Pseudomonas mosselii: a potential alternative for managing pyrethroid-resistant Aedes aegypti.

BACKGROUND: Aedes aegypti is a widespread mosquito in tropical and subtropical regions that causes significant mortality and morbidity in humans by transmitting diseases, such as dengue fever and Zika virus disease. Synthetic insecticides, such as pyrethroids, have been used to control Ae. aegypti, but these insecticides can also affect nontarget organisms and contaminate soil and water. This study aimed to investigate the mosquitocidal activity of Pseudomonas mosselii isolated from pond sludge against larvae of Ae. aegypti. RESULTS: Based on the initial results, similar time-course profiles were obtained for the mosquitocidal activity of the bacterial culture and its supernatant, and the pellet resuspended in Luria-Bertani (LB) medium also showed delayed toxicity. These results imply that the toxic component can be released into the medium from live bacteria. Further research indicated that the toxic component appeared in the supernatant approximately 4 h after a 3-mL stock was cultured in 200 mL of LB medium. The stabilities of the P. mosselii culture and supernatant stored at different temperatures were also evaluated, and the best culture stability was obtained at 28°C and supernatant stability at 4°C. The bacterial culture and supernatant were toxic to larvae and pupae of not only susceptible Ae. aegypti but also pyrethroid-resistant strains. CONCLUSION: This study highlights the value of the mosquitocidal activity of P. mosselii, which has potential as an alternative insecticide to control pyrethroid-resistant Ae. aegypti in the field. This article is protected by copyright. All rights reserved.

Correlation between pyrethroid knockdown resistance and mutation frequency of voltage-gated sodium channel and its application in Aedes aegypti management.

Aedes aegypti, the primary vector responsible for transmitting dengue fever in southern Taiwan, has developed a relatively high resistance to synthetic pyrethroids. It has evolved four amino acid substitutions in the voltage-gated sodium channel (VGSC), namely S996P, V1023G, F1565C, and D1794Y. To unveil the distribution and correlation of VGSC mutations and pyrethroid resistance among different field populations, Ae. aegypti collected from various districts in Kaohsiung and Tainan Cities underwent tests for resistance development against different pyrethroids and frequency of S996P, V1023G, F1565C, and D1794Y substitutions. The adult knockdown assay revealed a relatively high knockdown resistance in the Ae. aegypti populations from Kaohsiung and Tainan against permethrin, cypermethrin, and fenvalerate (averaging >50-fold). Conversely, less resistance was observed against α-cypermethrin, deltamethrin, λ-cyhalothrin, cyfluthrin, and etofenprox (averaging <35-fold). Using Polymerase Chain Reaction/restriction fragment length polymorphism analysis, four mutant haplotypes were identified in these field populations. Notably, the SIAVFD and SIBVFD wild haplotypes were absent. Analysis utilizing IBM SPSS Statistics 20.0 and Spearman's rank correlation coefficient indicated that Haplotype C (PIAGFD), especially P allele, frequency displayed a significant positive correlation with five Type II pyrethroid resistance, while 1023G and 1023G/G exhibited a significant association with permethrin and fevalerate resistance. Conversely, Haplotype E (SIBVCD) negatively correlated with pyrethroid resistance, particularly fenvalerate resistance (-0.776). Haplotype C and E were the most prevalent and widely distributed among the investigated field populations. This prevalence of haplotype C is likely tied to the extensive and excessive use of Type II pyrethroids for dengue control over the past three decades. Given the significant positive correlation, the best-fit lines and R2 values were established to facilitate the swift prediction of knockdown resistance levels to various pyrethroids based on VGSC mutation frequency. This predictive approach aims to guide insecticide usage and the management of pyrethroid resistance in the field populations of Ae. aegypti in Taiwan.

Joint influence of architectural and spatiotemporal factors on the presence of Aedes aegypti in urban environments.

