There are two different types of acetylcholinesterase (AChE1 and AChE2) in the western honeybee as in most of insects. It is suggested that soluble AmAChE1 might be related with a stress response as judged from its elevated expression level in honey bee workers when brood rearing was suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patterns of AmAChE1 following heat shock, brood rearing suppression and chemical treatments (Imidacloprid and fluvalinate) were investigated. Also, several heat shock protein (hsp) genes (hsp10, hsp60, hsp70 and hsp90) known as general stress markers were tested as positive references. Heat shock induced expression of every tested hsp along with AmAChE1. In brood rearing-suppressed worker bees, 7 days old bees showed much higher expression level of AmAChE1 and hsp90 compared to control honey bees. However, treatment of imidacloprid and fluvalinate did not induce any apparent overexpression of these genes. These results confirm that both HSP and AmAChE1 genes generally respond to temperature and brood rearing suppression and further suggest that AmAChE1 can serve as a potential biomarker along with hsps for the detection of stress in honey bee colonies.

Recently, the existence of non-neuronal, soluble AChEs with non-classical functions, such as stress response and chemical defense, has been reported in both vertebrates and invertebrates. With this in mind, it is intriguing to hypothesize that fat body is a main tissue to express non-neuronal AChE at least in some insects. As an initial step for the systematic approach to investigate the distribution of non-neuronal AChEs in insect fat body and to elucidate their physiological functions, we have selected 12 different insect species across different orders and isolated fat body tissues from them. Then, the presence or absence of AChE and its solubility nature were determined by native polyacrylamide gel electrophoresis in conjunction with western blot analysis insect-specific AChE1 and 2 antibodies. Among 12 insects examined, soluble AChE1 was determined to be expressed in fat bodies of insects involving honey bees, brown plant hoppers, dynastid beetles, lice, etc, AChE2 in fruit flies, bed bugs, mealworm beetles. However, no AChE was detected in fat bodies of the remaining two species American cockroaches and dragonflies Our findings clearly show that AChE is widely distributed in the fat body tissue of diverse insect species. More extensive investigation on in a wider variety of insect species would be necessary to deduce the evolutionary origin of fat body-specific AChE, which would be the ancestor of AChE with non-neuronal function.

To identify genes that commonly respond to the treatment of different insecticides, 3rd instar larvae of the diamondback moth, Plutella xylostella, were treated with sublethal doses (<LC10) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping. Then, transcriptomic profiles of treated larvae were compared with that of untreated control. A total of 117,181 transcripts in average with a mean length of 662 bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 207, 153, 336, 360, and 262 transcripts were determined to be up-regulated whereas 117, 47, 92, 115, and 81 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Finally, with the criteria of >10 X fold change (FC) and p < 0.05 or >4 X FC, p < 0.05 and q < 0.2, the genes commonly over-transcribed in all treated insects were selected and their over-transcription levels were confirmed by quantitative PCR. These commonly responding genes included three cytochrome P450 genes (Cyp303a1, Cyp6a20 and CYP9E2), three cuticle protein genes (LM-8, LM-19 and TM-A3A), lavesin-1, acyl-CoA D11 desaturase, glucose dehydrogenase, nose resistant to fluxetine protein 6, chorion peroxidase and protein yellow. As the five test insecticides have distinct structure and mode of action, the genes identified in this study were suggested to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in tolerance and resistance development were further discussed.

Abaecin is a proline-rich anti-bacterial peptide which is known to have high activity against Gram-negative bacteria and it is originally isolated from honeybee Apis mellifera. Since the anti-bacterial peptides including abaecin were identified in the hemolymph of A. mellifera infected with Gram-negative bacteria Escherichia coli, this result implies that honeybees express abaecin for defense against natural infection. Anti-microbial peptides including abaecin were also identified in the adult hemolymph of bumblebees. To investigate pharmacological and toxicological properties of abaecin from Bombus ussurensis, biological activities were evaluated by conducting anti-tumor, anti-microbial and cytotoxic activity assays. Abaecin exhibited significantly high anti-tumor activity against ovarian tumor cells SK-OV-3 and NIH; OVCAR-3 at 100 μM after 72 h treatment. Interestingly, abaecin showed none of anti-microbial activity against Gram-negative and Gram-positive bacteria. Since abaecin exhibited extremely low level of hemolytic activity against human erythrocytes, it may serve as a good model peptide for studying its potential as a selective anti-tumor drug.

