Acetylcholine titre regulation by non-neuronal acetylcholinesterase 1 and its putative roles in honey bee physiology.

Similar to other insects, honey bees have two acetylcholinesterases (AChEs), AmAChE1 and AmAChE2. The primary catalytic enzyme for acetylcholine (ACh) hydrolysis in synapses is AmAChE2, which is predominantly expressed in neuronal tissues, whereas AmAChE1 is expressed in both neuronal and non-neuronal tissues, with limited catalytic activity. Unlike constitutively expressed AmAChE2, AmAChE1 expression is induced under stressful conditions such as heat shock and brood rearing suppression, but its role in regulating ACh titre remains unclear. In this paper, to elucidate the role of AmAChE1, the expression of AmAChE1 was suppressed via RNA interference (RNAi) in AmAChE1-induced worker bees. The ACh titre measurement following RNAi revealed that the expression of AmAChE1 downregulated the overall ACh titre in all tissues examined without altering AmAChE2 expression. Transcriptome analysis showed that AmAChE1 knockdown upregulated protein biosynthesis, cell respiration, and thermogenesis in the head. These findings suggest that AmAChE1 is involved in decreasing neuronal activity, enhancing energy conservation, and potentially extending longevity under stressful conditions via ACh titre regulation.

Molecular, biochemical, and toxicological characterization of two acetylcholinesterases from the Western flower thrips, Frankliniella occidentalis.

We investigated the molecular and biochemical properties of two acetylcholinesterases (FoAChE1 and FoAChE2) from the Western flower thrips, Frankliniella occidentalis. Polyacrylamide gel electrophoresis and western blotting confirmed the membrane-anchored nature of both FoAChE1 and FoAChE2, which was further supported by hydrophobicity and glycophosphatidylinositol anchor predictions. High expression levels of both enzymes were observed in the head, indicating their predominant distribution in neuronal tissues. FoAChE1 exhibited significantly higher expression levels in all examined tissues compared to FoAChE2, suggesting its major role as a synaptic enzyme. Nonetheless, both recombinant enzymes displayed robust catalytic activity toward acetylthiocholine iodide, and FoAChE1 demonstrated nearly identical catalytic efficiency compared to FoAChE2. FoAChE1 exhibited slightly lower sensitivities to the cholinesterase inhibitors tested, including organophosphates (OPs) and carbamates (CBs), compared to FoAChE2. Field populations of F. occidentalis exhibited polymorphism of alanine vs. serine at position 197 of FoAChE1 within the conserved oxyanion hole. Contrary to common belief, however, functional analysis using recombinant enzymes revealed that neither A197 nor S197 residue was associated with FoAChE1 insensitivity to OPs and CBs. FoAChE2 did not exhibit any polymorphic amino acid substitutions at the positions known to be associated with resistance. Due to the absence of apparent resistance-associated mutations in field populations of F. occidentalis, the judicious use of some OPs or CBs can be suggested for controlling the highly resistant populations to other insecticides. Overall, our findings highlight the significance of both FoAChE1 and FoAChE2 as targets for toxicity assessment, while the specific contribution of each enzyme to toxicity remains unclear.

Inference of selection pressures that drive insecticide resistance in Anopheles and Culex mosquitoes in Korea.

Pyrethroids are primarily used for mosquito control in Korea. However, high frequencies of mutations conferring resistance to not only pyrethroids but also to other insecticides have been found in mosquito populations. This study aimed to examine the hypothesis that insecticides used outside of public health may play a role in selection. Briefly, the resistance mutation frequencies to three insecticide groups (pyrethroids, organophosphates, and cyclodienes) were estimated in two representative groups of mosquito species (Anopheles Hyrcanus Group and Culex pipiens complex). The relationship between these frequencies and the land-use status of the collection sites was investigated through multiple regression analysis. In the Anopheles Hyrcanus Group, the frequencies of both ace1 (organophosphate resistance) and rdl (cyclodiene resistance) mutations were positively correlated with 'proximity to golf course', possibly be due to the insecticides used for turf maintenance. They also showed positive correlations with field area and rice paddy area, respectively, suggesting the role of agricultural insecticides in the selection of these resistance traits. For the Cx. pipiens complex, the kdr (pyrethroid resistance), ace1, and rdl mutations were positively correlated with the residential area, field, and rice paddy, respectively. Therefore, pyrethroids used for public health could serve as a direct source of resistance selection pressure against kdr, whereas non-public health insecticides may pose primary selection pressure against the ace1 and rdl traits. The current findings suggest that the insecticides used in agriculture and the golf industry play a significant role in mosquito selection, despite variations in the extent of indirect selection pressure according to the mosquito groups and insecticide classes.

Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites.

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Exaptation of I4760M mutation in ryanodine receptor of Spodoptera exigua (Lepidoptera: Noctuidae): Lessons from museum and field samples.

Since 2007, diamide insecticides have been widely used in Korea to control various types of lepidopteran pests including Spodoptera exigua. For nearly a decade, diamide resistance in field populations of S. exigua across 18 localities has been monitored using bioassays. Despite their short history of use, resistance to diamide insecticides has emerged. Based on the LC50 values, some field populations showed a higher level of resistance to chlorantraniliprole, a diamide insecticide, compared to that of the susceptible strain, although regional and temporal variations were observed. To investigate resistance at a molecular level, we examined three mutations (Y4701C, I4790M, and G4946E) in the ryanodine receptor (RyR), which is the primary mechanism underlying diamide insecticide resistance. DNA sequencing showed that only the I4790M mutation was found in most field populations. As resistance levels varied significantly despite the uniform presence of the I4790M mutation, we considered the presence of another resistance factor. Further, the I4790M mutation was also found in S. exigua specimens collected prior to the commercialization of diamide insecticides in Korea as well as in other countries, such as the USA. This finding led us to hypothesize that the I4790M mutation were predisposed in field populations owing to selection factors other than diamide use. For further clarification, we conducted whole-genome sequencing of S. exigua (449.83 Mb) and re-sequencing of 18 individual whole genomes. However, no additional non-synonymous mutations were detected in the RyR-coding region. Therefore, we concluded that the high level of diamide insecticide resistance in Korean S. exigua is not caused by mutations at the target site, RyR, but is attributed to other factors that need to be investigated in future studies.

Biophysical properties of human body louse nit related proteins: LNSP1, Agp9 and Agp22.

The human parasitic head and body lice lay their eggs on either hair or clothing. Attachments of the eggs are possible because the female lice secret a glue substance from the accessory gland along with the egg, which hardens into a nit sheath that secures and protects the egg (The "nit" commonly refers to either the louse egg with an embryo or the empty hatched egg). Proteins called the louse nit sheath protein (LNSP) are suggested to be the major proteins of the nit sheath, but transcriptome profiling of the accessory glands indicated other proteins such as Agp9 and Agp22 are also expressed in the glands. In this study, human body louse LNSP1 (partial), Agp9, and Agp22 are recombinantly produced using the E. coli expression system, and the biophysical properties characterized. Circular dichroism analysis indicated that the secondary structure elements of LNSP1 N-terminal and middle-domains, Agp9, and Agp22 are prominently random coiled with up to 10-30% anti-parallel ß-sheet element present. Size-exclusion chromatography profiles of LNSP1 proteins further suggested that the ß-sheets made of the smaller N-terminal domain stacks onto the ß-sheets of the larger middle-domain.

Selection of stable reference genes for quantitative real-time PCR in the Varroa mite, Varroa destructor.

To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real-time polymerase chain reaction (qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase [NADHD], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], eukaryotic translation elongation factor 1 α 1 [eEF1A1], eukaryotic translation elongation factor 2 [eEF2], ribosomal protein L5 [RpL5], Actin, tubulin α-1D chain [α-tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α-tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α-tubulin and RpL5 are recommended for investigating the tissue-specific expression profiles of target genes.

Insecticide resistance in pepper greenhouse populations of Aphis gossypii (Hemiptera: Aphididae) in Korea.

