Wen-Chao Guo

Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)

BackgroundL. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato.ResultsThe expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1α EF1α) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR.ConclusionsThe expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.

RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say)

In Leptinotarsa decemlineata, juvenile hormones (JHs) play primary roles in the regulation of metamorphosis, reproduction and diapause. In JH biosynthetic pathway in insect corpora allata, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a potent feedback inhibitor S-adenosyl-L-homocysteine (AdoHcy). Rapid removal of AdoHcy is hypothesized to be essential for JH synthesis. AdoHcy hydrolase (SAHase) is the only eukaryotic enzyme catalyzing the removal. In the present paper, we firstly cloned a putative LdSAHase gene from L. decemlineata. The cDNA consists of 1806 bp and encodes a 525 amino acid protein. LdSAHase was expressed in all developmental stages. The gene had the highest and the lowest level of transcription respectively in the 3rd- and 4th-instars heads that contain corpora allata, which was positively correlated with JH titer in the haemolymph and the mRNA level of a JH early-inducible gene, the Krüppel homolog 1 gene (Kr-h1). Secondly, dietary ingestion of bacterially-expressed LdSAHase-dsRNA significantly decreased LdSAHase and LdKr-h1 mRNA levels, reduced JH titer, and caused the death of the larvae, and the failure of pupation and adult emergence. After continuous exposure for 12 days, 42% of the larvae died, 65% of the prepupae failed to pupate and 100% of the pupae failed to emerge. Moreover, RNAi-mediated LdSAHase knockdown also reduced larval developing time, and decreased larval weight. Lastly, application of JH analogue pyriproxyfen to LdSAHase-dsRNA-exposed larvae did not greatly increase LdSAHase expression level and JH content, but up-regulated LdKr-h1 mRNA level. Expectedly, pyriproxyfen application could partially rescue the negative effects on the survival and the development. Thus, our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Chlorantraniliprole Susceptibility in Leptinotarsa decemlineata in the North Xinjiang Uygur Autonomous Region in China

ABSTRACT n The Colorado potato beetle (Leptinotarsa decemlineata (Say)) in the north Xinjiang Uygur autonomous region has evolved resistance to various types of insecticides. Chlorantraniliprole is a novel anthranilic diamide insecticide that binds and activates ryanodine receptors. It exhibited excellent efficacy against L. decemlineata in several field trails in Europe. In the present paper, the susceptibility of L. decemlineata fourth-instar larvae derived from six field populations and L. decemlineata adults derived from three field populations to chlorantraniliprole was determined by a topical application. The fourth-instar larvae were substantially more susceptible to chlorantraniliprole than adults, although the range of susceptibility was far greater among the fourth-instar larvae. Regarding stomach toxicities, adult beetles were less susceptible to chlorantraniliprole than larvae. Chlorantraniliprole was most toxic to second-instar larvae, followed by third- and fourth-instar larvae. These data suggested that the appropriate timing for chlorantraniliprole spraying is the early larval stage. More-over, the synergistic activities of chlorantraniliprole in combination with triphenyl phosphate, diethyl maleate, or piperonyl butoxide against fourth-instar larvae from two field populations and adults from one field population were tested. Piperonyl butoxide had synergistic effects with chlorantraniliprole against fourth-instar larvae but not against adult beetles. Conversely, triphenyl phosphate and diethyl maleate exerted little synergistic effects. It appears that there is a potential risk of resistance against chlorantraniliprole resulting from cytochrome P450 monooxygenase activity.

Insecticide Resistance Status of Colorado Potato Beetle (Coleoptera: Chrysomelidae) Adults in Northern Xinjiang Uygur Autonomous Region

ABSTRACT Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), has become the economically most important insect defoliator of potatoes, Solanum tuberosum L., in northern Xinjiang Uygur autonomous region in China. Currently, control of Colorado potato beetle relies mainly on chemical insecticides. And this may result in insecticide resistance. In this study, LD50 values were measured by a topical bioassay for 14 conventional insecticides in seven local populations from Urumqi, Changji, Tacheng, Nilka, Gongliu, Qapqal, and Tekes counties (cities). The Tekes field population was the most susceptible population and was selected as a reference strain. Compared with the Tekes strain, the Changji, Qapqal, Nilka, Tacheng, and Gongliu populations exhibited moderate to very high levels of resistance to cyhalothrin. The Qapqal and Changji populations showed a moderate and a very high resistance to deltamethrin, respectively. And the Changji population developed a high resistance against &agr;-cypermethrin. Moreover, the Qapqal population had a moderate resistance to carbofuran, and the Urumqi population reached high level of resistance to endosulfan. Possible resistance mechanisms of the Changji and Qapqal populations were determined using three enzyme inhibitors. Triphenyl phosphate (TPP), diethylmeleate, and piperonyl butoxide (PBO) had little synergism to cyhalothrin in the two populations. In contrast, PBO and TPP exhibited some synergistic effects to carbofuran in the Qapqal population, indicating the involvement of monooxygenases and esterases in conferring carbofuran resistance. It seems that additional mechanisms, such as target site insensitivity, should play an important role in Colorado potato beetle resistances to cyhalothrin and carbofuran in northern Xinjiang local populations.

