Identification and expression patterns of voltage-gated sodium channel genes with intron retentions in different strains of Bactrocera dorsalis.

Pyrethroid are the primary insecticides used for controlling of Bactricera dorsalis, a highly destructive and invasive fruit pest. Field populations have developed serious resistance, especially to ß-cypermethrin. While mutations in the voltage-gated sodium channel (Vgsc) are a common mechanism of pyrethroid resistance, variations in BdVgsc associated with ß-cypermethrin resistance remain unclear. Here, we reported the resistance levels of five field populations from China, with resistance ratio ranging from 1.54 to 21.34-fold. Cloning the full length of BdVgsc revealed no specific or known amino acid mutations between the most resistant population and the susceptible strain. However, three types of partial intron retention (IRE4-5, IRE19-f and IREL-24) were identified in BdVgsc transcripts, with these intron retentions containing stop codons. The expression of IRE4-5 transcripts and total BdVgsc showed different trends across developmental stages and tissues. Exposure to ß-cypermethrin led to increased expression of IRE4-5. Comparison of genomic and transcriptional sequences reveled that IRE4-5 transcripts had two types (IRE4-5.5 T and IRE4-5.6 T) caused by genomic variations. Both field and congenic strains indicated that homozygotes for IRE4-5.5 T had lower IRE4-5 transcript levels than homozygotes for IRE4-5.6 T. However, congenic and field strains exhibited inconsistent results about the association of expression levels of IRE4-5 transcripts with sensitivity to ß-cypermethrin. In summary, this study is the first to identify intron retention transcripts in the Vgsc gene from B. dorsalis and to examine their expression patterns across different developmental stages, tissues, and strains with varying sensitivities to ß-cypermethrin. The potential role of the intron retentions of BdVgsc in insecticide toxicity is also discussed.

The Food Source and Gut Bacteria Show Effects on the Invasion of Alien Pests-A Case of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

How alien pests invade new areas has always been a hot topic in invasion biology. The spread of the Bactrocera dorsalis from southern to northern China involved changes in food sources. In this paper, in controlled conditions, we take Bactrocera dorsalis as an example to study how plant host transformation affects gut bacteria by feeding it its favorite host oranges in the south, its favorite host peaches and apples in the north, and feeding it cucumbers as a non-favorite host plant, thereby further affecting their fitness during invasion. The result showed that, after three generations of feeding on cucumbers, Bactrocera dorsalis took longer to develop as a larva while its longevity and fecundity decreased and pre-adult mortality increased. Feeding it cucumbers significantly reduced the overall diversity of gut microbiota of Bactrocera dorsalis. The relative abundance of Enterobacter necessary for survival decreased, while the Empedobacter and Enterococcus increased, resulting in decreased carbohydrate transport and metabolism and increased lipid transport and metabolism. Feeding Bactrocera dorsalis Empedobacter brevis and Enterococcus faecalis resulted in a 26% increase in pre-adult mortality and a 2-3 d increase in adult preoviposition period (APOP). Additionally, Enterococcus faecalis decreased the longevity of female and male adults by 17 and 12 d, respectively, and decreased fecundity by 11%. We inferred that the shifted plant hosts played an important role in posing serious harm to Bactrocera dorsalis invading from the south to the north. Therefore, after an invasion of Bactrocera dorsalis into northern China, it is difficult to colonize cucumbers for a long time, but there is still a risk of short-term harm. The findings of this study have established that the interactions between an insect's food source and gut bacteria may have an important effect on insect invasions.

Chitosan-Delivered Chlorantraniliprole for Pest Control: Preparation Optimization, Deposition Behavior, and Application Potential.

