Functional analysis of a chitinase gene during the larval-nymph transition in Panonychus citri by RNA interference.

Chitinases are hydrolytic enzymes that are required for chitin degradation and reconstruction in arthropods. In this study, we report a cDNA sequence encoding a putative chitinase (PcCht1) from the citrus red mite, Panonychus citri. The PcCht1 (564 aa) possessed a signal peptide, a conserver domain, and a chitin-binding domain. Structural and phylogenetic analyses found that PcCht1 had high sequence similarity to chitinases in Tetranychus urticae. Real-time quantitative PCR analyses showed that the transcript levels of PcCht1 peaked periodically in larval and nymph stages. Moreover, significant increase of PcCht1 transcript level in the larvae was observed upon the exposure of diflubenzuron. In contrast, exposures of the larvae to diflubenzuron resulted in the decreased chitin content. Furthermore, through a feeding-based RNA interference approach, we were able to reduce the PcCht1 transcript level by 59.7 % in the larvae, and consequently the treated larvae showed a very low molting rate compared with the control. Our results expanded the understanding of the important role of PcCht1 in the growth and development of P. citri.

Molecular characterization of vitellogenin and its receptor genes from citrus red mite, Panonychus citri (McGregor).

The production and uptake of yolk protein play an important role in the reproduction of all oviparous organisms. Vitellogenin (Vg) is the precursor of vitellin (Vn), which is the major egg storage protein, and vitellogenin receptor (VgR) is a necessary protein for the uptake of Vg into developing oocytes. In this paper, we characterize the full-length Vg and VgR, PcVg1 and PcVgR, respectively, of the citrus red mite Panonychus citri (McGregor). The PcVg1 cDNA is 5748 nucleotides (nt) with a 5553-nt open reading frame (ORF) coding for 1851 amino acids (aa), and the PcVgR is 6090 nt, containing an intact ORF of 5673 nt coding an expected protein of 1891 aa. The PcVg1 aa sequence shows a typical GLCG domain and several K/RXXR cleavage sites, and PcVgR comprises two ligand-binding domains, two epidermal growth factor (EGF)-like regions containing YWTD motifs, a transmembrane domain, and a cytoplasmic domain. An analysis of the aa sequences and phylogenetics implied that both genes were genetically distinct from those of ticks and insects. The transcriptional profiles determined by real-time quantitative PCR in different developmental stages showed that both genes present the same expressional tendencies in eggs, larvae, nymphs, and adults. This suggested that the biosynthesis and uptake of PcVg occurs coordinately. The strong reproductive capacity of P. citri has been hypothesized as an important factor in its resistance; consequently, understanding the molecular mechanisms regulating Vg and VgR are fundamental for mite control.

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.

Molecular characterization of two carboxylesterase genes of the citrus red mite, Panonychus citri (Acari: Tetranychidae).

The citrus red mite, Panonychus citri, is known for its ability to rapidly evolve resistance to insecticides/acaricides and to adapt to hosts that produce toxins. To get better insight into the detoxification mechanism of P. citri, two carboxylesterase (CarE) genes, PCE1 and PCE2, were isolated and characterized. PCE1 and PCE2 contained open reading frames of 1,653 and 1,392 nucleotides, encoding proteins of 550 and 463 amino acid residues, respectively. Phylogenetic analyses showed that PCE1 and PCE2 were most closely related to the CarE genes from other phytophagous mites. The transcriptional profiles of two CarE genes among developmental stages (egg, larva, nymph, adult female, and adult male), after exposing to four acaricides (avermectin, azocyclotin, pyridaben, and spirodiclofen) and acid rain were investigated using real-time quantitative PCR (qPCR). The results showed that during development, PCE1 was highly expressed at the egg stage, whereas PCE2 was abundantly expressed at the adult stage of males. The expression levels of PCE1 were highly induced upon exposure to acaricides and acid rain. On the other hand, the expression levels of PCE2 were increased after treatment with avermectin and pyridaben. These results suggest that PCE1 and PCE2 may have distinct roles in different developmental stages and participate in the detoxification of acaricides.

Identification and characterization of seven glutathione S-transferase genes from citrus red mite, Panonychus citri (McGregor).