RNA interference (RNAi) has been widely adopted as a primary reverse genetic tool to determine the physiological function of genes of interest. Nevertheless, the lack of optimized RNA delivery method has been a major obstacle for non-model organisms, such as Cimex lectularis. In this study, we have established a RNAi protocol for the silencing of C. lectularis salivary gland-specific cholinesterase (SChE) gene based on micro-injection of double stranded RNA (dsRNA). An aliquot (20 nl) of dsRNA solution (4.5 ng/nl) was injected to body cavity through the arthrodial membrane between metathoracic coxa and sternum of adult females. Observed mortality was less than 5% and at 6-day post injection, while the gene silencing efficiency reached 97~99% at 2-6 day post injection. This result demonstrates the efficacy of injection RNAi via the arthrodial membrane in C. lectularius.

Rapid resistance detection of resistance level is most important step to manage the resistant population in Tetranychus urticae in Korea. Residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods were employed to determine the resistance level and applied to measure the resistance level of 46 field population collected from rose, strawberry and apple trees. Most populations exhibited multiple resistance pattern to various types of acaricides. Especially, the resistance levels in mites from rose cultivation area were higher than those from strawberry and apple cultivation areas. The resistance level detection would provide a useful parameter enabling the proactive action for a proper resistant population management for T. urticae.

Ryanodine receptors (RyRs) regulate the contractions of insect muscles by altering intracellular Ca2+ concentration and are the targets of chlorantraniliprole. Here we established two resistant strains of Drosophila melanogaster, which were treated with low or high concentrations of chlorantraniliprole, and their resistance levels were determined on the basis of contact and ingestion toxicities. Compared with the wild type, the two resistant strains did not show any significant differences in contact toxicity. However, they showed significantly increased resistance ratios in ingestion toxicity than that by the wild type. The resistant strains had altered expression levels of RyRs and more enhanced Acetylcholinesterase and Glutathione-S-transferase activities than that by the non-selected strain. These results suggested that the resistance development of chlorantraniliprole in the two strains might be mediated by the activation of detoxification pathways in D. melanogaster.

A series of conserved point mutations in acetylcholinesterase (AChE) confer resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

The diamondback moth, Plutella xylostella (DBM) is a major pest of cruciferous vegetables. The insecticide resistance of this pest is very serious and has been reported in more than 600 cases. It is important to evaluate insecticide response to manage the resistant populations In this study, we investigated the insecticide susceptibility of local DBM strains collected from five major cabbage cultivation area in Korea to the ten commercial insecticides with leaf dipping method . As a result, the resistance ratio(R/R) of almost all the tested pesticides was 0.1∼3.3 (low resistant) and there was no differce within local regions. But dinotefuran showed middle resistance with regional differences (R/R , 5.9∼69.4) For the estimate the control effectiveness, we calculated the log value of the recommendation concentration divided by LC90 of pesticides. The result showed that the efficiency of neonicotinoids was generally low and that of emamectin benzoat every high. In conclusion, neonicotinoids was to use carefully and emamectin benzoate was very good alternative in DBM control in Korea

Two different types of acetylcholinesterae (AChE1 and AChE2) are present in majority of insects, including the Western honey bee. Out of the two honey bee AChEs (AmAChEs), the soluble AmAChE1 with little catalytic activity is widely distributed in both neuronal and non-neuronal tissues, including fat body. In this study, to identify stresss factors that can induce AmAChE expression, we tested various conditions that honey bees can encounter in natural setting, including heat shock, cold shock, bacterial challenge (Escherichia coli and Staphylococcus aureus) and Varroa mite infestations, and evaluated their effects on AmAChE expression. Among the stress factors tested, only heat shock condition induced AmAChE expression in a dose dependet manner. This finding suggests that one function of AmAChE1 is related with thermoregulations, especially against heat shock stress in honey bees.