Chitosan has emerged as a promising biopolymer carrier for the sustained release of pesticides owing to its good biocompatibility, biodegradability, and bioactivity. In this work, a controlled-release formulation of insecticide chlorantraniliprole was fabricated through coprecipitation-based synchronous encapsulation with chitosan, where the optimum preparation conditions, storage stability, deposition behavior, and application potential were investigated. Preparation of optimization data from response surface methodology showed high correlation coefficient (R2) of 0.9875 and adjusted coefficient (Radj2) of 0.9715. The resulting formulation displayed good loading content of 28.39%, high encapsulation efficiency of 75.71%, and good storage stability. Compared with the commercial suspension concentrate, the formulation exhibited better wettability and retention behaviors on plant leaves. Excitingly, effective control against one species of mealybug genus Paraputo Laing (outside the killing spectrum) on the Hippeastrum reticulatum plant was successfully achieved by spraying the controlled-release formulation at different time intervals. This work indicates the good potential of the developed formulation in expanding the application scope of chlorantraniliprole, which shows a new strategy for sustainable pest management.

Differences in morphology, mitochondrial genomes, and reproductive compatibility between two clades of parasitic wasps Aphelinus mali (Hymenoptera: Aphelindae) in China.

Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae) in China is comprised of two clades (termed, the Shandong and Liaoning clades). In order to clarify the genetic relationship between these two clades, we compared and analyzed the morphological characteristics and the mitochondrial genome of each, and performed a hybridization experiment. Morphological results showed that both males and females of the Liaoning clade were larger than Shandong clade, in terms of whole body, abdominal, wing and antennal lengths, however, there were no significant differences between clades for total length of the middle or hind leg of females. The length of the mitochondrial genome of the Shandong clade was 14415 bp and, for the Liaoning clade, it was 14804 bp. Each contained 31 genes, including 13 protein-encoded genes, 16 tRNA genes, and 2 rRNA genes. The highest AT level among the 13 protein-coding genes for the two clades were the same gene (ATP8) (Shandong clade, 91.52%; Liaoning clade, 90.91%). By hybridization and backcrossing, we found that there was no cross incompatibility between these two clades of A. mali. Our results indicate that the historic geographical isolation between these clades has not yet caused reproductive isolation of these populations, and they belong to the same species.

Monitoring the Methyl Eugenol Response and Non-Responsiveness Mechanisms in Oriental Fruit Fly Bactrocera dorsalis in China.

Bactrocera dorsalis is a notorious polyphagous pest in China, and its management strategies largely depend on methyl eugenol (ME), which has been widely used as an attractant to monitor and eradicate B. dorsalis populations for seven decades. However, the non-responsiveness levels in field B. dorsalis populations to ME is unknown. In this study, we monitored the response to ME in field populations from the four most heavily infested provinces in China, and the results showed that the populations had lower sensitivity to ME relative to GZS susceptible strain. The percent responsiveness of the lowest sensitivity population was 5.88-, 3.47-, and 1.47-fold lower relative to the susceptible strain at doses of 1, 10, and 100 µL of ME, respectively. Gene expression analysis and inhibitor assays further revealed that odorant binding protein (BdorOBP2, BdorOBP83b) and the P450 enzyme system may be associated with the lower response to ME. To our knowledge, this work is the first to report that the P450 enzyme system confers a lower responsiveness to lure insects. These findings provided valuable insights for exploiting ME non-responsiveness to protect sterile males from ME-based control strategies and the use of lures combined with insecticides.

Efficacy of RNA interference using nanocarrier-based transdermal dsRNA delivery system in the woolly apple aphid, Eriosoma lanigerum.

RNA interference (RNAi) is an essential approach for studying gene function and has been considered as a promising strategy for pest control. However, RNAi method has not been conducted in Woolly apple aphid (Eriosoma lanigerum Hausmann), one of the most damaging apple pests in the world. In the study, we investigated the efficacy of RNAi of V-ATPase subunit D (ATPD), an efficacious target for RNAi in other insects, in E. lanigerum by a transdermal double-stranded RNA (dsRNA) delivery system with nanocarriers. Our results showed although topical application of dsATPD in E. lanigerum for 24 h produced 40.5% gene silencing, the additional help of nanocarriers extremely improved the interference efficiency with 98.5% gene silencing. Moreover, a 55.75% mortality was observed 5 days after topical application of nanocarriers and dsATPD, relative to the control (topical application of nanocarriers and double-stranded green fluorescent protein [dsGFP]). The nanocarrier-based transdermal dsRNA delivery system will promote the development of functional analysis of vital genes and also provide a potential target for RNAi-based management of E. lanigerum.