The cotton aphid or melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous insect pest with a wide host range. Two distinct genetic clusters were found in A. gossypii populations in Korea. To determine whether the division of the genetic clusters was driven by insecticide selection pressure, the frequencies of insecticide resistance-associated mutations on three representative insecticide target genes [i.e., nicotinic acetylcholine receptor gene (nAChR), voltage-gated sodium channel gene (vgsc), and acetylcholinesterase 1 gene (ace-1)] were predicted in A. gossypii populations with known genetic structures. Most populations revealed heterozygosity-resistant alleles for the nAChR R81T and vgsc M918L mutations, but homozygous-resistant alleles for the ace-1 S431F mutation. However, assessment of the three mutation frequencies revealed no apparent correlation between the genetic structures and the resistance profiles. The regression analysis revealed no correlation between the genetic cluster ratios and resistance allele frequencies (R81T, S431F, and M918L). We used three insecticides that are commonly used in greenhouses: imidacloprid (neonicotinoid), acephate (organophosphate), and esfenvalerate (pyrethroid), to test resistance and susceptibility in A. gossypii populations. The bioassay results revealed that the BS_19 (Busan) and JE_19 (Jeongeup) populations were resistant to imidacloprid and acephate, the HS_19 (Honseong) population was resistant to acephate and esfenvalerate, and susceptible lab strains only exhibited resistance to acephate. The bioassay results were correlated with mutation frequency, but no correlation was detected among genetic clusters. These results suggest that the distinct genetic structure observed in the Korean populations of A. gossypii is not likely influenced by insecticide resistance traits, but rather by other factors.

Molecular and kinetic characterization of two acetylcholinesterases with particular focus on the roles of two amino acid substitutions (Y390N and F392W) in Bemisia tabaci.

Two acetylcholinesterases (AChEs) are present in Bemisia tabaci (BtAChE1 and BtAChE2). A conserved AChE mutation conferring organophosphate (OP) resistance (F392W in BtAChE1) is saturated in field populations despite its potential fitness cost, and a highly conserved amino acid residue forming the backdoor of AChE is substituted with a unique amino acid (Y390N in BtAChE1) in B. tabaci. Thus, the roles and relationships of the two amino acid substitutions in the evolutionary adaptation of B. tabaci remain to be elucidated, and little information is available on the catalytic and molecular properties of BtAChE1 and BtAChE2. To determine which AChE is a more relevant target of OPs and carbamates, the molecular and kinetic properties of BtAChE1 and BtAChE2 were investigated. Both BtAChE1 and BtAChE2 were exclusively expressed in head and thorax but not in abdomen, bound to the membrane via GPI anchoring, and present as dimeric forms. Soluble monomeric form was detected only in BtAChE2. The catalytic activity of baculovirus-expressed BtAChE1 was 19.5-fold higher than that of BtAChE2. The inhibition assay revealed that the F392W mutation in BtAChE1 enhanced resistance to OPs. The artificial substitution of N390 (wild form) to Y (putative ancient form) led to reduced catalytic efficiency and increased inhibition by glycoalkaloids, suggesting that the Y390N substitution in BtAChE1 may have been required for Solanaceae host adaptation. BtAChE1 was proven to function as a main catalytic enzyme for ACh hydrolysis, thus being the main target of OPs and carbamates.

Characterization of molecular and kinetic properties of two acetylcholinesterases from the Colorado potato beetle, Leptinotarsa decemlineata.

The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.

Molecular and kinetic properties of three acetylcholinesterases in the Varroa mite, Varroa destructor.

The Varroa mite, Varroa destructor, poses one of the most serious threats to honey bees worldwide. Although coumaphos, an anticholinesterase pesticide, is widely used for varroa mite control, little information is available on the properties of Varroa mite acetylcholinesterases (VdAChEs). In this study, three putative VdAChEs were annotated and named VdAChE1, VdAChE2, and VdAChE3. All VdAChEs possessed most of the functionally important signature domains, suggesting that they are catalytically active. Phylogenetic analysis revealed that VdAChE1 was clustered into a clade containing most arthropod AChE1s, whereas VdAChE2 and VdAChE3 formed a unique clade with other arachnid AChEs. VdAChE1 was determined to be membrane-anchored, but both VdAChE2 and VdAChE3 are soluble, as judged by electrophoresis in conjunction with western blotting. Tissue-specific transcription profiling revealed that VdAChE1 was most predominantly expressed in the synganglion. In contrast, VdAChE2 was most predominantly expressed in the legs and cuticle. VdAChE3 showed negligible expression levels in all the tissues examined. In a kinetic analysis using recombinant VdAChEs, VdAChE1 exhibited the highest catalytic efficiency, followed by VdAChE2 and VdAChE3. Inhibition experiments revealed that VdAChE1 was most sensitive to all tested inhibitors. Taken together, VdAChE1 appears to be the major synaptic enzyme with a more toxicological relevance, whereas VdAChE2 is involved in other noncatalytic functions, including chemical defense against xenobiotics. Current findings contribute to a more detailed understanding of the evolutionary and functional traits of VdAChEs and to the design of novel anticholinesterase varroacides.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing.

