The honey bee, Apis mellifera, has a defense system, including detoxification, antioxidation, and immunity pathways, against external stimulation such as chemicals, stress, and pathogens. However, pesticides, particularly neonicotinoids and butenolids, have been recently reported to alter physiological changes in honey bee. In this study, we investigated the expression levels of eight genes categorized into detoxification (CYPQ3), antioxidation (CAT and SOD2), and immune system (Abaecin, Apidaecin, Defensin1, Defensin2, and Hymenoptaecin), in five tissues (Head, Thorax, Gut, Fat body, and Carcass) of honey bee treated with three pesticides (Acetamiprid, Imidacloprid, and Flupyradifurone) using quantitative real-time PCR. Gene expression patterns was varied depending on the type of pesticides and tissues. However, among eight genes, the expression levels of CYPQ3 was notably induced, but those of AMPs were generally reduced by all pesticides tested in this study in five tissues. These suggest that CYPQ3-mediated detoxification pathway is induced, but AMP-mediated immune system might be disrupted when honey bee is exposed to neonicotinoids and butenolid.

Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatoninmediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) iodide-induced cytosolic free Ca2+ concentration ([Ca2+]i) increase and norepinephrine secretion in a concentrationdependent manner. The inhibitory effect of melatonin on the DMPP-induced [Ca2+]i increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.

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.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this paper, we investigated the molecular structure, tissue distribution patterns, and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was detected by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of sequestration and hydrolysis of acetylcholine (ACh)/choline (Ch) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. Kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions as a sequestering enzyme specific to ACh (not to Ch) by having a very strong affinity to ACh but an extremely long turnover time.

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 in both the head (central nervous system) and abdomen (peripheral nervous system) 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. Interestingly, the expression levels of choline acetyltransferase (AmChAT) and molecular marker genes of immune systems were significantly reduced in honey bee head under the same conditions. Taken together, ACh titer appears to be reduced via a cooperative interaction of the AmAChE1 overexpression and AmChAT underexpression and to be linked to reduced inmmune responses under the overwintering and brood rearing-suppressed conditions. The roles of AmAChE1 (with little catalytic activity) and AmChAT in the ACh homeostasis and signaling was discussed in the contexts of immune response and longevity regulation in honey bees.

We evaluated the larvicidal activity of 12 Apiaceae plant essential oils and their components against the Asian tiger mosquito, Aedes albopictus, and the inhibition of acetylcholine esterase with their components. Of the 12 plant essential oils tested, ajowan (Trachyspermum ammi), caraway seed (Carum carvi), carrot seed (Daucus carota), celery (Apium graveolens), cumin (Cuminum cyminum), dill (Anethum graveolens), and parsley (Petroselinum sativum) resulted in >90% larval mortality when used at 0.1 mg/mL. Of the compounds identified, α-phellandrene, α-terpinene, p-cymene, (-)-limonene, (+)-limonene, γ-terpinene, cuminaldehyde, neral, (S)-+-carvone, trans-anethole, thymol, carvacrol, myristicin, apiol, and carotol resulted in >80% larval mortality when used at 0.1 mg/mL.

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic Ca2+ ([Ca2+]i), transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular Ca2+ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent Ca2+ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced [Ca2+]i increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

The striped fleas beetle (SFB), Phyllotreta striolata is a notorious and widely distributed pest in the world including Korea. We surveyed SFB insecticide resistance in Pyeongchang and Hoengseng population. Leaf dipping bioassay with recommended concentration of insecticides was performed using SFB adults. Two populations showed high level of resistance in various insecticides particularly neonicotinoids. Nicotinic acetylcholinesterase receptor (nAChR) is well known target site in neonicotinoid. Therefore, we tried to identify the nAChR from the SFB. Primary, partial contig sequence obtained by RNAseq from Hoengseng population and additional gap fillings were conducted. Total 12 nAChR subunits (10 alpha subunits and 2 beta subunits) were identified and phylogenetic relationships were predicted. Earlier studies reported that point mutations contribute to develop the imidacloprid resistance in aphids and brown planthopper, however we did not find it. Further studies relative to neonicotinoid insecticide resistance mechanism are demanded.