BACKGROUND: Urbanization has led to the proliferation of high-rise buildings, which have substantially influenced the distribution of dengue vectors, such as Aedes aegypti (L.). However, knowledge gaps exist regarding the individual and combined effects of architectural and spatiotemporal factors on dengue vector. This study investigated the interrelationship between Ae. aegypti presence, building architectural features, and spatiotemporal factors in urban environments. RESULTS: The mosquito Ae. aegypti presence varied by location and seasons, being higher in outdoor environments than in indoor environments. Lingya (Kaohsiung City, Taiwan) had the highest mosquito numbers, particularly in basement and first floor areas. Ae. aegypti was found on multiple floors within buildings, and their presence was greater in summer and autumn. The XGBoost model revealed that height within a building, temperature, humidity, resident density, and rainfall were key factors influencing mosquito presence, whereas openness had a relatively minor impact. CONCLUSION: To effectively address the problems caused by urbanization, the three-dimensional distribution of Ae. aegypti, including their spatial distribution across heights and areas within the urban environment, must be considered. By incorporating these multiple factors, this approach provides valuable insights for those responsible for urban planning and disease management strategies. Understanding the interplay between architectural features, environmental conditions, and the presence of Ae. aegypti is essential for developing targeted interventions and mitigating the adverse impacts of urbanization on public health. © 2023 Society of Chemical Industry.

Insecticide Resistance and Control Failure Likelihood Analysis in Plutella xylostella (Lepidoptera: Plutellidae) Populations From Taiwan.

The status of insecticide resistance levels is important for applying suitable pest management approaches. The present study investigated the insecticide resistance of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) populations from five major cruciferous growing areas in Taiwan. The surveyed locations were distributed from central to southern Taiwan and included Taichung, Changhua, Yunlin, Chiayi, and Kaohsiung. High levels of resistance to spinosad, indoxacarb, metaflumizone, and chlorantraniliprole were recorded among the surveyed populations except in Taichung. The resistance ratios ranged from 2.376 to 1,236-fold for spinosad and 24.63-1,511-fold for indoxacarb. Similarly, those for metaflumizone and chlorantraniliprole, were 2.563-76.21- and 4.457-683.0-fold, respectively. However, Bacillus thuringiensis subsp. aizawai, Bacillus thuringiensis subsp. kurstaki, emamectin benzoate, and diafenthiuron were still relatively effective against most field populations of P. xylostella. After approximately 10 generations of being maintained in the laboratory without exposure to insecticides, the resistance ratios of the Kaohsiung and Changhua populations declined to approximately 1.4-10-fold, and insecticides with control failure likelihood also began to show a negligible risk of control failure. Although spinosad, indoxacarb, metaflumizone, and chlorantraniliprole have lost their effectiveness in most field populations of P. xylostella in Taiwan, their effectiveness may be recovered in the absence of insecticide-selection pressure for approximately 10 generations. Therefore, we suggest that a constant survey of insecticide resistance and well-designed insecticide rotation based on the survey results are necessary for the effective control and insecticide resistance management of P. xylostella.