BACKGROUND: Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. RESULTS: A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. CONCLUSIONS: This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Transcriptomic identification and characterization of genes commonly responding to sublethal concentrations of six different insecticides in the common fruit fly, Drosophila melanogaster.

Pretreatment with sublethal concentrations (LC10) of six insecticides (chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, ivermectin, and spinosad) significantly elevated tolerance of the common fruit fly Drosophila melanogaster to lethal concentration of the respective insecticide. Commonly responding genes to sublethal treatments of the six insecticides were identified by transcriptome analysis based on a fold change >1.5 or < -1.5, and p < 0.05 as selection criteria. Following treatment with all the six insecticides, 26 transcripts were commonly over-transcribed, whereas 30 transcripts were commonly under-transcribed. Reliability of the transcriptome data was confirmed by quantitative PCR. A majority of the over-transcribed genes included those related to olfactory behavior, such as odorant-binding proteins, as well as immune-related genes, including attacin, diptericin, and immune-induced molecule 18. In contrast, genes belonging to the mitochondrial respiratory chain, such as mitochondrial NADH-ubiquinone oxidoreductase chain 1/3/4/5 and mitochondrial cytochrome b/c, were commonly under-transcribed. Furthermore, genes related to eggshell formation and motion were also under-transcribed, which may indicate a possible energy trade-off for xenobiotic stress. In summary, most of the differentially expressed genes were not directly related to well-known detoxification genes, suggesting that the roles of commonly expressed tolerance-related genes are not likely related to direct metabolic detoxification, but rather are associated with restoration of homeostasis.

Transcriptomic identification and characterization of genes responding to sublethal doses of three different insecticides in the western flower thrips, Frankliniella occidentalis.

Pretreatment with sublethal concentrations (LC10) of three insecticides (chlorfenapyr, dinotefuran, and spinosad) enhanced tolerance to a lethal dose of the respective insecticide in the Western flower thrips, Frankliniella occidentalis. To identify genes responding to sublethal treatment with insecticides, transcriptome analysis was conducted for thrips treated with LC10 of the three insecticides. When based on a fold change >1.5 or < -1.5 as a selection criterion, 199 transcripts were commonly up-regulated, whereas 31 transcripts were commonly down-regulated following all three insecticide treatments. The differential expression levels of representative genes were validated by quantitative PCR. Most over-transcribed transcripts could be categorized as basic biological processes, such as proteolysis and lipid metabolism. Detoxification genes, such as one glutathione S transferase S1, three UDP-glucuronosyltransferases, four CYP450s, and one ABC transporter G family member 20, were commonly overexpressed in all three insecticide-treated groups. Knockdown of the five representative commonly overexpressed genes via ingestion RNA interference increased mortalities to all the three test insecticides, supporting their common role in tolerance induction. In contrast, three C2H2-type zinc finger-containing proteins were significantly down-regulated in all insecticide-treated thrip groups. Since the tested insecticides have distinct structures and modes of action, the roles of commonly expressed genes in tolerance were discussed.

Regional and seasonal detection of resistance mutation frequencies in field populations of Anopheles Hyrcanus Group and Culex pipiens complex in Korea.

Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May-October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies.

Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea.

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (Cimex lectularius) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery.