The effects of a newly developed flower thinning formulation (FTF) on the vitality of the honey bee Apis mellifera were examined by measuring the activities of various digestive enzymes in adult worker bees. First, direct spraying of the FTF solution did not cause any behavioral changes or lethal effects for the honey bees based on 24 h observation. Second, oral ingestion of a sugar solution containing the FTF did not produce any significant change in the activities of amylase, proteinases, lipase, or acetylcholine esterase (AChE) in the worker bees 6 h or 24 h after treatment. Meanwhile, a commercial formulation containing sulfur compounds showed slightly reduced activities for several digestive enzymes and AChE, although no behavioral disturbance. Thus, the results of the present study suggest that the FTF is not toxic for honey bees, in terms of contact and ingestion. Therefore, this newly developed FTF can be used for flower thinning without any detrimental effects on pollinating insects.

We evaluated insecticidal and acetylcholine esterase inhibition activity of 11 Apiaceae plant essential oils and their constituents against male and female adults of Blattella germanica. Of 11 Apiaceae plant essential oils tested, dill (Anethum graveolens), carvi (Carum carvi), and cumin (Cuminum cyminum) essential oils demonstrated > 90 % fumigant toxicity against male adults of German cockroach at 5 mg/filter paper. In contact toxicity test, dill (Anethum graveolens), carvi (Carum carvi), cumin (Cuminum cyminum), and ajowan (Trachyspermum ammi) essential oils produced strong insecticidal activity against male and female adults of German cockroach. Among test compounds, carvone, 1,8-cineole, trans-dihydrocarvone, cuminaldehyde, trans-anethole, p-cymene, and γ-terpinene demonstrated strong fumigant toxicity against male and female adults of B. germanica. In a contact toxicity test, carveol, cuminaldehyde, (S)-(+)-carvone, trans-anethole, thymol, and p-cymene showed the strong contact toxicity against B. germanica male and female adults. IC50 values of α-pinene, carvacrol, and dihydrocarvone against female acetylcholine esterase were 0.28, 0.17, and 0.78 mg/mL, respectively. The toxicity of blends of constituents identified in 4 active oils indicated that carvone, cuminaldehyde, and thymol were major contributors to the fumigant or contact toxicity of the artificial blend.

아세틸콜린 분해효소(acetylcholine esterase, AChE) 억제제는 아세틸콜린 함량을 높여 콜린성 neuron 을 활성화함으로써 기억 능력의 개선 및 치매 개선을 가져와 현재 다양한 AChE 억제제들이 개발되어 사용되고 있다. 본 연구에서는 AChE에 대한 억제 활성을 갖는 천연물을 다양한 식물추출물 및 에센스오일로부터 탐색하였으며, 탐색한 추출물의 scopolamine으로 기억손상을 유발한 쥐의 기억력 개선 활성을 치매 치료제로 사용하고 있는 donepizil과 비교 분석하였다. 그 결과 자몽(Citrus paradisi) 유래의 에센스 오일이 AChE 억제 활성이 가장 높아 20 ug/ml의 농도로 처리하였을 때 90% 이상의 효소 억제 활성을 나타내었다. 수동회피 실험 결과, 자몽 유래의 에센스오일(100 mg/kg, p.o.)을 투여한 쥐는 치매 치료제로 사용하고 있는 donepizile (0.5 mg/kg)을 투여한 쥐와 유사한 latency time을 나타내어 인지기능이 개선되었다. 또한, 수중미로 시험 결과, 자몽 유래 에센스오일(100 mg/Kg, p.o.)을 투여한 쥐는 donepizile(0.5 mg/kg)을 투여한 쥐와 유사한 latency time을 나타내어 인지기능이 개선되었다. 이상의 결과로부터 자몽 유래 에센스오일은 매우 효과적으로 기억력을 개선하여 인지기능을 개선해 줄 수 있는 안전하고 효과적인 후보물질이라고 사료된다.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the geneitself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface asa GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by α3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing α3β4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

체외 배양 과정 중에 나타나는 생쥐 초기 2-세포 배의 "in vitro 2-cell block" 현상은 세포내 농도 변화와 밀접한 관련이 있다. 다양한 종류의 세포에서 acetylcholine은 세포막에 존재하는 muscarnic acetylcholine receptor를 통해 세포내 농도 증가를 유도한다. 본 실험에서는 생쥐 "in vitro 2-cell block" 현상에 있어서 ACh의 영향을 알아보기 위해 세포 내 농도 조절 물질을 처리한 후,