Hu Wan

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

Insecticide resistance monitoring and correlation analysis of insecticides in field populations of the brown planthopper Nilaparvata lugens (stål) in China 2012–2014

The brown planthopper is a serious rice pest in China. Chemical insecticides have been considered a satisfactory means of controlling the brown planthopper. In the present study, we determined the susceptibility of twenty-one populations of Nilaparvata lugens to eleven insecticides by a rice-stem dipping method from 2012 to 2014 in eight provinces of China. These field-collected populations of N. lugens had developed high levels of resistance to imidacloprid (resistant ratio, RR=233.3-2029-fold) and buprofezin (RR=147.0-1222). Furthermore, N. lugens showed moderate to high levels of resistance to thiamethoxam (RR=25.9-159.2) and low to moderate levels of resistance to dinotefuran (RR=6.4-29.1), clothianidin (RR=6.1-33.6), ethiprole (RR=11.5-71.8), isoprocarb (RR=17.1-70.2), and chlorpyrifos (RR=7.4-30.7). In contrast, the susceptibility of N. lugens to etofenprox (RR=1.1-4.9), thiacloprid (RR=2.9-8.2) and acetamiprid (RR=2.7-26.2) remained susceptible to moderate levels of resistance. Significant correlations were detected between the LC50 values of imidacloprid and thiamethoxam, dinotefuran, buprofezin, and etofenprox, as well as between clothianidin and thiamethoxam, dinotefuran, ethiprole, acetamiprid, and thiacloprid. Similarly, significant correlations were observed between chlorpyrifos and etofenprox, acetamiprid and thiacloprid. Additionally, the activity of the detoxification enzymes of N. lugens showed a significant correlation with the log LC50 values of imidacloprid, dinotefuran and ethiprole. These results will be beneficial for effective insecticide resistance management strategies to prevent or delay the development of insecticide resistance.

Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme

In this study, we describe the cloning and characterization of a Prx from the common cutworm Spodoptera litura (SlPrx5). The SlPrx5 cDNA contains an open reading frame of 477 bp encoding a predicted protein of 159 amino acid residues, 16.902 kDa, and an isoelectric point of 7.68. Furthermore, the deduced amino acid sequence of the SlPrx5 cDNA showed 86% identity to Papilio xuthus Prx5, 72% to Aedes aegypti Prx5, and 64-67% to other insect Prxs. A phylogenetic analysis further revealed that the deduced amino acid sequence of SlPrx5 groups within the atypical 2-Cys Prx cluster. Recombinant SlPrx5 (20 kDa) purified from baculovirus-infected insect cells was found to reduce H2O2 in the presence of electrons donated by dithiothreitol and protect super-coiled DNA from damage by metal-catalyzed oxidation in vitro. During S. litura development, SlPrx5 is constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the fat body and midgut. Additionally, SlPrx5 mRNA expression was up-regulated after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. A disc diffusion assay indicated that recombinant SlPrx5 can play a functional role in protecting cells from oxidative stress in vivo. These results provide insight into the role of SlPrx5 during development and the oxidative stress response of S. litura.

A spider (Araneus ventricosus) chymotrypsin inhibitor that acts as an elastase inhibitor and a microbial serine protease inhibitor

Spider-derived Kunitz-type serine protease inhibitors have been shown to exhibit plasmin and elastase inhibition activity and potassium channel blocking activity, but thus far, no additional roles for spider-derived chymotrypsin inhibitors have been elucidated. In this study, a spider (Araneus ventricosus) chymotrypsin inhibitor (AvCI) that acts as an elastase inhibitor and a microbial serine protease inhibitor was identified. AvCI is a 70-amino acid mature peptide that displays eight conserved cysteine residues and a P1 lysine residue. Recombinant AvCI expressed in baculovirus-infected insect cells demonstrated inhibitory activity against chymotrypsin (Ki 49.85 nM), but not trypsin, which defines a role for AvCI as a spider-derived chymotrypsin inhibitor. AvCI also exhibited inhibitory activity against microbial serine proteases such as subtilisin A (Ki 20.51 nM) and proteinase K (Ki 65.42 nM). Furthermore, AvCI exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or plasmin; however, AvCI strongly inhibited human neutrophil elastase (Ki 8.74 nM) and porcine pancreatic elastase (Ki 11.32 nM), indicating that AvCI acts as an anti-elastolytic factor. These findings constitute molecular evidence that AvCI acts as an inhibitor against chymotrypsin, microbial serine proteases, and elastases. This paper provides a novel view of the functions of a spider-derived chymotrypsin inhibitor.

Developmental regulation and antifungal activity of a growth-blocking peptide from the beet armyworm Spodoptera exigua

Insect cytokine growth-blocking peptides (GBPs) are involved in growth regulation and the innate immune response. However, the microbial binding and antimicrobial activities of GBPs remain unclear. Here, we investigate the developmental role and antifungal activity of a GBP from the beet armyworm Spodoptera exigua (SeGBP). Sequence analysis predicted that mature SeGBP consists of 24 amino acid residues, including 2 cysteine residues. During S. exigua development, SeGBP is constitutively expressed in the fat body during the larval and adult stages but not in pupae. SeGBP expression is up-regulated by 20-hydroxyecdysone and down-regulated by juvenile hormone analog. Recombinant SeGBP purified from baculovirus-infected insect cells retards the growth of S. exigua larvae. Additionally, SeGBP expression is acutely induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. Recombinant SeGBP can bind to B. bassiana but not to E. coli or B. thuringiensis. Consistent with these findings, SeGBP shows antifungal activity against B. bassiana. Therefore, these results provide insight into the role of SeGBP during the innate immune response following microbial infection, and furthermore, they suggest a novel function for SeGBP as a direct antifungal agent against entomopathogenic fungi, such as B. bassiana.

