To identify and compare the venom components and expression patterns of some bees/wasps, venom gland-specific transcriptome analyses were conducted for 14 Aculeate bees/wasps. Most of the allergens and pain-producing factors showed extremely high expression levels in social wasps, implying that social wasps have evolved to use venom to defend the colony against intruders. Acid phosphatase and tachykinin, which are known as allergens and neurotoxic peptides, were found with high frequencies in the venom glands of solitary wasps. This suggests that solitary wasps might use their venom for catching and preserving prey. In the venom glands of bumblebees, little or no transcripts of major allergens or pain producing factors were identified, implying that bumblebees venoms are relatively less toxic than those of social or solitary wasps. Taken together, the differential expression patterns of venom genes in some Aculeate bees/wasps implies that bees/wasps have unique groups of highly expressed venom components, which appear to have evolved in response to both ecological and behavioral influences.

When treating the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, with sublethal doses (LC10) of chlorantraniliprole, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. By analyzing the differentially expressed genes (DEGs), we found a number of genes that respond commonly or specifically to the test insecticides. With the criteria of p value < 0.05 and Log2FC > 1/ < -1, a total of 476, 367 and 410 genes were determined to respond specifically to chlorantraniliprole, indoxacarb and spinosad, respectively. Gene Ontology (GO) analysis revealed that the cuticle reorganization is commonly associated in all treatments and the oxidative stress-related process is also shown in all insecticides except spinosad. Finally, the DEGs seemingly related with enhanced tolerance were chosen for further characterization, and reliability of the transcriptome data were confirmed by quantitative PCR. The functional categories of these DEGs included mostly detoxification related genes, cuticle proteins, energy metabolism and transcriptional regulation. While the commonly responding DEGs suggest that they are likely involved in defense against common intoxication process, the DEGs specifically responded to each insecticide suggests the presence of unique tolerance mechanisms to each insecticide depending on their different structure and mode of action. Their possible roles in the tolerance/resistance development were discussed.

Frankliniella occidentalis is a major pest in agriculture. Following overuse of insecticides, high resistance has developed due to its high reproduction rate and short generation time. To control the resistant strains of the thrips, the ingestion RNAi- based control was established. A total of 67 genes were selected, and their double-stranded RNAs (dsRNA) were delivered to thrips via the leaf disc-feeding method. Among the genes screened, the dsRNA of Toll-like receptor 6 (TLR6) and coatomer protein subunit epsilon (COPE) resulted in the highest mortality (3.8- and 2.8-fold faster LT50 compared to control, respectively) when ingested by thrips. The dsRNA-fed thrips showed 53% and 83% reduced transcription levels of TLR6 and COPE, respectively. This result demonstrates that the observed mortality of thrips following dsRNA ingestion was due to RNAi, and this lethal genes can be employed as a practical tool to control thrips in the field.

Mosquitoes are primary medical insect pests due to their diseases transmission as vectors. In Korea, the insecticide-resistant populations of disease vector mosquito species, such as Anopheles sinensis, Culex pipiens and Culex tritaeniorhynchus, have constantly increased. Thus, management of insecticide resistance to major insecticides including pyrethroids and organophosphates is required for more efficient control of resistant populations. In this study, the quantitative sequencing (QS) protocols were established to detect the frequencies of three mutations (the L1014F on voltage sensitive sodium channel and the G119S and F331W on acetylcholinesterase 1) that are associated with either pyrethroids or organophosphates. Based on the QS protocol using newly designed non-polymorphic primers, resistance allele frequencies (RAFs) were estimated in field populations of An. sinensis, Cx. pipiens and Cx tritaeniorhynchus collected from an identical site in Korea. The dynamics of each resistance allele frequency over time in the same populations were also evaluated.

A residual contact vial plus water (RCVpW) bioassay method was established to monitor insectiside resistance in field populations of the melon thrips, Thrips palmi. Resistance level against six major insecticides were evalutated in five regions to test applicability of RCVpW as an on-site resistance monitoring tool. Reduced mortality in response to six test insecticides were exhibited compared to the RDA susceptable strain showing 100 % mortality, indicating different degree of resistance. An apparently reduced mortality to emamectin benzoate and chlofenapyr was observed in some field populations, suggesting uneven distribution of resistance to these insecticides in field populations. In addition, spinosad resistance was high and widely distributed in the test regions. Synergistic bioassay revealed that cytochrome P450-mediated metabolic factor is involved in spinosad resistance in the Korean population.