Knowledge about, attitude and acceptance towards, and predictors of intention to receive the COVID-19 vaccine among cancer patients in Eastern China: A cross-sectional survey.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has had a serious impact on health all over the world. Cancer patient, whose immunity is often compromised, faces a huge challenge. Currently, some COVID-19 vaccines are being developed and applied on general population; however, whether cancer patients should take COVID-19 vaccine remains unknown. Our study aimed to explore the knowledge, attitude, acceptance, and predictors of intention to receive the COVID-19 vaccine among cancer patients in Eastern China. METHODS: A cross-sectional study was conducted in Eastern China from June 17th to September 3rd, 2021. Patients were selected using a convenience sampling method. A self-report questionnaire was developed to assess knowledge about the COVID-19 vaccine, attitude towards the vaccine and acceptance of the vaccine; following a review of similar studies previously published in the scientific literature, multivariate logistic regression analysis was used to determine the predictors associated with COVID-19 vaccine acceptance. RESULTS: A total of 2158 cancer patients were enrolled in this study. The rate of vaccine hesitancy was 24.05% (519/2158); further, among the participants of vaccine acceptance, 767 had taken COVID-19 vaccine (35.54%), and 872 were willing to get vaccinated (40.01%). A total of 24 variables including demographic characteristics, clinical status of cancer, impact of COVID-19 pandemic on study participants, patients' knowledge about the COVID-19 vaccine, and attitude towards the vaccine, had significant differences between the "vaccine hesitancy" population and "vaccine acceptance" population. Multivariate logistic regression analysis indicated that parameters including alcohol consumption (odds ratio [OR] = 1.849; 95% confidence interval [CI]: 1.375-2.488; P-reference [P-Ref] < 0.001 vs non-drinkers), income impacted by COVID-19 pandemic (OR = 1.930, 2.037 and 2.688 for mild, moderate, and severe impact, respectively; all P-Ref < 0.01 vs no impact), knowledge of how the vaccine was developed (OR = 1.616; 95% CI: 1.126-2.318; P-Ref = 0.009 vs unknown), believing in the safety of the vaccine (OR = 1.502; 95% CI: 1.024-2.203; P-Ref = 0.038 vs denying the safety of vaccine), willingness to pay for the vaccine (OR = 3.042; 95% CI: 2.376-3.894; P-Ref < 0.001 vs unwilling), and willingness to recommend families and friends to get vaccinated (OR = 2.744; 95% CI: 1.759-4.280; P-Ref < 0.001 vs do not recommend) were contributors to vaccine acceptance. While such as being retired (OR = 0.586; 95% CI: 0.438-0.784; P-Ref < 0.001 vs unemployed), undergoing multiple therapies of cancer (OR = 0.408; 95% CI: 0.221-0.753; P-Ref = 0.004 vs no ongoing treatment), and worrying that the vaccine might deteriorate the prognosis of cancer (OR = 0.393; 95% CI: 0.307-0.504; P-Ref < 0.001 vs might not) were contributors to vaccine hesitancy. CONCLUSION: This study provided preliminary estimates of the rates of vaccine acceptance and vaccine hesitancy among cancer patients in Eastern China. The intention to receive the COVID-19 vaccine was impacted by factors such as patient occupation, alcohol consumption, and some parts of knowledge about and attitude towards COVID-19 vaccine. It is recommended to develop individualized vaccination plans that meet the healthcare needs of cancer patients.

Introduction of a cold sensitivity-conferring mutation into the RTA-Bddsx hybrid system of Bactrocera dorsalis for establishment of a thermally controllable homozygous line.

BACKGROUND: For efficient control of the economically important fruit pest Bactrocera dorsalis, a hybrid system combining ricin toxicity and sex-related alternative splicing of the doublesex gene has been developed. This system exhibits the expected female-specific lethal effect; however, the transgenic females do not survive, making it difficult to raise stable homozygous lines. Since modification of ricin toxin A chain (RTA) through a single-residue change (Gly212 > Arg212 ) leads to cold-sensitive posttranslational repression of its toxicity, we utilized this unique property to obtain RTA-Bddsx females that survive at low temperature for line maintenance. RESULTS: In transient expression experiments using embryonic injection, two groups treated with RTAcs-derived DNA (LERQcs and RTAcs) exhibited temperature-dependent effects. The toxicity was higher at 29 °C than at 18 °C. The proportion of males was close to 50% at 18 °C in all the tested groups except LERQcs-treated flies, which exhibited a high proportion of males (over 70%) at 29 °C. The results indicate the cold-sensitive responses of RTA and further suggest a female-specific lethal effect. Subsequently, 14 putative RTAcs-Bddsx transgenic Ds-Red+ G1 males were identified, and female-specific lethal effects were observed in Ds-Red+ G2 and G3 lines under cultivation at 29 °C but not at 18 °C. The male ratio can be increased to up to 95% in G3 line 001, indicating that RTAcs functions well in B. dorsalis. CONCLUSION: The improved RTAcs-Bddsx system with conditional toxicity represents a novel and promising step toward the practical control of B. dorsalis. © 2021 Society of Chemical Industry.

Effect of imidacloprid exposure on life history traits in the agricultural generalist predator Paederus beetle: Lack of fitness cost but strong hormetic effect and skewed sex ratio.

