Guo-Rui Yuan

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.

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.

Regulation of three isoforms of SOD gene by environmental stresses in citrus red mite, Panonychus citri

Abstract Superoxide dismutase (SOD) is a family of enzymes with multiple isoforms that possess antioxidative abilities in response to environmental stresses. Panonychus citri is one of the most important pest mites and has a global distribution. In this study, three distinct isoforms of SOD were cloned from P. citri and identified as cytoplasmic Cu-ZnSOD (PcSOD1), extracellular Cu-ZnSOD (PcSOD2), and mitochondrial MnSOD (PcSOD3). mRNA expression level analysis showed that all three isoforms were up-regulated significantly after exposure to the acaricide abamectin and to UV-B ultraviolet irradiation. In particular, PcSOD3 was up-regulated under almost all environmental stresses tested. The fold change of PcSOD3 expression was significantly higher than those of the two Cu-ZnSOD isoforms. Taken together, the results indicate that abamectin and UV-B can induce transcripts of all three SOD isoforms in P. citri. Furthermore, PcSOD3 seems to play a more important role in P. citri tolerance to oxidative stress.

The effect of dietary restriction on longevity, fecundity, and antioxidant responses in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Recent studies in fruit flies have imposed dietary restriction (DR) by diluting yeast and have reported increased lifespan as the yeast-to-sugar ratio decreased. In this study, the effects of DR on the lifespan of Bactrocera dorsalis were investigated using constant-feeding diets with different yeast:sugar ratios and an intermittent-feeding diet in which flies ate every sixth day. Antioxidant enzyme activities and the malondialdehyde concentration were also measured in virgin females under constant-feeding DR protocols to investigate their relationships with lifespan. The results showed that B. dorsalis lifespan was significantly extended by DR, and carbohydrate-enriched diet may be important for lifespan-extension. Female flies lived significantly longer than males at all dietary levels under both feeding regimes, indicating no interaction between diet and sex in determining lifespan. Antioxidant enzyme activities increased with the amount of yeast increased in the diets (0-4.76%) between starvation and DR treatments, indicating that the antioxidants may have influences in determining lifespan in B. dorsalis under starvation and DR treatments. However, antioxidants cannot keep up with increased oxidative damage induced by the high yeast diet (25%). These results revealed that the extension of lifespan by DR is evolutionarily conserved in B. dorsalis and that yeast:sugar ratios significantly modulate lifespan in this species.

Antimicrobial peptide gene cecropin-2 and defensin respond to peptidoglycan infection in the female adult of oriental fruit fly, Bactrocera dorsalis (Hendel).

Cecropins and defensins are important antimicrobial peptides in insects and are inducible after injection of immune triggers. In this study, we cloned the cDNAs of two antimicrobial peptides (AMPs), cecropin-2 (BdCec-2) and defensin (BdDef) from Bactrocera dorsalis (Hendel), a serious pest causing great economic losses to fruits and vegetables. The BdCec-2 sequence of 192bp encodes a protein of 63 amino acids residues with a predicted molecular weight of 6.78kD. The 282bp cDNA of BdDef encodes a protein of 93 residues with a predicted molecular weight of 9.81kD. Quantitative real-time PCR analyses showed that BdCec-2 and BdDef had similar expression profiles among development stages, the highest mRNA levels of these two AMP genes were observed in the adult stage. Among different adult body segments and tissues, both genes had similar transcriptional profiles, the highest mRNA levels appeared in abdomen and fat body, which was consistent with the reported fact that fat body was the main organ synthesizing AMPs in insects. The expression of BdCec-2 and BdDef were up-regulated after challenge with peptidoglycans from Escherichia coli (PGN-EB) and Staphylococcus aureus (PGN-SA), respectively, suggesting their antimicrobial activity against Gram-negative and Gram-positive microorganisms. These results describe for the first time the basic properties of the cecropin-2 and defensin genes from B. dorsalis that probably play an important role in the defense response against invading microbes.

Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.