One of major mechanisms of insecticide resistance is the reduced susceptibility caused by mutations on the main target sites, such as sodium channel and acetylcholinesterase. Bioassay is a useful method to diagnose insecticide resistance of mosquitoes; however, it is hard to establish regional/annual resistance database based on bioassay. Recently, various markers of mutation and copy number variation have been identified through insecticide mechanism studies. Thus, molecular detection of resistance based on the resistance marker is now feasible, which can be readily implemented as a novel resistance monitoring tool to complement or replace the conventional bioassay method. In Korea, the density of vector mosquitoes native to the subtropical areas has increased due to climate change. Therefore, it is required to establish an efficient resistance monitoring system based on molecular markers to facilitate the construction of a nation-wide resistance map for more effective control of mosquitos. In addition, alternative insecticides should be introduced to the areas where mosquitoes develop high levels of resistance to maximize control efficacy against resistant populations

Honey bee, Apis mellifera L., have been widely used as a model organism for biological science because of its highly developed sociality, specialized labor division and passive population management. In order to examine the expression patterns of genes putatively involved in social development in honey bee, quantitative real-time PCR (qRT-PCR) that has been widely used to investigate the expression level of target gene can be used in honey bee study. However, the selection and validation of optimal reference genes is a crucial step prior to running qRT-PCR. In the present study, therefore, the seasonal expression stability of five candidate reference genes in the abdomen of forager and nurse was investigated using three programs (geNorm, NormFinder and BestKeeper), and selected reference genes were validated by the normalization of expression level of vg encoding vitellogenin. Although three programs revealed slightly different gene stability values, overall the combination of two genes (rpS18 and gapdh encoding ribosomal protein S18 and glyceraldehyde-3-phosphate dehydrogenase, respectively) was resulted in the most suitable use for normalization of the target gene in forager. However, a single gene, either rpL32 or rpS18 in the nurse or either rpL32, rpS18, or gapdh in the comparison between foragers and nurses, were suggested to be applied for normalization of seasonal and labor-specific gene expression by qRT-PCR.

Molecular diagnostic markers are necessary for establishing highthroughput screening systems to support insecticide-resistant population management. Here, we identified single amino acid substitution mutations related to carbamate resistance in Laodelphax striatellus Fallén type-1 acetylcholinesterase (Lsace1) using carbofuran-selected strains. The phenotypic resistance profiles of the final selection strain (SEL9) compared to the susceptible strain revealed a 14-fold higher resistance ratio based on topical application, 1.2-fold higher general esterase activity, and 4.3- fold higher acetylcholinesterase insensitivity based on the 50% inhibitory concentration (I50), suggesting that insensitivity of the target site could occur as a resistance factor. Comparison of the nucleotide sequences of Lsace1 of five strains (SUS, SEL0, SEL3, SEL6, and SEL9) revealed two amino acid substitutions (F330Y and F331H). To understand the roles of these mutations, we determined the allele frequency of both point mutations in the selected strains using quantitative sequencing methods. In addition, several quantitative genotypic traits (e.g., gene copy numbers and transcript levels of Lsace1, Lsace2, and LS.CarE1) were assessed. A correlation analysis of genotypic and phenotypic traits revealed strong correlations between resistance level and I50 with F331H allele frequency. Interestingly, the F331H mutation was negatively correlated with transcript levels of Lsace1, suggesting that selection pressure might result in a reduction of the target gene. Overall, the F331H mutation and reduced mRNA are important factors in the development of carbamate resistance. Furthermore, the point mutation can be used to monitor rapid carbofuran resistance in conjunction with molecular diagnostic methods such as quantitative sequencing.

To identify the venom components and their expression patterns of some Aculeata bees/wasps, venom gland-specific transcriptome analysis was conducted. FPKM values were normalized with the average of the transcription level of reference gene (a-tubulin). Common components in both solitary and social wasp venoms include hyaluronidase, phospholipase A2, metalloendopeptidase, etc. Although it has been expected that more diverse bioactive components with the functions of prey inactivation and physiology manipulation are present in solitary wasps, the information on venom compositions of solitary wasps obtained in this study was not sufficient to generalizae this notion. Nevertheless, some neurotoxic peptides (e.g., pompilidotoxin and dendrotoxin-like peptide) and proteins (e.g., insuline-like peptide binding protein) appear to be specific to solitary wasp venom. In contrast, several proteins, such as venom allergen 5 protein, venom acid phosphatase, and various phospholipases, appear to be relatively more abundant in social wasp venom. In the venom gland trancsriptome of bumblebees, major allergens or pain producing factors were barely identified, implying that bumblebee venoms are relatively less toxic than those of social or solitary wasps.

The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations both in the head (neuronal) and abdomen (non-neuronal) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. The expression levels of another two factors that regulate ACh titer, choline acetyltransferase (AmAChT) and acetylcholinesterase 2 (AmAChE2), were not altered as judged by qPCR and native PAGE, suggesting that the lower ACh titer was mainly regulated by AmAChE1. For precise verification of AmAChE1 as an ACh titer regulator, honey bees were put under brood rearing-suppressed condition to induce AmAChE1 and injected AmAChE1 dsRNA to knock down the gene. The ACh titer of AmAChE1-knocked down honey bees was 1.9 and 2.6 folds higher than that of control bees in head and abdomen, respectively. Taken together, in spite of its extremely low catalytic activity, the overexpression of AmAChE1 is likely to be related with the low level of ACh homeostasis, perhaps via ACh sequestration, under brood rearingsuppressed condition, and likely induce metabolic changes through ACh receptors-related pathways.