A trade-off between life history traits in the evolution of insecticide resistance is common in insects because energy acquisition is mainly channeled for detoxification enzyme production. In addition, sublethal exposure to insecticides may have an effect on the physiology and behaviors of surviving insects. Similar to other agricultural pests, pesticide use may have led to insecticide resistance in populations of Paederus fuscipes Curtis. In this study, we determined the median lethal time of 10 field-collected strains in Taiwan for three insecticides that are commonly employed to manage agricultural pests. We determined that the susceptibility of these strains to cyhalothrin and fenitrothion were similar, with resistance ratios (RRs) ranging from 1 to 4; however, significantly different to imidacloprid (RRs: 1-16). The effect of imidacloprid resistance on the life history traits studied of Paederus beetles was limited; only a prolonged egg incubation period, and adult longevity decreased as imidacloprid resistance increased. Regarding sublethal exposure to imidacloprid, adult sex ratios were female biased in most combinations, though nonsignificant. The quality of offspring, particularly the length of eggs significantly decreased. In addition, a hormetic effect was apparent when the individual was exposed to LT25 and LT50; mean fecundity per female increased from 12.80 ±â€¯8.95 (±â€¯standard error [SE]) to 42.70 ±â€¯13.77 eggs compared with that of the control (7.10 ±â€¯1.32). However, the hormetic effect was inconsistent among the tested strains, possibly because of the difference in insecticide resistance levels given that reproductive compensation was absent among the resistant population.

Inheritance and stability of mevinphos-resistance in Plutella xylostella (L.), with special reference to mutations of acetylcholinesterase 1.

Diamondback moth (Plutella xylostella L.) causes enormous damage on cruciferous vegetables and can rapidly develop resistance to all kinds of insecticides. To effectively manage the insecticide resistance of P. xylostella, an understanding of its inheritance and stability is essential. Here we investigated the phenotypic and genotypic basis of mevinphos resistance by crossing two genetically pure lines of P. xylostella, an SHggt wild-type strain and an SHMTCN resistant strain carrying 892T/T, 971C/C, and 1156T/G (TCN) mutations of the acetylcholinesterase 1 gene (Pxace1). Similar median lethal concentrations and degrees of dominance in the reciprocal cross progeny, and no plateau on the log concentration-probit line of F1 backcross and self-cross progeny, suggest that the mevinphos-resistance in P. xylostella is inherited as an autosomal and incomplete dominant trait governed by more than one gene. In the absence of mevinphos exposure, the resistance ratio and Pxace1 mutation frequency declined concomitantly in the SHMTCN strain. After 20-generation relaxation, the mevinphos resistance decreased from 52- to 6-fold and the Pxace1 mutation frequency of the TCN haplotype pair decreased from 100% to 0%. A good correlation was found between the resistance ratio and TCN frequency within the range of 12.5- to 25-fold resistance. Since there was no TCN haplotype pair detected below a resistance level of 12.5-fold, we speculate that resistance mechanisms other than target site insensitivity may exist. These observations are important for the prediction and management of mevinphos and related organophosphate resistance in field populations of P. xylostella.

Genetically Engineered Ricin Suppresses Bactrocera dorsalis (Diptera: Tephritidae) based on Demographic Analysis of Group-Reared Life Table.

The oriental fruit fly, Bactrocera dorsalis (Hendel), reduces the quantity and quality of many host fruits through the process of oviposition and larval feeding, and this insect has been considered a major insect pest in several Asian countries for decades. Using an earlier-developed, female-specific system that combines the toxicity of the ricin A chain (RTA) and the alternative RNA splicing property of doublesex ( Bddsx ), we show that transgenic male flies harboring the RTA-Bddsx transgene unevenly repress the pest population through inheritable effects. In age-stage, two-sex life-table analyses, high larval mortality and a delay in pupation were observed after introducing the transgene. The high male to female ratio in DsRed + flies demonstrates the lethal effect of ricin on females. The fitness of both the DsRed + - and DsRed - -transformed females was reduced as shown in the decrease of the net reproductive rate ( R0 ), intrinsic rate ( r ), and finite rate (λ) values compared with the wild-type populations. The integrity of the RTA-Bddsx transgene remained in more than 80% of DsRed + males after ten generations, supporting the stable inheritance of the transgene. All of the data from this study support the proposed RTA-Bddsx SIT approach, which provides a species-specific and environmentally friendly method of suppressing, rather than eradiating, B. dorsalis.