The citrus red mite, Panonychus citri (McGregor), is a global citrus pest, and has developed severe resistance to several types of acaricides. However, the molecular mechanisms of resistance in this mite remain unknown. In this study, seven full-length cDNAs encoding glutathione S-transferases (GSTs) genes were identified and characterized in P. citri. The effects of pyridaben and fenpropathrin exposure on the expression of these genes were also investigated. Phylogenetic analysis revealed that the seven GSTs genes in P. citri cloned in this study belong to three different cytosolic classes, including four in mu, two in delta and one in zeta. Among these seven GSTs genes, the relative expression level of PcGSTm1 was significantly higher in adult than in the other life stages (egg, larvae and nymph). Compared with the control, the mRNA levels of the seven GST genes did not change significantly following exposure to pyridaben at LC10. However, RT-qPCR results showed that, when exposed to LC10 of fenpropathrin, six GSTs gene (PcGSTm1, PcGSTm3, PcGSTm4, PcGSTd1, PcGSTd2 and PcGSTz1) transcripts increased in a time-dependent manner. This is the first insight into the molecular characteristics of GSTs gene cDNAs in P. citri. The elevated GSTs gene transcripts following exposure to fenpropathrin might be one of the mechanisms involved in detoxification of this acaricide.

Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae).

Quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR) is preferred for gene expression analysis in living organisms. Currently, it is a valuable tool for biological and ecological studies as it provides a relatively straightforward way to assess the relevance of transcriptional regulation under developmental and stress tolerance conditions. However, studies have shown that some commonly used reference genes varied among different experimental treatments, thus, systematic evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of arthropods. The aim of this study is to identify the suitable reference genes for RT-qPCR experiments involving various developmental stages and/or under abiotic stresses in citrus red mite Panonychus citri, a key pest in citrus orchards worldwide. GeNorm, NormFinder, and Bestkeeper software analysis indicates that elongation factor-1 alpha (ELF1A), RNA polymerase II largest subunit, alpha tublin, and glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are the most stable reference genes in various developmental stages, meanwhile, ELF1A and GAPDH were the most stable reference genes under various abiotic stresses. Furthermore, this study will serve as a resource to screen reference genes for gene expression studies in any other spider mite species.

Synergistic Effects of a Tomato chlorosis virus and Tomato yellow leaf curl virus Mixed Infection on Host Tomato Plants and the Whitefly Vector.

In China, Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV) are widely present in tomato plants. The epidemiology of these viruses is intimately associated with their vector, the whitefly (Bemisia tabaci MED). However, how a ToCV+TYLCV mixed infection affects viral acquisition by their vector remains unknown. In this study, we examined the growth parameters of tomato seedlings, including disease symptoms and the heights and weights of non-infected, singly infected and mixed infected tomato plants. Additionally, the spatio-temporal dynamics of the viruses in tomato plants, and the viral acquisition and transmission by B. tabaci MED, were determined. The results demonstrated that: (i) ToCV+TYLCV mixed infections induced tomato disease synergism, resulting in a high disease severity index and decreased stem heights and weights; (ii) as the disease progressed, TYLCV accumulated more in upper leaves of TYLCV-infected tomato plants than in lower leaves, whereas ToCV accumulated less in upper leaves of ToCV-infected tomato plants than in lower leaves; (iii) viral accumulation in ToCV+TYLCV mixed infected plants was greater than in singly infected plants; and (iv) B. tabaci MED appeared to have a greater TYLCV, but a lower ToCV, acquisition rate from mixed infected plants compared with singly infected plants. However, mixed infections did not affect transmission by whiteflies. Thus, ToCV+TYLCV mixed infections may induce synergistic disease effects in tomato plants.

Transcriptome Profiling of the Whitefly Bemisia tabaci MED in Response to Single Infection of Tomato yellow leaf curl virus, Tomato chlorosis virus, and Their Co-infection.

Tomato yellow leaf curl virus (TYLCV) and Tomato chlorosis virus (ToCV) are two of the most devastating cultivated tomato viruses, causing significant crop losses worldwide. As the vector of both TYLCV and ToCV, the whitefly Bemisia tabaci Mediterranean (MED) is mainly responsible for the rapid spread and mixed infection of TYLCV and ToCV in China. However, little is known concerning B. tabaci MED's molecular response to TYLCV and ToCV infection or their co-infection. We determined the transcriptional responses of the whitefly MED to TYLCV infection, ToCV infection, and TYLCV&ToCV co-infection using Illumina sequencing. In all, 78, 221, and 60 differentially expressed genes (DEGs) were identified in TYLCV-infected, ToCV-infected, and TYLCV&ToCV co-infected whiteflies, respectively, compared with non-viruliferous whiteflies. Differentially regulated genes were sorted according to their roles in detoxification, stress response, immune response, transport, primary metabolism, cell function, and total fitness in whiteflies after feeding on virus-infected tomato plants. Alterations in the transcription profiles of genes involved in transport and energy metabolism occurred between TYLCV&ToCV co-infection and single infection with TYLCV or ToCV; this may be associated with the adaptation of the insect vector upon co-infection of the two viruses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses demonstrated that the single infection with TYLCV or ToCV and the TYLCV&ToCV co-infection could perturb metabolic processes and metabolic pathways. Taken together, our results provide basis for further exploration of the molecular mechanisms of the response to TYLCV, ToCV single infection, and TYLCV&ToCV co-infection in B. tabaci MED, which will add to our knowledge of the interactions between plant viruses and insect vectors.

