Lixin Bai

Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses.

Heat shock cognate protein 70 (Hsc70) is a very important stress‐resistance protein of insects against environmental stresses. We employed fluorescent real‐time quantitative polymerase chain reaction and Western‐blot techniques to analyze the transcriptional and translational expression profiles of AlHSC70 under extreme temperature (4°C and 40°C) or 4 pesticide stresses in Apolygus lucorum. The results showed that the expression of AlHSC70 were significantly induced by cyhalothrin or extremely high temperature (40°C) in both transcriptional and translational levels (P < 0.05), while the transcriptional and translational level of AlHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature (4°C) (P < 0.05). Moreover, after Apolygus lucorum treated by imidacloprid or emamectin benzoate, the expression of AlHSC70 was only up‐regulated significantly at the transcriptional level (P < 0.05), although obviously up‐regulated at the translational level of AlHSC70. Therefore, this study confirmed that the Alhsc70 gene played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature (40°C). In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were shown in all the nymph and adult stages (P < 0.01), indicating temperature was an important factor to affect the relative expression of Alhsc70.

Sublethal effects of the chitin synthesis inhibitor, hexaflumuron, in the cotton mirid bug, Apolygus lucorum (Meyer-Dür).

Hexaflumuron is a type of benzoylphenylurea insecticide which is highly toxic for many insects. Sublethal doses of hexaflumuron have been shown to significantly affect insect growth and development. However, the action mechanism of hexaflumuron is not well understood. In the present study, first instar Apolygus lucorum nymphs were exposed to sublethal doses of hexaflumuron based on the estimated 120h acute LC50 valve of 20.53mg/ml. We found that exposure to sublethal hexaflumuron doses resulted in a significant increase in development time and reduced the weights of fifth instar A. lucorum nymphs. We also measured trehalose, which is a primary blood sugar in insects, and the enzyme trehalase that is involved in energy metabolism. Trehalose content in first instar nymphs significantly increased following hexaflumuron treatment while the glucose content, soluble trehalase activity and expression levels of ALTre-1 mRNA decreased significantly. However, no significant changes in membrane-bound trehalase activity and ALTre-2 mRNA expression were observed. In addition, these decreases or increases could be correlated to increases in treatment time or concentration of hexaflumuron, respectively. The present findings indicated that sublethal doses of hexaflumuron could interfere the normal carbohydrate metabolism by depressing the expression of ALTre-1 in A. lucorum, which provide valuable information on the physiology and molecular mechanisms for the toxicity of hexaflumuron.

Molecular characterisation of the vitellogenin gene (AlVg) and its expression after Apolygus lucorum had fed on different hosts.

BACKGROUND The polyphagous pest Apolygus lucorum is now the dominant pest of Bacillus thuringiensis (Bt) cotton in China. In this study, the transcriptional and translational profiles of AlVg influenced by different hosts were identified, and then the correlations between AlVg gene or AlVg protein expression and key population proliferation parameters of A. lucorum were further clarified. RESULTS AlVg or AlVg expression can be significantly regulated by different host nutrients (P < 0.05). AlVg or AlVg expression was significantly higher in A. lucorum reared on Bt and conventional cotton than in A. lucorum reared on garland chrysanthemum and broad bean (P < 0.05), but there was no significant difference between AlVg or AlVg expression in A. lucorum reared on Bt and conventional cotton (P > 0.05). In addition, there were significant linear regression correlations between AlVg or AlVg expression and total mortality rate of nymphs, female lifespan, per female fecundity and egg hatching rates (P < 0.05). CONCLUSION Our results confirm that AlVg or AlVg is the key parameter affecting female fertility of A. lucorum. AlVg and AlVg expression can be influenced by different host nutrients except for Bt toxin.