Establishment of an RTA-Bddsx hybrid system for female-specific splicing that can affect the sex ratio of Bactrocera dorsalis (Hendel) after embryonic injection.

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel), a very destructive insect pest in many areas of Asia, including Taiwan, can cause significant damage by ovipositing in and larval feeding on many kinds of fruit. A female lethal system, combining the splicing property of doublesex (dsx) with the toxicity of ricin A chain (RTA), has been developed. In this system, a modified RTA is separated by Bddsx intron 3; the expressed RNA can only be spliced in females, with toxic effects, whereas the immature RTA in males is harmless. RESULTS: Two RTA-Bddsx constructs, clone BE 24-7 and clone CF 26-21, containing Bddsx intron 3 and its flanking exonic sequences, with four nucleotides at the 5'-end and five nucleotides at the 3'-end, correctly spliced in a sex-specific manner. Wild-type and modified RTAs expressed in an Escherichia coli system retained their ability to suppress protein synthesis: 90.4% for Ricin-WT, 71.3% for Ricin-LERQ and 58.0% for Ricin-FEGQ. Embryonic injection of Acp-CF26-21, the RTA-Bddsx gene driven by the actin 5C promoter, resulted in a significant increase in male percentage in the eclosed adults. CONCLUSION: Our results indicate that the RTA-Bddsx hybrid system offers a novel and promising approach for oriental fruit fly control.

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. © 2015 Society of Chemical Industry.

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. © 2015 Society of Chemical Industry.

Amino acid substitutions and intron polymorphism of acetylcholinesterase1 associated with mevinphos resistance in diamondback moth, Plutella xylostella (L.).

The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field.

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.

Lipase catalyzed acetylation of 3,5,4'-trihydroxystilbene: optimization and kinetics study.

The use of immobilized lipase from Candida antarctica (Novozym(®) 435) to catalyze acetylation of trans-3,5,4'-trihydroxystilbene was investigated in this study. Response surface methodology and 5-level-4-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, including reaction time (24-72 h), temperature (25-65 °C), substrate molar ratio (1:15-1:75), and enzyme amount (600-3,000 PLU) on the percentage molar conversion of trans-4'-O-acetyl-3,5-dihydroxystilbene. The results showed that reaction temperature and enzyme amount were the most important parameters on percentage molar conversion. Based on ridge max analysis, the optimum conditions for synthesis were: reaction time 60 h, reaction temperature 64 °C, substrate molar ratio 1:56 and enzyme amount 2,293 PLU. The molar conversion of actual experimental values was 95% under optimal conditions. The synthesis product was analyzed using HPLC, mass and NMR. The results revealed that the major product was trans-4'-O-acetyl-3,5-dihydroxystilbene. The reaction kinetics was found to follow the Ping-Pong mechanism; substrate inhibition was not found at high vinyl acetate concentration.

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.

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.

A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti.

To identify pertinent mutations associated with knockdown resistance to permethrin, the entire coding sequence of the voltage-gated sodium channel gene Aa-para was sequenced and analyzed from a Per-R strain with 190-fold resistance to permethrin and two susceptible strains of Aedes aegypti. The longest transcript, a 6441bp open reading frame, encodes 2147 amino acid residues with an estimated molecular mass of 241kDa. A total of 33 exons were found in the Aa-para gene over 293kb of genomic DNA. Three previously unreported optional exons were identified. The first two exons, m and n, were located within the intracellular domain I/II, and the third, f', was found within the II/III linkers. The two mutually exclusive exons, d and l, were the only alternative exons in all the cDNA clones sequenced in this study. The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain. The high frequency and coexistence of the two mutations in the Per-R strain suggest that they might exert a synergistic effect to provide the knockdown resistance to permethrin. Furthermore, both cDNA and genomic DNA data from the same individual mosquitoes have demonstrated that RNA editing was not involved in amino acid substitutions of the Per-R strain.

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.