Comparative study of two thioredoxins from common cutworm (Spodoptera litura): cloning, expression, and functional characterization.

Thioredoxins (Trxs) are a ubiquitous family of antioxidant enzymes that are involved in protecting organisms against various oxidative stresses. Here, we cloned and characterized two thioredoxins, named SlTrx1 and SlTrx2, from the common cutworm Spodoptera litura. SlTrx1 and SlTrx2, respectively, consist of 988 and 606 bp full-length cDNA with 318 and 447 bp open reading frames encoding 106 and 149 amino acid residues. Furthermore, the N-terminal region of SlTrx2 contains a predicted mitochondrial localization signal (33 amino acids). A phylogenetic relationship analysis revealed that SlTrx1 is in the cytosolic thioredoxin Trx1 cluster, whereas SlTrx2 is in the mitochondrial thioredoxin Trx2 cluster. Recombinant SlTrx1 (14 kDa) and SlTrx2 (16 kDa), expressed in baculovirus-infected insect Sf9 cells, demonstrated insulin disulfide reductase activity at the same optimum temperature and pH value of 35 °C and 7.0, respectively, in vitro. During S. litura development, we found that SlTrx1 and SlTrx2 had similar transcript expression patterns and were constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the epidermis and midgut. In addition, both SlTrx1 and SlTrx2 mRNA were up-regulated in S. litura after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. These results suggest that SlTrx1 and SlTrx2 function as potent antioxidant enzymes, and provide a molecular basis for the roles SlTrx1 and SlTrx2 during development and the oxidative stress response of S. litura.

Sublethal effects of buprofezin on development and reproduction in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae)

In the present study, the effects of sublethal concentrations of buprofezin on life-table traits of S. furcifera were evaluated for two consecutive generations (F0 and F1). Our results exhibited that the fecundity, life span (longevity) and hatchability of the F0 and F1 generations were significantly decreased at LC30 compared to the control. However, copulation was not significantly affected for the F0 or F1 generations at sublethal concentrations. The female life span was affected negatively at both treatments in F0 and at LC30 in F1, compared to the control. Furthermore, significant effects of the sublethal concentrations were found on the developmental rate of all instars except the 3rd instar of F1. However, the pre-adult period, total pre-oviposition period (TPOP) and adult pre-oviposition period (APOP) significantly increased in F1 individuals at LC30 and LC10 compared to the control. Our findings revealed that demographic characters (survival rate, intrinsic rate of increase (ri), finite rate of increase (λ), net reproductive rate (R0), and gross reproductive rate (GRR)) of the F1 generation (from F0 parents) significantly decreased compared to the untreated group; however, the generation time (T) increased at LC10. Therefore, the results suggested that buprofezin could adversely affect individuals in the successive generation.

Fitness and inheritance of metaflumizone resistance in Plutella xylostella

The diamondback moth, Plutella xylostella (L.) has developed resistance to many types of insecticides in the field. To study inheritance and fitness cost of metaflumizone resistance, a susceptible strain of diamondback moth was continuously selected with metaflumizone during 37 generations under laboratory conditions. The resistance to metaflumizone was at a high level (resistance ratios from 250.37 to 1450.47-fold). We investigated a metaflumizone resistance strain (G27) and a susceptible strain of P. xylostella, using the age-stage, two-sex life table approach. Compared to the susceptible strain, egg duration, the developmental time of the first and second instar larvae, pupae duration, adult preoviposition period (APOP), total preoviposition period (TPOP), egg hatchability, the survival rate of second instar larva and the mean generation time (T) were significantly differences in the resistant strain. The resistant strain had a relative fitness of 0.78. The inheritance of metaflumizone resistance was also studied by crossing the metaflumizone resistant and susceptible populations. Results revealed an autosomal and incompletely recessive mode of inheritance for metaflumizone resistance in the resistant population of P. xylostella. The present study provided useful information for planning potential management strategies to delay development of metaflumizone resistance in P. xylostella.

Cloning, expression, and functional analysis of two acetylcholinesterase genes in Spodoptera litura (Lepidoptera: Noctuidae)

Two acetylcholinesterase genes (SlAce1 and SlAce2) were cloned from Spodoptera litura, which is an important pest that causes widespread economic damage to vegetables and ornamental plants. We analyzed their expression patterns and compared their biological functions by using RNA interference. Our results showed that SlAce1 and SlAce2 cDNA contains 2085bp and 1917bp nucleotides and encoding proteins of 694 and 638 amino acid residues, respectively. Phylogenic analysis indicated that the lineage of SlAce genes and SlAce1 was completely different from SlAce2. Although both genes were expressed in all developmental stages and majorly in the brain. The expression levels of the both genes were suppressed by inserting their related dsRNA in the 6th instar larvae, which led to 47.3% (SlAce1) and 37.9% (SlAce2) mortality. Interestingly, the suppression of the SlAce2 transcripts also led to significant reductions in the fecundity, hatching, and offspring in the parental generation of S. litura. It is concluded that SlAce2 is responsible for the hydrolysis of acetylcholine and also plays role in female breeding, embryo progress, and the development of progeny. Considerable larval mortality was observed after both AChE genes (i.e. Ace1 and Ace2) were silenced in S. litura confirms its insecticidal effectiveness, which provided a molecular basis in biological pest control approach.