The ectoparasitic mite, Varroa destructor is one of the most destructive pests of the honeybee (Apis mellifera) leading to the collapse honey bee colony in many regions of the world. RNA interference (RNAi) is a novel approach recently proposed for insect pest control. However, the efficiency of RNAi in insects is low due to the lack of effective delivery methods for dsRNA and sensitivity to nuclease degradation. Therefore, the success of RNAi technology largely depends on the stability of dsRNA. To explore the possibility of using RNAi to control varroa mite, we determined the effects of dsRNA targeting a subunit of the cytoplasmic coatomer protein complex B2, D, and E subunits on target gene expression for varroa mite. We observe that dsRNA ingested by bees is transferred to the varroa mite, resulting in knockdown of COPB2 expression. Furthermore, we demonstrate that chitosan nanoparticles-dsRNA complexes were more stable for 7 days in honeybee tissue fluids. The dsRNA-conjugated with chitosan was protected from degradation in hemolymph, fat body, and midgut extracts collected from the honeybee. These results possibly suggest that nanoparticles-dsRNA complexes might be horizontally transferred from treated honeybee to varroa mite, in which case the honeybees could serve as RNAi vectors. We confirmed, moreover, dsRNA fed nontarget insects, honeybee, were unaffected, and no toxicity was observed for honeybee. Overall, these data suggest that dsRNA-conjugated with chitosan help escape effectively from degradation by honeybee tissue fluids and could improve RNAi efficiency in varroa mite.

지난 10년 동안, 이중 가닥 RNA (double-stranded RNA, dsRNA)를 이용한 특정 유전자 발현 간섭(RNA interference, RNAi) 기술은 의약품 개발뿐만 아니라 작물보호 분야에 해충방제부터 익충보호까지 다양하게 그 기술이 사용되어 왔다. 그동안 학계 및 산업체에서 활발히 연 구되어 온 RNAi기술을 이용한 작물 및 익충보호제는 상용화를 눈앞에 두고 있다. 미래 농업 시장에서 해충방제제와 익충보호제로써의 개발을 위한 RNAi의 기술적 응용은 상당한 잠재력을 가지고 있지만, 현장에 직접 사용되기에는 아직 여러 가지 한계점이나 극복해야 할 과제가 남아있 다. 본 리뷰에서는 최근에 활발히 진행되고 있는 작물보호제 및 익충보호제(protection of crops and beneficial insects)로써의 dsRNA의 다양 한 활용과 그 잠재성(potential)을 소개하고자 한다.

ATP-binding cassette (ABC) transporter는 다양한 기질을 세포 밖과 세포 안으로 수송하는 대표적인 수송단백질이다. 곤충에서 ABC transporter는 살충제에 대한 저항성을 발달시키는 중요한 역할을 한다. 현재까지 모델곤충인 초파리를 대상으로 ABC transporter의 살충제 교차저항성에 관한 연구는 많이 수행되어오지 않았다. 본 연구에서는 ABC transporter에 속하는 Mdr49A 유전자가 여섯 종류의 살충제에 보이는 교차저항성 기작을 형질전환 초파리를 이용하여 구명하였다. 초파리 91-R과 91-C 계통은 공통된 조상으로부터 유래되었으며 91-R은 60년 이상 DDT에 노출되었지만 91-C는 어떠한 살충제에도 노출되지 않고 유지되어 왔다. 91-R 계통의 MDR49A 단백질에서 유래된 3개의 아미노산 돌연변이를 형질전환 초파리에 과발현 시켰을 때 carbofuran에 대해서 2.0~6.7배 그리고 permethrin에 대해서 2.5~10.5배의 교차저항성을 나타 낸 반면 다른 약제, abamectin, imidacloprid, methoxychlor, prothiofos에 대해서는 어떠한 교차저항성도 나타내지 않았다. 이상의 결과는 Mdr49A 유전자의 과발현과 더불어 3개의 아미노산 돌연변이는 두 개 약제, carbofuran과 permethrin에 대해 교차저항성 기능을 한다고 제시하고 있다

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 common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is an ectoparasitic pest that feeds on humans as well as other mammals. We investigate that point mutations on the voltage-sensitive sodium channel are associated with the resistance to pyrethroids. Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel (VSSC) α-subunit gene have been identified in deltamethrin-resistant bed bugs. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly. V419L, located in the IS6 transmembrane segment, is a novel mutation. To establish a population-based genotyping method as a molecular resistance monitoring tool, a quantitative sequencing (QS) protocol was developed. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.993). In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to pyrethroids. A pyrethroid-resistant strain showed > 9375- and 6990-fold resistance to deltamethrin and λ-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB method should greatly facilitate the detection and monitoring of pyrethroid resistant bed bugs in the field.

We analyzed molecular and enzymatic properties of three cholinesterases (ChEs; ClAChE1, ClAChE2 and ClSChE) from Cimex lectularius. The ClAChE1 and ClAChE2 were generally present as a membrane-anchored dimeric insoluble form in the brain and ganglia. In the case of ClSChE, monomeric and dimeric soluble forms were observed. To investigate enzymatic properties, three ChEs were functionally expressed using baculovirus expression system. ClAChE1 revealed a significantly higher activity than ClAChE2 to acetylthiocholine iodide (ATChI) substrate. Kinetic analysis using two choline substrates (ATChI and butyrylthiocholine iodide) demonstrated that ClAChE2 had higher catalytic efficiency but lower substrate specificity than ClAChE1. Inhibition assay was conducted by using three inhibitors (BW284C51, eserine, Iso-OMPA) and two insecticides (chlorpyrifos-methyl and carbaryl). Two ClAChEs revealed high sensitivities to BW284C51, eserine, chlorpyrifos-methyl and carbaryl, but were not sensitive to Iso-OMPA. This inhibition profile confirmed that both ClAChEs are categorized as ChEs. Interestingly, the salivary specific cholinesterase did not show any measurable activities to choline substrates, confirming its non-synaptic function in C. lectularius

We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting its paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland in much higher levels (>3000 fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies in conjunction with activity staining revealed that ClAChE1 is more active than ClAChE2 whereas ClSChE has little enzyme activity. Three-dimensional structure modeling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.