The western flower thrips, Frankliniella occidentalis, is a major pest that damages a wide variety of crops and vegetables. Following extensive use of insecticides, it has developed high levels of resistance to almost all groups of insecticides due to its high reproduction rate and short generation time. Therefore, an alternative pest control strategy, such as RNA interference (RNAi)-based control, is essential. To establish an ingestion RNAi-based control, a total of 57 genes involved in various biological processes were selected, and their double-stranded RNAs (dsRNA) were delivered to an insecticide-susceptible strain of F. occidentalis via the leaf disc-feeding method using a bioassay chamber optimized by 3D printing. The mortality of dsRNA-ingested thrips was examined every 24 h until 120 h post-treatment. Of the 57 genes screened, dsRNAs of the Toll-like receptor 6, apolipophorin, coatomer protein subunit epsilon and sorting and assembly machinery component were most lethal when ingested by thrips. The dsRNA-fed thrips showed substantially reduced transcription levels of target genes, demonstrating that the observed mortality was likely due to RNAi. When these genes were tested for ingestion RNAi against an insecticide-resistant strain of F. occidentalis, bioassay results were similar. In conclusion, this study provides the first evidence that ingestion RNAi can be lethal to F. occidentalis, a mesophyll sucking pest, and further suggests that transgenic plants expressing hairpin RNA of these essential genes can be employed to control insecticide-resistant thrips.

Pathway profiles based on gene-set enrichment analysis in the honey bee Apis mellifera under brood rearing-suppressed conditions.

Perturbation of normal behaviors in honey bee colonies by any external factor can immediately reduce the colony's capacity for brood rearing, which can eventually lead to colony collapse. To investigate the effects of brood-rearing suppression on the biology of honey bee workers, gene-set enrichment analysis of the transcriptomes of worker bees with or without suppressed brood rearing was performed. When brood rearing was suppressed, pathways associated with both protein degradation and synthesis were simultaneously over-represented in both nurses and foragers, and their overall pathway representation profiles resembled those of normal foragers and nurses, respectively. Thus, obstruction of normal labor induced over-representation in pathways related with reshaping of worker bee physiology, suggesting that transition of labor is physiologically reversible. In addition, some genes associated with the regulation of neuronal excitability, cellular and nutritional stress and aggressiveness were over-expressed under brood rearing suppression perhaps to manage in-hive stress under unfavorable conditions.

Transcriptome-based identification and characterization of genes commonly responding to five different insecticides in the diamondback moth, Plutella xylostella.

When the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, were pretreated with sublethal doses (LC10) and then subsequently exposed to lethal doses (LC50) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. To identify genes that commonly respond to the treatment of different insecticides and are responsible for the tolerance enhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared with that of untreated control. A total of 117,181 transcripts with a mean length of 662bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined to be up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealed little differences in their GO profiles between treatments with different insecticides except for spinosad. Finally, the DEGs commonly responding to all insecticides were selected for further characterization, and some of their over-transcription levels were confirmed by quantitative PCR. The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantly down-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested, the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

Agroinfiltration-based expression of hairpin RNA in soybean plants for RNA interference against Tetranychus urticae.

The coatomer subunit alpha (COPA) and aquaporin 9 (AQ9) genes from the two-spotted spider mite, Tetranychus urticae, were previously determined to exhibit RNA interference (RNAi)-based lethality when their double-stranded RNAs were systemically delivered via multi-unit chambers (Kwon et al., 2016 [8]). In current study, the hairpin RNAs of the COPA and AQ9 were transiently expressed in soybean plants by agroinfiltration. When T. urticae was fed with the soybean plants agroinfiltrated with the COPA and AQ9 hairpin RNA cassettes, the cumulative mortality increased significantly at 6days post-infestation. Quantitative PCR analysis revealed that the transcript level of both COPA and AQ9 was significantly reduced in T. urticae after 2days post-infestation, thereby confirming that the significant increases in mortality resulted from the knockdown of COPA and AQ9 transcripts. Our findings demonstrate the utility of COPA and AQ9 as potential genes for plant host-mediated RNAi control of T. urticae. In addition, we proved the usefulness of agroinfiltration as a rapid validation tool for confirming the RNAi-based lethality of target genes against arthropod pests before producing transgenic plants as agroinfiltration requires less time and skill to validate transgene function. Furthermore, these findings prove the concept that hairpin RNA expressed in plant hosts can also induce RNAi and eventually kill T. urticae, a sap-sucking pest.