Shu-Mei Dai

Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel).

A homologue of the doublesex gene (Bddsx) has been cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). Northern analysis indicates a differential expression of Bddsx in male and female flies, as reported for other dsx genes. A structural conservation of DNA binding domain/oligomerization domain 1 and oligomerization domain 2 suggests that the doublesex protein (BdDSX) of this fruit fly serves as a transcriptional factor for downstream sex-specific gene expression. The putative transformer/transformer-2 protein binding sequence in female-specific transcript suggests that a preserved alternative splicing process found in other flies mediates the synthesis of Bddsx transcript. RNA interference (RNAi) data from adult abdominal dsRNA injection assays indicate that female-specific dsx dsRNA reduces specifically its own transcript, inhibits selectively expression of the yolk protein gene (Bdyp1), and delays ovary development. The number of matured eggs is significant reduced after RNAi treatment, but the sex ratio of offspring is not biased. Moreover, 27% of female progeny with RNAi show deformed ovipositor, but male flies are not affected. Although this is a transient treatment, the specific Bddsx(f) interference offers a promising and novel approach to oriental fruit fly control in the future.

Responses of striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), from Taiwan to a range of insecticides

BACKGROUND Information on the insecticide susceptibility of striped stem borer, Chilo suppressalis (Walker), is essential for an effective pest management programme. An early detection of resistance development can prompt the modification of current control methods and increase the lifespan of insecticides through the rotation of chemicals with different modes of action. In this study, the susceptibility of this pest in Taiwan to four classes of insecticides has been examined. RESULTS Over 1000-fold resistance to carbofuran was detected in C. suppressalis collected from Chiayi and Changhua prefectures, with estimated LC(50) values of > 3 mg cm(-2). In addition, 61-fold resistance to cartap was found in the Chiayi population. On the other hand, all tested populations of rice stem borer were still relatively susceptible to chlorpyrifos, fipronil and permethrin, with LC(50) values ranging from 30 to 553 ng cm(-2). Chilo suppressalis populations collected from the central parts of Taiwan have a higher degree of resistance to the tested insecticides than those from northern areas. CONCLUSION The occurrence of high resistance to carbofuran in the Chiayi and Changhua areas suggests that this compound should be replaced with chemicals having a different mode of action, such as chlorpyrifos, fipronil and permethrin, to which low cross-resistance has been detected.

Display female-specific doublesex RNA interference in early generations of transformed oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most destructive pests in many Asian countries. An effective strategy to reduce fly density in the field is urgently required. Recently, the doublesex of B. dorsalis (Bddsx(f) ) has been cloned, and RNA interference (RNAi) indicates that it can reduce the offspring in vitro. In this study, a piggyBac-based construct that generates short hairpin RNA (shRNA) against the female-specific region of Bddsx was introduced into the pest to test the RNAi effects on reproductive functions in vivo. RESULTS After embryonic injection and backcross, 21 transgenic lines with germline transformation were identified. Genomic DNA analysis showed that the exogenous transgene including short hairpin Bddsx(f) and a DsRed marker had integrated into the genomes of 11 transformed lines. Northern blot analysis indicated the presence of Bddsx(f) short interfering RNA (siRNA) under the control of a U6 promoter in transformed flies. As expected, the specific effects of RNAi led to the delay of egg maturation, and the offspring was significantly reduced. Reverse transcription real-time PCR further demonstrated that in vivo interference not only specifically inhibited the Bddsx(f) transcript but also repressed expression of the downstream yolk protein gene (Bdyp1). CONCLUSION The results clearly indicate that RNAi is heritable through the expression of specific siRNA in early generations of transformed oriental fruit fly. These results can broaden the understanding of sex-related developmental mechanisms in the fly, and also offer a possible molecular approach for pest control in the future.