Use of Age-Stage, Two-Sex Life Table to Compare the Fitness of Bactrocera dorsalis (Diptera: Tephritidae) on Northern and Southern Host Fruits in China.

Bactrocera dorsalis (Hendel), as a quarantine pest in many countries and regions, has shown a trend of northward diffusion in the past century in China. In order to determine whether B. dorsalis will cause great harm to the dominant northern fruits, the age-stage two-sex life tables of peaches and apples were constructed, with oranges as the control. The results showed that the developmental rate, intrinsic rate of increase (r), and finite rate of increase (λ) on oranges and peaches were significantly greater than on apples. Additionally, the prediction of population growth 90 days after oviposition revealed that the whole population on oranges and peaches increased by 13,667.3 and 12,112.1 times, respectively, indicating that B. dorsalis is very likely to endanger peach orchards. The population increased on apples by 4311 times, though this is lower than that on oranges and peaches. Overall, peaches with high fitness similar to oranges are very suitable as a host for B. dorsalis and are likely to become a new favorable host, while apples may also become a potentially new host, though with lower fitness. Therefore, the most pressing solutions to take are population monitoring, comprehensive prevention, and control in the case of any potential large-scale outbreak of B. dorsalis in northern China.

Slow resistance evolution to neonicotinoids in field populations of wheat aphids revealed by insecticide resistance monitoring in China.

BACKGROUND: The wheat aphids, Rhopalosiphum padi and Sitobion miscanthi, are serious agricultural insect pests of many crops. Neonicotinoid insecticides are commonly used as alternatives to organophosphate and pyrethroid insecticides for controlling wheat aphids. RESULTS: Long-term monitoring of resistance to imidacloprid and acetamiprid in R. padi and S. miscanthi was carried out between 2007 and 2019. For this study, 344 specimens of the two wheat aphids were collected from field populations found in the main wheat production areas in China, from 2007 to 2019. In R. padi, the fluctuation in resistance was 14.7 times for imidacloprid and 1.4 times for acetamiprid; in S. miscanthi, it was 9.7 times for imidacloprid and 6.5 times for acetamiprid. CONCLUSION: Our results demonstrated that both R. padi and S. miscanthi tended to have higher resistance to imidacloprid compared with acetamiprid. However, it is difficult for wheat aphids to develop a high level of neonicotinoid resistance given the pest control practices used in China. These results should be useful for the biorational application and resistance management of neonicotinoid insecticides. © 2021 Society of Chemical Industry.

Fitness studies of insecticide resistant strains: lessons learned and future directions.

The evolution of insecticide resistance is generally thought to be associated with a fitness cost in the absence of insecticide exposure. However, it is not clear how these fitness costs manifest or how universal this phenomenon is. To investigate this, we conducted a literature review of publications that studied fitness costs of insecticide resistance, selected papers that met our criteria for scientific rigor, and analyzed each class of insecticides separately as well as in aggregate. The more than 170 publications on fitness costs of insecticide resistance show that in 60% of the experiments there is a cost to having resistance, particularly for measurements of reversion of resistance and reproduction. There were differences between classes of insecticides, with fitness costs seen less commonly for organochlorines. There was considerable variation in the experiments performed. We suggest that future papers will have maximum value to the community if they quantitatively determine resistance levels, identify the resistance mechanisms present (and the associated mutations), have replicated experiments, use related strains (optimally congenic with the resistance mutation introgressed into different genetic backgrounds) and measure fitness by multiple metrics. Studies on the fitness costs of insecticide resistance will continue to enlighten our understanding of the evolutionary process and provide valuable information for resistance management. © 2021 Society of Chemical Industry.

Evidence for both sequential mutations and recombination in the evolution of kdr alleles in Aedes aegypti.