Involvement of Met and Kr-h1 in JH-mediated reproduction of female Bactrocera dorsalis (Hendel)

Juvenile hormone (JH) prevents metamorphosis during insect larval stages and promotes adult reproductive processes. Krüppel-homolog 1 (Kr-h1), a zinc finger transcription factor assumed to be induced by JH via the JH receptor methoprene-tolerant (Met), mediates the antimetamorphic effect of JH in insects, but its function in JH-mediated reproductive processes has not been fully explored. In this study, Met and Kr-h1 involved in the JH signaling pathway were first cloned and identified from the oriental fruit fly, Bactrocera dorsalis, an important pest infesting fruit and vegetables worldwide. Subsequent spatiotemporal expression analysis revealed that Met and Kr-h1 were both highly expressed in 7-day-old adults and fat body of female adults, respectively. Treatment with a JH analog (methoprene) significantly induced the expression of JH signaling and vitellogenin (Vg) genes and accelerated ovary development. RNA interference (RNAi) further revealed that either Met or Kr-h1 depletion at the adult stage of B. dorsalis impeded ovary development, with significantly lower egg production noted as well. In addition, rescue through methoprene application after RNAi stimulated the expression of JH signaling and Vg genes. Although there were still differences in ovary phenotype between rescued insects and the pre-RNAi control, ovary redevelopment with a larger surface area was observed, consistent with the spatiotemporal expression and phenotypes recorded in the original methoprene experiment. Our data reveal the involvement of Met and Kr-h1 in insect vitellogenesis and egg production, thus indicating the crucial role of the JH signaling pathway in insect reproduction.

Genome-wide identification, phylogenetic analysis, and expression profiles of ATP-binding cassette transporter genes in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

The ATP-binding cassette (ABC) is the largest transporter gene family and the genes play key roles in xenobiotic resistance, metabolism, and development of all phyla. However, the specific functions of ABC gene families in insects is unclear. We report a genome-wide identification, phylogenetic, and transcriptional analysis of the ABC genes in the oriental fruit fly, Bactrocera dorsalis (Hendel). We identified a total of 47 ABC genes (BdABCs) from the transcriptomic and genomic databases of B. dorsalis and classified these genes into eight subfamilies (A-H), including 7 ABCAs, 7 ABCBs, 9 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 15 ABCGs, and 3 ABCHs. Comparative phylogenetic analysis of the ABCs suggests an orthologous relationship between B. dorsalis and other insect species in which these genes have been related to pesticide resistance and essential biological processes. Comparison of transcriptome and relative expression patterns of BdABCs indicated diverse multifunctions within different B. dorsalis tissues. The expression of 4, 10, and 14 BdABCs from 18 BdABCs was significantly upregulated after exposure to LD50s of malathion, avermectin, and beta-cypermethrin, respectively. The maximum expression level of most BdABCs (including BdABCFs, BdABCGs, and BdABCHs) occurred at 48h post exposures, whereas BdABCEs peaked at 24h after treatment. Furthermore, RNA interference-mediated suppression of BdABCB7 resulted in increased toxicity of malathion against B. dorsalis. These data suggest that ABC transporter genes might play key roles in xenobiotic metabolism and biosynthesis in B. dorsalis.

Molecular cloning, mRNA expression and alternative splicing of a ryanodine receptor gene from the citrus whitefly, Dialeurodes citri (Ashmead)

Insect ryanodine receptors are the main targets of diamide insecticides that have highly selective insecticidal activity but are less toxic to mammals. Therefore, these insecticides are ideal for pest control. Ryanodine receptors (RyRs) play a critical role in Ca2+ signaling in muscle and non-muscle cells. In this study, we cloned the complete cDNA (DcRyR) of the RyR from the citrus whitefly, Dialeurodes citri, a serious pest of citrus orchards in China. The open reading frame of RyR is 15,378bp long and encodes a protein with 5126 amino acids with a computed molecular weight of 579.523kDa. DcRyR shows a high amino acid sequence identity to RyRs from other insects (76%-95%) and low identity to those from nematodes and mammals (44%-52%). DcRyR shares many features of insect and vertebrate RyRs, including a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeat domain, RIH-associated domain at the N-terminus, two consensus calcium-binding EF-hands and six transmembrane domains at the C-terminus. The expression of DcRyR mRNA was the highest in the nymphs and lowest in eggs; DcRyR mRNA was 1.85-fold higher in the nymphs than in the eggs. Among the tissues, DcRyR mRNA expression was 4.18- and 4.02-fold higher in the adult head and thorax than in the abdomen. DcRyR had three alternative splice sites and the splice variants showed body part-specific expression and were developmentally regulated. These results may help investigate target-based resistance to diamide insecticides in D. citri.