Amino acid substitutions of acetylcholinesterase associated with carbofuran resistance in Chilo suppressalis

BACKGROUND Over 1000-fold carbofuran resistance has been observed in Chilo suppressalis (Walker) collected from the Changhua (CH) and Chiayi (CY) prefectures of Taiwan. An understanding of the pertinent mechanisms will benefit effective insecticide resistance management of C. suppressalis. RESULTS Among the five amino acid substitutions of acetylcholinesterase (AChE) identified in C. suppressalis, A314S and H668P had been reported and E101D, F402V and R667Q were novel. Substitution frequencies in AChE of CH and CY populations were much higher than in the susceptible Hsinchu (HC) population. Significantly negative correlations were observed between the frequencies of E101D, A314S and R667Q and the kinetic parameters of AChEs in these populations. AChE from the resistant CH population was less susceptible to the inhibition of carbofuran, with an I50 that was 3.6-fold higher than that of the susceptible HC population. Although Km and Vmax of AChE from the CH and CY populations were reduced to 72-87% of those from the HC population, the overall catalytic efficiency (Vmax /Km ) remained constant for all three populations. CONCLUSION Amino acid substitutions identified in the AChE of C. suppressalis are associated with changes in AChE kinetics and its insensitivity to carbofuran. These observations are helpful for rapid monitoring, prediction and management of OP and CB resistance in the field.

Introduction of the RTA‐Bddsx gene induces female‐specific lethal effects in transformed Bactrocera dorsalis (Hendel)

BACKGROUND The oriental fruit fly, Bactrocera dorsalis (Hendel), can reduce fruit production and quality and is considered to be a major insect pest in many Asian countries. A system combining the toxicity of ricin and the alternative RNA splicing properties of doublesex (RTA-Bddsx) has been proposed that results in differential sexual processing in vitro. A transgenic approach was used in this study to confirm the existence of female-specific lethal effects in vivo. RESULTS The piggyBac-based vector PB-Acp-CF21-26, which carries the actin 5C promoter and RTA-Bddsx, was used to establish transgenic lines. Five surviving male flies (F1) demonstrated the presence of selection marker Ds-Red((+)) throughout their entire bodies following single-pair mating with wild-type females, indicating germline transmission. A high percentage of males (59.6-100%) were observed in transformed F3 offspring, and this skewed sex ratio indicated that the female-lethal effects of the RTA-Bddsx system were heritable and functioned well in B. dorsalis. Some transformed female flies were observed, and these unexpected results were attributed to the loss of the intact transgene after genomic PCR analyses. CONCLUSION This transgenic study provides direct evidence for the female-specific lethal effects of RTA-Bddsx in B. dorsalis and offers a novel and promising approach for the control of B. dorsalis in the future.

Distinct contributions of A314S and novel R667Q substitutions of acetylcholinesterase 1 to carbofuran resistance of Chilo suppressalis Walker

BACKGROUND In the striped stem borer, Chilo suppressalis, A314S, R667Q and H669P substitutions in acetylcholinesterase 1 (CsAChE1) have been associated with >1000-fold resistance against carbofuran. In this study, eight variants of CsAChE1 carrying different combinations of these substitutions were cloned and expressed using the Bac-to-Bac expression system to verify their contributions. RESULTS The expressed AChE1s had molecular weights of ca 160 kDa per dimer and 80 kDa per monomer. AChE kinetics and inhibition analysis showed that the A314S mutation was the key substitution responsible for a 15.1-fold decrease in hydrolytic activity to acetylthiocholine iodide and a 10.6-fold increase in carbofuran insensitivity of CsAChE. Compared with wild-type CsAChE1, this substituted CsAChE1 also showed 23.0-, 3.3- and 2.6-fold insensitivity to methomyl, triazophos and chlorpyrifos-oxon respectively. It should be noted that the R667Q substitution conferred a capability to increase the activity of wild-type and A314S-substituted CsAChE, while the A314S substitution reduced Km and compensated for overall catalytic efficiency. CONCLUSION With the enhancing activity of the R667Q substitution, A314S is the major CsAChE1 substitution responsible for fitness-cost compensation and increased insensitivity to AChE inhibitors. The lower insensitivity of A314S-substituted CsAChE1 to chlorpyrifos-oxon suggests that chlorpyrifos could be an alternative insecticide for managing carbofuran-resistant field C. suppressalis in Taiwan.