BACKGROUND: Aedes aegypti is a globally distributed vector of human diseases including dengue, yellow fever, chikungunya, and Zika. Pyrethroid insecticides are the primary means of controlling adult A. aegypti populations to suppress arbovirus outbreaks, but resistance to pyrethroid insecticides has become a global problem. Mutations in the voltage-sensitive sodium channel (Vssc) gene are a major mechanism of pyrethroid resistance in A. aegypti. Vssc resistance alleles in A. aegypti commonly have more than one mutation. However, our understanding of the evolutionary dynamics of how alleles with multiple mutations arose is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We examined the geographic distribution and association between the common Vssc mutations (V410L, S989P, V1016G/I and F1534C) in A. aegypti by analyzing the relevant Vssc fragments in 25 collections, mainly from Asia and the Americas. Our results showed all 11 Asian populations had two types of resistance alleles: 1534C and 989P+1016G. The 1534C allele was more common with frequencies ranging from 0.31 to 0.88, while the 989P+1016G frequency ranged from 0.13 to 0.50. Four distinct alleles (410L, 1534C, 410L+1534C and 410L+1016I+1534C) were detected in populations from the Americas. The most common was 410L+1016I+1534C with frequencies ranging from 0.50 to 1.00, followed by 1534C with frequencies ranging from 0.13 to 0.50. Our phylogenetic analysis of Vssc supported multiple independent origins of the F1534C mutation. Our results indicated the 410L+1534C allele may have arisen by addition of the V410L mutation to the 1534C allele, or by a crossover event. The 410L+1016I+1534C allele was the result of one or two mutational steps from a 1534C background. CONCLUSIONS/SIGNIFICANCE: Our data corroborated previous geographic distributions of resistance mutations and provided evidence for both recombination and sequential accumulation of mutations contributing to the molecular evolution of resistance alleles in A. aegypti.

miR-147b-modulated expression of vestigial regulates wing development in the bird cherry-oat aphid Rhopalosiphum padi.

BACKGROUND: Most aphids exhibit wing polyphenism in which wingless and winged morphs produce depending on the population density and host plant quality. Although the influence of environmental factors on wing polyphenism of aphids have been extensively investigated, molecular mechanisms underlining morph differentiation (i.e. wing development /degeneration), one downstream aspect of the wing polyphenism, has been poorly understood. RESULTS: We examined the expression levels of the twenty genes involved in wing development network, and only vestigial (vg) showed significantly different expression levels in both whole-body and wall-body of third instar nymphs, with 5.4- and 16.14- fold higher expression in winged lines compared to wingless lines, respectively in Rhopalosiphum padi. vg expression was higher in winged lines compared to wingless lines in third, fourth instar nymphs and adults. Larger difference expression was observed in third (21.38-fold) and fourth (20.91-fold) instar nymphs relative to adults (3.12-fold). Suppression of vg using RNAi repressed the wing development of third winged morphs. Furthermore, dual luciferase reporter assay revealed that the miR-147 can target the vg mRNA. Modulation of miR-147b levels by microinjection of its agomir (mimic) decreased vg expression levels and repressed wing development. CONCLUSIONS: Our findings suggest that vg is essential for wing development in R. padi and that miR-147b modulates its expression.

The F1534C voltage-sensitive sodium channel mutation confers 7- to 16-fold resistance to pyrethroid insecticides in Aedes aegypti.