Honey bee swarming is a natural phenomenon that occurs when the colony encounters changes in the in-hive (i.e. population size and queen condition) and environmental conditions. To better understand the molecular basis of swarming, we conducted the transcriptomic profiles of worker bees between before swarming [pre-swarming colony (PSC)] and after swarming [swarming group (SG) and remaining group (RG)]. Based on the gene set enrichment analysis (GSEA), we predicted the biological processes associated with swarming. In addition, we analyzed the composition of cuticular hydrocarbons (CHCs) by gas chromatography-mass spectrometry and compared their profiles between different bee groups. GSEA results showed that there were a little differences between PSC and RG while many of the pathways related with metabolism and protein processing were down regulated in SG relative to PSC and RG. CHCs profiling revealed a similar CHCs composition between PSC and RG but some differences in CHCs composition (i.e. heneicosane, octacosane, octacosanol) were detected between SG and RG. These differences in gene pathway and CHC composition were discussed with respect to physiological changes and social communication.

In our previous study, COPA (coatomer subunit alpha) gene from the two-spotted spider mite, Tetranychus urticae, exhibited RNA interference (RNAi)-based lethality when its double-stranded RNA (dsRNA) was systemically delivered via multi-unit chambers or its hairpin RNA was in planta-expressed by agroinfiltration. The cumulative mortality of T. urtcae was 55.0 ±14.2% in soybean plants agroinfiltrated with COPA gene. To investigate the temporal expression profiles of hairpin RNA following agroinfiltration, the amount of hairpin RNA expressed in plants was quantified over time by quantitative real-time PCR. Relative transient expression levels of T. urticae COPA hairpin RNA was highest at 46 h post-agroinfiltration and the extent of COPA gene knockdown was lowest at 12 h post-infestation on soybean plants. To investigate small interference RNA (siRNA) profiling, northern blot assay is currently under progress.

Acetylcholinesterase (AChE) is an enzyme for hydrolyzing neurotransmitter acetylcholine. Soluble form of AChE is generated via alternative splicing and functions as a bioscavenger in Dropsophila melanogaster. In this study, effects of ethanol and acetic acid on the soluble AChE expression were investigated. Treatment of ethanol and acetic acid results in over-expression of soluble AChE in the abdomen in a dose-dependent manner. However, no apparent change in AChE expression was observed in the head. Our finding suggests that the soluble AChE is involved in chemical defense against high concentration of ethanol, which is a common by-product from fermented food，and acetic acid, the main metabolite of ethanol. Thus, high level of ethanol and acetic acid resistance in D. melanogaster appears to be evolved via the induction mechanism of soluble AChE expression.

Colony collapse disorder (CCD), a phenomenon of honeybees disappearance, has been reported since 2006. Chronic exposure to neonicotinoid insecticides, particularly imidacloprid, has been suggested to impair forager’s ability for foraging and be a main cause of CCD. Recently, it has been reported that imidacloprid induces insulin resistance in animal cell line by blocking glucose uptake. Similarly to human insulin, insulin-like peptide (ILP) of insects is involved in maintaining blood glucose contents in hemolymph by regulating the concentration of trehalose and glycogen. Therefore, we have hypothesized that sublethal concentration of neonicotinoid may affect the metabolic pathway of honey bees as well. We investigated the transcription levels of the genes involved in the insulin/insulin-like signaling (IIS) pathway, such as AmILP and AmInR, following an acute or a chronic dietary exposure of sublethal concentrations of imidacloprid to foragers. In both experiments, honeybees showed increased expression levels of ILP and InR in a dose-dependent manner. Our results suggest that sublethal dose of imidacloprid likely upregulates IIS pathway, thereby rendering honey bees to become resistant to insulin.

Bombolitin is a venom peptide originally isolated from bumblebees and possesses various biological activities, including hemolytic activity. Bombolitins exhibit amphipathic α-helical structure in lipid-membrane-mimicking environments. To investigate their pharmacological and toxicological properties, anti-tumor, anti-microbial and cytotoxic activities of bombolitins from Bombus ardens and Bombus ussurensis were evaluated. Bombolitins of the two species exhibited extremely high anti-tumor activity against ovarian tumor cells SK-OV-3 and NIH; OVCAR-3 at 25-50 μM, which is 2-fold more potent than other wasp venom peptides studied to date (Yoon et al., 2015; Yoon et al., 2016). The two bombolitins showed significantly high antimicrobial activity. However, bombolotin of B. ussurensis showed no antimicrobial activity against Escherichia coli. In addition to their high levels of anti-tumor activity, bombolitins showed considerable levels of hemolytic activity. Thus, to utilize bombolitins as a potential candidate for anti-tumor peptide drugs, further studies for reducing cytotoxic properties of bombolitns is essential.

