gene expression studies in Toxoptera citricida (Kirkaldy), an important citrus pest and a main vector of the Citrus tristeza virus, we used five tools (GeNorm, NormFinder, BestKeeper, ΔCt methods, and Ref Finder) to evaluate seven candidate reference genes (elongation factor-1 alpha [EF1&agr;], beta tubulin [β-TUB], 18S ribosomal RNA [18S], RNA polymerase II large subunit (RNAP II), beta actin (β-ACT), alpha tubulin, and glyceraldhyde-3-phosphate dehydrogenase) under different biotic (developmental stages and wing dimorphism) and abiotic stress (thermal, starvation, and UV irradiation) conditions. The results showed that EF1&agr; and 18S were the most stable genes under various biotic states, β-ACT and β-TUB during thermal stress, EF1&agr; and RNAP II under starvation stress, and RNAP II, β-ACT, and EF1&agr; under UV irradiation stress conditions. This study provides useful resources for the transcriptional profiling of genes in T. citricida and closely related aphid species.

De novo characterization of the Dialeurodes citri transcriptome: mining genes involved in stress resistance and simple sequence repeats (SSRs) discovery

The citrus whitefly, Dialeurodes citri (Ashmead), is one of the three economically important whitefly species that infest citrus plants around the world; however, limited genetic research has been focused on D. citri, partly because of lack of genomic resources. In this study, we performed de novo assembly of a transcriptome using Illumina paired‐end sequencing technology (Illumina Inc., San Diego, CA, USA). In total, 36 766 unigenes with a mean length of 497 bp were identified. Of these unigenes, we identified 17 788 matched known proteins in the National Center for Biotechnology Information database, as determined by Blast search, with 5731, 4850 and 14 441 unigenes assigned to clusters of orthologous groups (COG), gene ontology (GO), and SwissProt, respectively. In total, 7507 unigenes were assigned to 308 known pathways. In‐depth analysis of the data showed that 117 unigenes were identified as potentially involved in the detoxification of xenobiotics and 67 heat shock protein (Hsp) genes were associated with environmental stress. In addition, these enzymes were searched against the GO and COG database, and the results showed that the three major detoxification enzymes and Hsps were classified into 18 and 3, 6, and 8 annotations, respectively. In addition, 149 simple sequence repeats were detected. The results facilitate the investigation of molecular resistance mechanisms to insecticides and environmental stress, and contribute to molecular marker development. The findings greatly improve our genetic understanding of D. citri, and lay the foundation for future functional genomics studies on this species.

Genetic Diversity and Population Structure of Panonychus citri (Acari: Tetranychidae), in China Based on Mitochondrial COI Gene Sequences

ABSTRACT The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is regarded as one of the most important citrus pests in many countries, such as Japan, Spain, and China. In this study, the mitochondrial cytochrome oxidase subunit I gene was used to analyze genetic diversity and population structure of 15 P. citri populations collected from three citrus (Citrus spp.) planting regions of China. Our analysis found that these populations had relatively low genetic diversities. Bayesian tree and haplotype network showed that the 22 haplotypes of P. citri formed two lineages with low Bayesian posterior probabilities (0.55 and 0.63), and haplotypes in a sample were scattered throughout the Bayesian tree, indicating that there may be no significant genealogical structure among populations. However, when Yuxi and Danjiangkou populations (both without H7 haplotype) were excluded from the analysis, analysis of molecular variance found a weak, but significant, geographic structuring. Mantel test indicated a significant and positive correlation between genetic and geographical distances, reflecting certain degree of isolation by distance. The genetic differentiation based on pairwise FST was not significant between most populations, and some FST were even negative, indicating remarkable gene flow among these populations. The weak population structure of P. citri in this study was probably influenced by high gene flow between some populations due to long-distance dispersal of this species, which may be largely relied on the movement of plants between populations. The mismatch distribution analysis showed that no signal of population growth, but the Fus Fs value was significantly negative for total populations and the star-like shape of haplotype network, suggesting a history of population expansion of P. citri in China.

Female remating inhibition and fitness of Bactrocera dorsalis (Diptera: Tephritidae) associated with male accessory glands

Abstract Polyandry Is widespread among insects but male accessory gland products can influence the propensity of former mates to copulate later with other males. In addition, females may receive nutritional supplements in accessory gland fluids that substantially increase fitness regardless of whether remating has been inhibited. In this study, we investigated polyandry in female Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) and the relationship between remating and male accessory gland contents. Similar to other fruit flies, 2 kinds of accessory glands were observed in male B. dorsalis. Male mesodermal accessory glands expanded significantly in length and area when copulation finished. A remating refractory period occurred in B. dorsalis females, but It did not differ in duration following copulations with either virgin or non-virgin males. Besides, we found that virgin females lived longer, but produced much fewer eggs than mated females. Remating with a refractory period resulted In more eggs being laid and offspring produced than continuous exposure to 2 mates. In addition, females In the continuous presence of 2 males produced significantly more offspring than females with only 1 male present. We also observed that increases in male and female age reduced the rate of fertilization. These results indicated that multiple matings increased the fitness of B. dorsalis females, although remating inhibition existed in B. dorsalis fruit flies. It is the great reproductive ability of S. dorsalis that enable flourishing populations to occur in wild.