BACKGROUND: Recent outbreaks of dengue and Zika have emphasized the importance to effectively control Aedes aegypti, which vectors the viruses causing these diseases. Pyrethroid insecticides are primarily used to control adult A. aegypti, especially during disease outbreaks. However, pyrethroid resistance in A. aegypti is an increasing problem. Mutations in the voltage-sensitive sodium channel (Vssc) are a common mechanism of pyrethroid resistance. The F1534C mutation is common and distributed globally in A. aegypti populations, but previous studies disagree about the role of this mutation in conferring resistance to pyrethroid insecticides. RESULTS: We isolated a congenic strain (1534C:ROCK) which was closely related to a susceptible strain Rockefeller (ROCK), but was homozygous for the 1534C Vssc allele. We determined resistance levels against eight insecticides that target the VSSC: six pyrethroids, DDT and DCJW (the bioactivated metabolite of indoxacarb). The resistance levels ranged from 7- to 16-fold, and resistance was inherited as an incompletely recessive trait. We also found a novel 367I+1520I+1534C allele, in addition to the 1534C and 1520I+1534C alleles, in mosquitoes from Thailand. The T1520I mutation did not increase pyrethroid resistance beyond what was conferred by the F1534C mutation alone. CONCLUSION: The F1534C Vssc mutation is common in A. aegypti populations and confers 7- to 16-fold resistance to pyrethroids, DDT, and DCJW in Aedes aegypti. These resistance levels are considerably less than previously reported for the S989P+V1016G mutations. Our results provide useful information for resistance management, specifically the levels of resistance conferred by the most common Vssc mutation in A. aegypti. © 2020 Society of Chemical Industry.

Potential for insecticide-mediated shift in ecological dominance between two competing aphid species.

Competition is a key structuring component of biological communities, which is affected by both biotic and abiotic environmental stressors. Among the latter, anthropic stressors and particularly pesticides are noteworthy due to their intrinsic toxicity and large use in agroecosystems. However this issue has been scarcely documented so far. In this context, we carried out experiments under laboratory conditions to evaluate stress imposed by the neonicotinoid insecticide imidacloprid on intra and interspecific competition among two major wheat pest aphids. The bird cherry-oat aphid Rhopalosiphum padi L. and the English grain aphid Sitobion avenae F. were subjected to competition on wheat seedlings under varying density combinations of both species and subjected or not to imidacloprid exposure. Intraspecific competition does take place without insecticide exposure, but so does interspecific competition between both aphid species with R. padi prevailing over S. avenae. Imidacloprid interfered with both intra and interspecific competition suppressing the former and even the latter for up to 14 days, but not afterwards when a shift in dominance takes place favoring S. avenae over R. padi, in contrast with the interspecific competition without imidacloprid exposure. These findings hinted that insecticides are indeed able to mediate species interaction and competition influencing community structure and raising management concerns for favoring potential secondary pest outbreaks.

Characterization of the metabolic transformation of thiamethoxam to clothianidin in Helicoverpa armigera larvae by SPE combined UPLC-MS/MS and its relationship with the toxicity of thiamethoxam to Helicoverpa armigera larvae.

In order to characterize the metabolic transformation of thiamethoxam (TMX) to clothianidin (CLO) in Helicoverpa armigera larvae and clarify its relationship with the insecticidal toxicity of TMX, method for determination of TMX and its metabolite clothianidin (CLO) residues in H. armigera larvae by solid phase extraction (SPE) combined UPLC-MS/MS was established. Following acetonitrile extraction and purification by SPE on florisil cartridge and C18 cartridge sequently, and cleanup by PSA adsorption, TMX and CLO residues in H. armigera larvae were successfully determined by UPLC-MS/MS. By using the established method, the concentration-time curves of TMX and its metabolite CLO in H. armigera larvae in vivo and metabolism of TMX by microsome of H. armigera larvae midguts in vitro were studied. TMX was quickly eliminated from H. armigera larvae with the elimination half-life as 4.2h. Meanwhile, only a small amount of CLO was formed from TMX metabolism, with the maximum CLO level in H. armigera larvae only accounts for the metabolic transformation of 7.99% of TMX, at 10h after intravenous TMX administration. Our results suggested that the low insecticidal efficacy of TMX against H. armigera larvae was related with the rapidly elimination of TMX from H. armigera larvae, meanwhile, CLO as TMX metabolite at a very low level in vivo didn't contribute to TMX toxicity to H. armigera larvae. In H. armigera larvae, TMX didn't act as proinsecticide for CLO in insecticidal efficacy of TMX.