Molecular characterization and expression of vitellogenin gene from Spodoptera exigua exposed to cadmium stress

In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in Spodoptera exigua. The complete Vg cDNA consists of 5694 nucleotides with a long open reading frame encoding 1761 amino acid residues. The deduced amino acid sequence shared high similarity with the Vgs of other lepidopteran insects, particularly in the C-terminal region including the GL/ICG motif, five cysteine residues and DGXR motif. We analyzed the spatiotemporal expression of Vg transcripts in S. exigua by RT-PCR and Real-time Quantitative PCR. The results revealed that Vg was expressed specifically in the female fat body and was detectable after 5th day female pupae. The maximum level of Vg mRNA appeared in 48-h-old female adults and started to decrease quickly. When exposed to cadmium (Cd) (25-100mg/kg), there was a significantly decreased Vg level in female adults. The information presented in this study suggested the Vg in S. exigua could be significantly affected by Cd pressure.

Complete mitochondrial genome of the pink bollworm Pectinophora gossypiella (Lepidoptera: Gelechiidae).

Abstract Pectinophora gossypiella (Lepidoptera: Gelechiidae) is a key pest in many cotton-growing countries of the world. In this study, the complete mitochondrial (mt) genome of the pink bollworm P. gossypiella was determined, which is 15,202 bp in length (GenBank accession number: KM225795) containing 37 typical animal mitochondrial gene and an A + T-rich region. The gene order of P. gossypiella mtDNA was different from the insect ancestral gene order in the translocation of trnM, as shared by previously sequenced lepidopteran mtDNAs. The protein-coding genes (PCGs) have typical mitochondrial start codons ATN, with the exception of COI, Nad5, which uses the start codons CGA, GTT. Eight PCGs stop with complete termination codons (TAA), whereas five PCGs use incomplete stop codon T. All of the tRNA genes had typical cloverleaf secondary structures except for trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. Like other insects, the control region is located between rrnS and trnM with a length of 309 bp and an A + T content of 94.8%, which is the most AT-rich region and comparatively simple, with little evidence of long tandem repeats, but harbors a conserved structure combining the motif ATAGA and a 18-bp poly-T stretch.

MOLECULAR CHARACTERIZATION OF SOLUBLE AND MEMBRANE-BOUND TREHALASES IN THE COTTON MIRID BUG, Apolygus lucorum

Trehalose, a major hemolymph sugar in insects, is hydrolyzed by trehalase. We identified a soluble and a membrane-bound form of trehalase and isolated the corresponding mRNA, ALTre-1, and ALTre-2 in the cotton mirid bug, Apolygus lucorum. The deduced amino acid sequences of ALTre-1 and ALTre-2 revealed mature proteins with 643 and 617 amino acids, respectively. ALTre-1 and ALTre-2 contained trehalase signature motifs, and ALTre-2 contained a putative transmembrane domain near the C-terminus, suggesting that ALTre-1 and ALTre-2 encoded a soluble trehalase and a membrane-bound trehalase, respectively. Comparison of trehalase activity at different developmental stages and in six tissues indicated that soluble trehalase activity accounted for the majority of total trehalase activity in A. lucorum. ALTre-1 and ALTre-2 were expressed in all tissues and stages, with the highest expression of both in the second instar nymphs, ALTre-1 in the ovary and malpighian tubules, ALTre-2 in the flight muscles and fat body. Following the exposure of second instar nymph to 20-E, the soluble trehalase activity increased gradually while the membrane-bound trehalase activity remained at its initial level. Similarly, 20-E upregulated ALTre-1 expression but had no effect on ALTre-2 expression. These results suggest that an increase of this soluble trehalase activity was upregulated by ALTre-1 gene.

Complete mitochondrial genome of Cotton Leaf Roller Haritalodes derogata (Lepidoptera: Crambidae).