There are many other detection methods for Bursaphelenchus xylophilus. However, Baermann funnel method, PCR-based methods, which are laborious and time consuming processes that are unsuitable to rapid diagnose the Pine Wilt Disease (PWD) on the field. For these reasons, the aim of our experiment is not only to apply field diagnostic for pine wood nematode (PWN) but also to reduce total time for detection PWN in the pine trees by loop-mediated isothermal amplification (LAMP) with phosphate (Pi)-induced coloration reaction which could be yes or no answer for detection of PWN has not required UV detector but it just could be discriminated by naked eyes within 30 min. Genomic DNA was directly extracted from pine wood chips by Wood Chips Direct Lysis procedure which can be used for LAMP only after simple dilution. Our results suggest that LAMP-Pi detection method, simple and rapid method for detection of PWN, could be applied to the field diagnostic for PWD.

Both Nilaparvata lugens Stål and Laodelphax striatellus Fallén has been known as the major rice pests which were irregularly migrated into Korea by wind current from China. We re-arranged and compared the resistance profiles to detect the existence convergent adaptation by using the previously published dataset (N. lugens at 2014, 17:711-716 and L. Striatellus at 2016, 19:247-252 in J. Asia Pac. Entomol., respectively). Both species commonly exhibited the high level of resistance to imidacloprids. Especially, the immigrant populations of L. Striatellus exhibited higher resistance level to indigenous populations, suggesting that they would settle down in Korea with insecticide resistance traits being predisposed. To detect the convergent adaptation to insecticide, pearson correlation analysis were employed by comparing mean dosage of LD50 and resistance ratio to nine insecticides. Strikingly, high and similar dosage responses were observed between the two species with high correlation coefficients of 0.928 (df=7, P <0.001) and 0.950 (df=7, P <0.001), respectively, in the comparison of LD50 vs. resistance ratio. This finding indicates that convergent adaption has occurred in both species through consistent selection by insecticides with similar usage patterns.

RNA interference (RNAi) has been proven as an operative technique for efficient gene silencing in many organisms. In our study, Tetranychus urticae, an extremely polyphagous and rapidly resistance developing mite against acaricides, was screened by double-stranded RNA (dsRNA) delivery method using multi-unit chambers. Among several lethal genes of T.urticae, COPA (the coatomer subunit alpha), a gene involved in membrane transport between the endoplasmic reticulum (ER) and the Golgi complex, showed the highest mortality rate [median lethal time (LT50)=54h]. To investigate the effect of dsCOPA treatment to lysosome formation, we used the Lysotracker green DND26 dye, selective to acidic cellular compartments such as lysosome. The result revealed that the dsEGFP-treated T. urticae has 1.3-fold more of lysosome than that of dsCOPA treated, indicating that downregulation of COPA affected lysosomes function and autophagy, thereby resulting in lethality. To investigate the further detailed toxic mechanism of COPA knockdown, investigation on histological changes in T. urticae fed COPA dsRNA is currently on going.

Honey bee swarming is a naturally occurring phenomenon under the conditions of population increase, climate change and pollen deficit. However, unexpected swarming usually results in loss of bee colony, it poses a considerable trouble in bee keeping. In an attempt to search for molecular markers that can predict the swarming behavior, transcriptional profiling was conducted and compared between the heads of swarming group and the remaining group in the same honey bee colonies. A total of 25,551 transcripts were initially identified and 1,144 differentially expressed genes between the two groups were sorted by FC2 (fold change) cut-off value. Several transcripts, including 6 apidermin (structurally novel cuticular protein)-related, 16 cuticular and 3 odorant binding proteins, showed lower expression levels in the swarming group compared with the remaining group (FC range of –2.17 to –667.48, -2.04 to –54.34 and -2.08 to –21.34 respectively). Pathway analyses are currently in progress to understand the physiological and metabolic differences between swarming and remaining groups of honey bees.