Guo-Qing Li

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.

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

ABSTRACT 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.

A Spodoptera exigua cadherin serves as a putative receptor for Bacillus thuringiensis Cry1Ca toxin and shows differential enhancement of Cry1Ca and Cry1Ac toxicity.

ABSTRACT Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.

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.

RNA Interference Depletion of the Halloween Gene Disembodied Implies its Potential Application for Management of Planthopper Sogatella furcifera and Laodelphax striatellus

Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

RNAi suppression of the ryanodine receptor gene results in decreased susceptibility to chlorantraniliprole in Colorado potato beetle Leptinotarsa decemlineata.

Leptinotarsadecemlineata is the most important pest in potato and causes serious yield loss each year. Chlorantraniliprole acts on insect ryanodine receptors (RyRs) and is among the most active compounds against L. decemlineata. Here we cloned and characterized a 15,792-bp full-length LdRyR cDNA that encoded a 5128-amino acid protein. LdRyR shares 85-92% amino acid similarities with other insect RyR homologues, and 59-61% similarities with those from Caenorhabditis elegans and Homo sapiens. All hallmarks of the RyR proteins are conserved in LdRyR. LdRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR domain and a RIH-associated domain in the N-terminus, and it possesses two consensus calcium ion-binding EF-hand motifs and six predicted transmembrane helices in the C-terminus. Temporal, spatial and tissue-specific expression patterns of LdRyR were evaluated. LdRyR expression level was increased constantly from egg to wandering stages, dropped in pupal stage and was increased again in the adult stage. It was widely expressed in the head, thorax and abdomen of day 3 fourth-instar larvae. Moreover, it was ubiquitously expressed in all inspected tissues including epidermis, foregut, midgut, ileum, rectum, fat body, ventral ganglia and Malpighian tubules in day 3 fourth-instar larvae. Dietary introduction of double-stranded RNA of LdRyR significantly reduced the mRNA levels of the target gene in the larvae and adults, respectively, and significantly decreased chlorantraniliprole-induced mortalities. Thus, our results suggested that LdRyR encoded a functional ryanodine receptor in L. decemlineata.

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.