Abstract Haritalodes derogata (Lepidoptera: Crambidae) has been recorded as an important pest of cotton in many countries of the world. In this study, the complete mitochondrial (mt) genome of Cotton Leaf Roller Haritalodes derogata is determined, which is 15,253 bp in length (GenBank accession number: KC515397) containing 37 typical animal mitochondrial gene and an A + T-rich region. The gene order of H. derogata mtDNA was different from the insect ancestral gene order in the translocation of trnM, as shared by previously sequenced lepidopteran mtDNAs. The protein-coding genes (PCGs) have typical mitochondrial start codons ATN, with the exception of COI, Nad5, which uses the start codons CGA, GTT. In addition, five of 13 PCGs harbor the incomplete termination codons, a single T. All of the tRNA genes had typical cloverleaf secondary structures except for trnS1(AGN). Like other lepidopteran mtgenomes, the control region is located between rrnS and trnM with a length of 329 bp and an A + T content of 96%, which is the most AT-rich region and habors a conserved structure combining the motif ATAGA and a 14-bp poly-T stretch. Both maximum likelihood and Bayesian inference analyses highly support a sister-group relationship: Pyraustinae + (Spilomelinae + (Acentropina (Crambine + Schoenobiine))).

Molecular and functional characterization of the ecdysone receptor isoform-A from the cotton mirid bug, Apolygus lucorum (Meyer-Dur).

We cloned the cDNA of the ecdysone receptor (EcR) isoform-A from the mirid bug, Apolygus lucorum (AlEcR-A). The AlEcR-A cDNA has an open reading frame of 1410 bp with a conserved sequence of approximately 20 amino acids at the carboxyl-end of its A/B-specific domain. Phylogenetic analysis showed that AlEcR-A is very similar to the EcR-A genes of other Hemiptera species. AlEcR-A mRNA was detected at all developmental stages of A. lucorum with peaks correlating to ecdysteroid pulses. AlEcR-A was also expressed in all analyzed tissues with maximum expression in the epidermis and fat body. An AlEcR-A mRNA of size 1.8 kb was detected in all tissues by northern blot analysis. We investigated the functions of AlEcR-A in A. lucorum growth and development using RNAi in vivo. Weights of fifth instar nymphs were significantly decreased in insects treated with AlEcR-A specific anti-sense RNA. Mortality from third instar nymphs to adults increased significantly along with a significant increase in instar duration.

Discovery of Vg and VgR gene expression profiles related to host‐plant suitability of Spodoptera exigua

BACKGROUND The beet armyworm Spodoptera exigua, a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and the host plant is a basic and important component of early forecasting of beet armyworm outbreaks. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. RESULTS In this study, key life-history parameters of S. exigua reared on distinct hosts were investigated; the host plants could be ranked as lettuce > shallot > tomato > celery in their order of suitability. Full-length S. exigua vitellogenin receptor (SeVgR) cDNA was cloned, and sex-, stage- and tissue-specific expression characteristics were assessed. Spodoptera exigua vitellogenin (SeVg) and SeVgR expression levels were markedly modulated by host nutrients (P < 0.05). SeVg and SeVgR expression levels were significantly higher in S. exigua reared on lettuce, the most preferred and most nutritive host, than in those reared on tomato and celery. Interestingly, significant linear regression correlations were found between SeVg and SeVgR expression levels and key S. exigua life-history parameters, especially life span, pupa weight, and female fecundity (P < 0.01). CONCLUSION Host plant type and suitability could affect the expression pattern of SeVg and SeVgR, which influenced S. exigua fecundity. Vg and VgR have the potential to be used as molecular markers of S. exigua fecundity and for forecasting outbreaks of S. exigua on different hosts.

Phenotypic variation and identification of Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) in China.

Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is an invasive mealybug that seriously damages cotton and other important crops. In previous studies in China, the presence of two submedian longitudinal lines of pigmented spots on the dorsum of adult females frequently has been used to identify this species. However, the present study records the occasional absence of pigmented spots in a sample from Guangxi province, China. Specimens without pigmented spots showed all the molecular and morphological characters that separate P. solenopsis from the similar species P. solani Ferris, especially the distribution of multilocular disc pores. In different geographic populations of P. solenopsis in China, mitochondrial COI and nuclear 28SrDNA genes are very similar (99.8-100%), indicating that they are conspecific. For COI, the genetic distance between P. solenopsis and P. solani is more than 3%. A map of the distribution of P. solenopsis in China is given. To help identify both pigmented and non-pigmented P. solenopsis accurately, an identification key to the 16 species of Phenacoccus found in China is provided. The key also identifies five potentially invasive Phenacoccus species not yet established in China, in case they get introduced there.