Secretory laccase 1 in Bemisia tabaci MED is involved in whitefly-plant interaction.

The whitefly Bemisia tabaci is a phloem-feeding pest that lives predominantly on herbaceous species and causes serious damage to hosts. Whitefly saliva is thought to contain proteins that modulate plant defences and facilitate feeding. A predicted secreted protein, laccase 1 (LAC1), was found in the salivary gland transcriptome of B. tabaci and might be existed in the watery saliva of B. tabaci. As LAC1 has a potential role in detoxification of secondary plant compounds in insects, we speculated that it may participate in the insect's response to plant defences. Here, we cloned the complete cDNA of LAC1 and found that (1) LAC1 was highly expressed in the salivary gland (SG) and midgut; (2) LAC1 transcript level in head (containing SG) was 2.1 times higher in plant-fed than in diet-fed whiteflies and 1.6 times higher in the head and 23.8 times higher in the midgut of whiteflies that fed on jasmonic acid (JA)-sprayed plants than on control plants; and (3) silencing LAC1 decreased the survival rate of plant-fed whiteflies but had a marginal effect on whiteflies raised on an artificial diet. These results indicate that LAC1 enables whiteflies to overcome the chemical defences of host plants and might act as an effector in saliva.

Identification of responsive proteins in Panonychus citri exposed to abamectin by a proteomic approach.

Abamectin is a microbial-derived pesticide widely used for control of agricultural pests. However, sustained use of abamectin has led to the development of resistance in some target species. Previous studies on arthropod resistance to abamectin have mainly used traditional biochemical and molecular approaches. To understand the responses of citrus red mite, Panonychus citri, exposed to abamectin, comparative proteomic analysis was conducted using two-dimensional electrophoresis (2-DE). A total of 26 distinct protein spots were present in response to abamectin exposure. Tandem mass spectrometry (MS/MS) identified 16 proteins that were mainly involved in energy metabolism and detoxification. Some remaining proteins were not identifiable, suggesting that they may be novel. The expression levels of transcripts associated with proteins were analyzed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, to validate the proteomic data obtained in the present study, Western-blot experiment was performed and the expression of sHsp and PcE1 proteins were confirmed, respectively. BIOLOGICAL SIGNIFICANCE: The citrus red mite has developed resistance to many acaricides, including abamectin. In the current study, we used the proteomic approaches involving 2-DE, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and MS/MS to document changes in adult P. citri during 24h of abamectin exposure. Abamectin stress induced a total of 16 differentially regulated proteins. The proteomic results were validated in mRNA expression patterns using qRT-PCR. This is the first analysis of differentially expressed proteins in P. citri exposed to abamectin. The results help clarify the physiological mechanisms of P. citri responses to abamectin exposure.

Molecular characterisation of a sodium channel gene and identification of a Phe1538 to Ile mutation in citrus red mite, Panonychus citri.

BACKGROUND: The citrus red mite, Panonychus citri (McGregor), is regarded as one of the most serious citrus pests in many countries and has developed high resistance to pyrethroids as a result of the intensive use of these acaricides. RESULTS: The para sodium channel gene of P. citri (named PcNav ), containing an entire coding region of 6729 bp, was cloned in this study. Three alternative splicing sites and 12 potential RNA editing sites were identified in PcNav . Thus, exons alt 1 and alt 3-v3 were found to be unique to PcNav . Comparison of field fenpropathrin-resistant (WZ) and susceptible (LS) strains identified the point mutation F1538I in IIIS6 of the sodium channel, which is known to confer strong resistance to pyrethroids in mites. Moreover, it was also found that the PcNav mRNA was present during all life stages, and the transcript seems to be more abundant in larvae than in other developmental stages. CONCLUSION: These results suggest that the F1538I mutation plays an important role in fenpropathrin resistance in citrus red mites. This is the first study of the sodium channel in P. citri and provides abundant information for further research on the mechanism of pyrethroid resistance.

Mining genes involved in insecticide resistance of Liposcelis bostrychophila Badonnel by transcriptome and expression profile analysis.

BACKGROUND: Recent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method. CONCLUSION: The L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids.

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.