Colorado Potato Beetle Leptinotarsa decemlineata (Say)

Colorado potato beetle (CPB) was naturally dispersed from Kazakhstan into Xinjiang Autonomous Region of China in 1993. Since then, it has been widely distributed all over the southern region of the Tianshan Mountain. Chinese scientists focused on its monitoring and invasion risk management in China, as well as its invasion biology and ecology related to rapid dispersal, such as developmental threshold and cumulative temperature, diapause condition, and influence factors for flight. In invaded regions of China, several techniques such as improved crop cultivation techniques, friendly environmental chemical control (low or none toxic insecticides), biological control, physical techniques, ecological regulations etc. can be combined into an integrated pest management of CPB. However, some questions still remain in theses fields, for example, the genetic variations, environment (hosts, habitats, climates, soil etc.) adaptabilities and geographical populations of CPB are still unclear, and mechanisms of CPB’s rapid resistance development to pesticides have not been well understood. The concerned diapause mechanisms are still unknown. Moreover, the interactions between CPB, hosts, pathogens, predators and parasitoids, environments have not been studied, and the resistance or tolerance of potato plants to CPB and their related mechanisms all need to be understood in order to breed CPB-resistant potato crops.

Identification of cytochrome P450 monooxygenase genes and their expression profiles in cyhalothrin-treated Colorado potato beetle, Leptinotarsa decemlineata.

Based on a Leptinotarsa decemlineata transcriptome dataset and the GenBank sequences, a total of 74 cytochrome P450 monooxygenase genes (Cyps) were identified. These genes fell into CYP2 clan, mitochondrial clan, CYP3 clan and CYP4 clan, and were classified into 19 families and 35 subfamilies according to standard nomenclature. Two new families were discovered in CYP4 clan, and were named CYP412 and CYP413 respectively. Four new families that were recently discovered in Tribolium castaneum, including mitochondrial family CYP353, CYP3 clan families CYP345 and CYP347, and CYP4 clan family CYP350, were also found in L. decemlineata. The phylogenetic trees of CYPs from L. decemlineata and other representative insect species were constructed, and these trees provided evolutionary insight for the genetic distance. Our results facilitate further researches to understand the functions and evolution of L. decemlineata Cyp genes. In order to find cyhalothrin-inducible Cyp genes, the expression levels of Cyps belonging to CYP12, CYP6, CYP9 and CYP4 families were determined by quantitative reverse transcriptase-PCR in cyhalothrin-treated and control fourth-instar larvae. Nine Cyp genes, i.e., Cyp12H2, Cyp6BH2, Cyp6BJ1, Cyp6BQ17, Cyp6EG1, Cyp6EH1, Cyp6EJ1 Cyp4BN13v1 and Cyp4BN15, were highly expressed in cyhalothrin-treated larvae. These CYPs are the candidates that are involved in cyhalothrin detoxification.

Efficacy of endosulfan and fipronil and joint toxic action of endosulfan mixtures against Leptinotarsa decemlineata (Say)

The Colorado potato beetle (Leptinotarsa decemlineata [Say]) in the north Xinjiang Uygur autonomous region in China has evolved resistance to pyrethroids and carbamates. Mutations resulting in target site insensitivity, namely L1014F in LdVssc1 voltage-sensitive sodium channel and S291G in acetylcholine esterase, confer, at least partially, the resistance. Insecticides with different modes of action may serve as likely replacements. Endosulfan and fipronil are GABA-gated chloride channel-blocking insecticides. In this article, we found that the contact toxicities of the two compounds were among the highest ever estimated to the 4th-instar larvae and the adults and affirmed that they were useful for L. decemlineata control in north Xinjiang. Regarding stomach toxicities of the two compounds, adult beetles were less sensitive than 2nd-, 3rd-, and 4th-instar larvae, suggesting that the appropriate timing for spraying is the early larval stage. Mixtures of endosulfan and α-cypermethrin at 1:160, 1:80, and 1:40 ratios, of endosulfan and phoxim at 1:24 ratio, and of endosulfan and isocarbophos at 1:72 and 1:288 ratios, significantly increased toxicity in a field population. The combination indices were significantly below 1 at both LD50 and LD90 levels, revealing synergistic effects. Our results demonstrated that endosulfan and fipronil could be applied alone and endosulfan may also be used in binary mixtures to restore pyrethroid susceptibility. These findings may have considerable practical implications for L. decemlineata resistance management.

Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata

Leptinotarsa decemlineata adults exhibit a season‐dependent activity. In spring, post‐diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5′UTR and coding region, but differed in the lengths of 3′UTRs (515, 1 092 and 1 242 bp for isoforms‐1, ‐2 and ‐3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH‐like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH‐dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3′UTR of isoform‐3. The first, second and third polyadenylation sites gave rise to isoforms‐1, ‐2 and ‐3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform‐1, which expressed at low levels in pre‐diapause adults, became abundant in post‐diapause beetles. It is indicated that the LdProDH expression is fine‐tuned through 3′UTR to control proline catabolism for the season‐dependent activity of L. decemlineata adults.

Knockdown of juvenile hormone acid methyl transferase severely affects the performance of Leptinotarsa decemlineata (Say) larvae and adults

BACKGROUNDnJuvenile hormone (JH) plays a critical role in the regulation of metamorphosis in Leptinotarsa decemlineata, a notorious defoliator of potato. JH acid methyltransferase (JHAMT) is involved in one of the final steps of JH biosynthesis.nnnRESULTSnA putative JHAMT cDNA (LdJHAMT) was cloned. Two double-stranded RNAs (dsRNAs) (dsJHAMT1 and dsJHAMT2) against LdJHAMT were constructed and bacterially expressed. Experiments were conducted to investigate the effectiveness of RNAi in both second- and fourth-instar larvae. Dietary introduction of dsJHAMT1 and dsJHAMT2 successfully knocked down the target gene, lowered JH titre in the haemolymph and reduced the transcript of Krüppel homologue 1 gene. Ingestion of dsJHAMT caused larval death and weight loss, shortened larval developmental period and impaired pupation. Moreover, the dsJHAMT-fed pupae exhibited lower adult emergence rates. The resulting adults weighed an average of 50 mg less than the control group, and the females did not deposit eggs. Application of pyriproxyfen to the dsJHAMT-fed insects rescued all the negative effects.nnnCONCLUSIONSnLdJHAMT expresses functional JHAMT enzyme. The RNAi targeting LdJHAMT could be used for control of L. decemlineata.

Susceptibilities of Leptinotarsa decemlineata (Say) in the North Xinjiang Uygur Autonomous Region in China to Two Biopesticides and Three Conventional Insecticides

Abstract The Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) in the northern part of the Xinjiang Uygur Autonomous Region has evolved resistance to several pyrethroid and carbamate insecticides. Biological control methods should be a major component of integrated pest management for L. Decemlineata. Spinosad and abamectin are two biopesticides that have unique mechanisms of action. In this study, the contact toxicities of spinosad and abamectin to L. Decemlineata fourth instars and adults were determined by topical applications to several field populations. The average LD50 values of spinosad and abamectin for adults were 0.1275 and 0.0101 µg (a.i.) per individual, and for fourth instars they were 0.0181 and 0.0016 µg (a.i.) per individual, respectively. These data were among the lowest LD50s ever estimated, which affirmed that the two biopesticides are useful for L. Decemlineata control in north Xinjiang. Susceptibilities to the two biopesticides varied slightly but significantly among tested field populations and the variations did not result from cross-resistance to conventional insecticides. Regarding stomach toxicities, the LC50 values of spinosad applied to excised potato leaves for second instars, third instars, fourth instars, and adults were 0.2840, 0.4093, 1.2413, and 2.3783 mg/L (a.i.), respectively. The LC50 values of abamectin for second instars, third instars, fourth instars, and adults were 0.0036, 0.0088, 0.0177, and 0.2591 mg/L (a.i.), respectively. The two biopesticides were most toxic to second instars, followed by third, then fourth instars, and they were least toxic to adults. These data suggested that the appropriate timing for spinosad or abamectin spraying is to early larval stages.