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

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 RefFinder) to evaluate seven candidate reference genes (elongation factor-1 alpha [EF1α], beta tubulin [ß-TUB], 18S ribosomal RNA [18S], RNA polymerase II large subunit (RNAP II), beta actin (ß-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 EF1α and 18S were the most stable genes under various biotic states, ß-ACT and ß-TUB during thermal stress, EF1α and RNAP II under starvation stress, and RNAP II, ß-ACT, and EF1α under UV irradiation stress conditions. This study provides useful resources for the transcriptional profiling of genes in T. citricida and closely related aphid species.

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.

The Essential Role of Vitellogenin Receptor in Ovary Development and Vitellogenin Uptake in Bactrocera dorsalis (Hendel).

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

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

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.

Influence of various stressors on the expression of core genes of the small interfering RNA pathway in the oriental fruit fly, Bactrocera dorsalis.

RNA interference (RNAi)-based technology has emerged as a potential tool for controlling insect pests, however, previous studies found that the efficiency of RNAi in Bactrocera dorsalis was variable. In nature, insects often meet various challenges, such as pathogen infections, extreme temperatures, lack of nutrition and heavy metals. To better understand the association of the stressors with efficiency of RNAi, in the current study we tested the expression of three core genes, dicer2 (Bddcr2), r2d2 (Bdr2d2) and argonaute2 (Bdago2), of the small interfering RNA (siRNA) pathway of B. dorsalis upon various stressors. Our results showed that all three genes were upregulated by the infection of invertebrate iridescent virus 6, which suggested a function of the siRNA pathway against viral infection. The loading of FeCl3 could also increase the expression of Bddcr2. The treatments of Escherichia coli, extremely high (40°C) and low (0°C) temperatures, as well as starvation, could negatively influence the expression of Bddcr2 and/or Bdago2. In total, our results showed that various stressors could influence the expression of core components of B. dorsalis siRNA pathway. This highlights further speculation on the RNAi efficiency upon these stressors. Considering the complexity and variation of RNAi efficiency in different conditions, these results provide initial aspects in possible environmental stressors to influence the activity of the siRNA pathway, but the real impact of RNAi efficiency posed by these stressors requires further studies.

Characterization of an insect heterodimeric voltage-gated sodium channel with unique alternative splicing mode.

Recent discovery of the heterodimeric voltage-gated sodium channels (Nav) in two aphid species, Acyrthosiphon pisum and Myzus persicae, aroused interest in exploring whether this kind of channel is conserved for aphids. Herewith, we aim to provide evidence for the conservation of heterodimeric Navs in aphids and investigate whether they have unique splicing patterns. We found that the only identifiable Nav from Toxoptera citricida consisted of two subunits, forming a heterodimeric Nav, which carried an atypical "DENS" ion selectivity filter and a conventional "MFM" inactivation gate, confirming the heterodimeric Navs' conservation within aphids. These unique heterodimeric channels may form a new Nav subfamily, specific to aphids. A more ancient member of four-domain Nav homolog was well preserved in T. citricida, carrying a typical "DEEA" and "MFL" motif. The presence of "DENS" in mammalian Naxs and "DEKT" in a fungus Nav suggested that the heterodimeric Navs may still preserve Na+ permeability. Sequencing 46 clones from nymphs and adults exposed unique splicing patterns for this heterodimeric Nav from T. citricida, revealing 7 alternatively spliced exons, evidencing that exon 5 was no longer unique to Bombyx mori, and exon k/l was semi-mutually exclusive. Two previously undescribed optional exons and a SNP site seemingly unique to aphids were identified. In conclusion, the dimeric Navs might form a new aphids-specific heterodimeric Nav subfamily. This dimeric Nav from T. citricida was characterized with distinguishable alternative splicing modes, exemplified by the discovery of two novel alternative exons and unique usage patterns of alternative exons.

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.

Alternative splicing contributes to the coordinated regulation of ferritin subunit levels in Bactrocera dorsalis (Hendel).

A constant ratio of ferritin heavy chain homolog (HCH) and light chain homolog (LCH) subunits seems to be required to compose the ferritin heteropolymer protein in insects. However, the mechanism by which insect LCH genes regulate protein levels remains unclear. We report that alternative promoters and alternative splicing contribute to maintaining a constant ratio of the two subunits, BdFer1HCH and BdFer2LCH (ferritin 1 HCH and ferritin 2 LCH), in Bactrocera dorsalis, a notorious quarantine pest. The genes BdFer1HCH and BdFer2LCH were identified with a series of potential transcription factor binding sites and were shown to be clustered within the genome in a "head to head" fashion. Thus, we unearthed a potential post-transcriptional mechanism to regulate the levels of LCH subunits, and confirmed that the expressions of BdFer1HCH and BdFer2LCH were induced by 20-hydroecdysone, iron overload, and immune challenge.

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

Molecular characterization and alternative splicing of a sodium channel and DSC1 ortholog genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels.