Morphology of antennal sensilla and their distribution were investigated in male and female adults of Gymnosoma rotundatum, a parasitic fly to hemipteran species, using scanning electron microscopy. The overall length of antenna was not different between male and female. Antenna of G. rotundatum was composed of scape, pedicel and funiculus in both sexes. Three types of sensilla (sensillum basiconica, s. chaetica and s. coeloconica) were identified from both sexes, in varying numbers and distribution along the antennae. The two sensillum types were further divided into different subtypes; s. basiconica into three subtypes and s. chaetica into two subtypes. Among sensilla, s. basiconica subtype 1, 2 and 3 were multiporous, indicating that the ir primary function is olfactory, and others were not. The s. basiconica was most numerous on the antennae in both sexes. The number of subtype 1 of s. basiconica was different between male and female. The morphological information obtained in our study provides a basis for electrophysiological and behavioral studies of the olfactory sensory function of each morphological type of sensilla. (Supported by PJ011756022018, RDA)

The Korean rhinoceros beetle, Allomyrina dichotoma (Coleoptera: Scarabaeidae), has been traditionally used for the treatment of liverrelated diseases. Recently A. dichotoma was registered as a food ingredient in Korea. Given the increasing interest in this beetle, studies on the development of safe and nutritious feed sources for its commercial production are needed. In this study, we compared the effects of by-products on the growth of third instar larvae of A. dichotoma. An abundance of food by-products have been found to serve as an alternative energy source in animal feeds with the reduced cost. We fed third instar A. dichotoma larvae feeds consisting of fermented sawdust with nine different combinations of citrus peel, soybean curd cake, soybean oil meal, and brewers’ dried grain, and measured their effects on growth until the larvae pupated. The highest survival rate was in feed supplemented with 10% of brewers’ dried grain (66.7%). Especially, larvae fed on 10% of brewers’ dried grain were 26% heavier than the control (with no supplement). For the group of 10% brewers’ dried grain, the larval period of third instar was shortened by almost 28 days compared to the control group. Of the experimental and control groups, only the group of 10% brewers’ dried grain showed more than 90% of pupation rate. Therefore, brewers’ dried grain may be sufficiently useful as a source of feed for A. dichotoma.

Insect peptides have been extensively studied due to beneficial effects in the treatment of infectious diseases. Melittin, a fundamental component of honeybee venom produced by European honeybee Apis mellifera, has applied to prevent various inflammatory disease and bacterial infections in human. However, the therapeutic application of melittin is limited due to its low stability, hemolytic activity and expensive manufacturing costs. In this study, we aimed to discovery unknown peptides from the Apis mellifera and evaluate its antibacterial activity against Escherichia coli KACC 10005. A total 15,853 peptide sequences were diciphered using Illumina HiSeq 2500 next-generation sequencing (NGS) platform and analyzed based on the Apis mellifera official Gene Set Version 3.2 (amel_OGSv3.2) and the Collection of Anti-Microbial Peptides (CAMPR3) database. All the peptide sequences and annotation data sets were combined and sorted by physicochemical features of antimicrobial peptides (AMPs), such as short peptide length <=50, positive charge, isoelectric point (8.0<=pl<=12), and aggregation propensity (in-vitro: <=500, in-vivo: –40<= Na4vSS <=60). Among the screened peptides, four unknown peptide candidates, named AMP1-4, were chemically synthesized and tested for antimicrobial activity in comparison with a reference peptide, melittin. Inhibition of bacterial growth was observed in the AMP4 treated group from 6 hours to 48 hours post-treatment against E. coli. These results suggest that honeybee-derived peptide sequences can be applied as natural resources to acquire novel AMPs and the peptide sequences derived parameters are enough to recognize antibacterial peptides. In addition, the selected novel peptide candidate, AMP4, has antibacterial activity.

The red imported fire ant (Solenopsis invicta) is a species of ant native to South America. The fire ant was inadvertently introduced into USA, Australia, New Zealand, and other Asian countries including China and Taiwan. Since the first report of the fire ant in port city of Busan, Korea in 2017, it was found in many other cities of Korea in following year. To obtain the molecular information of this invasive species, total RNA was extracted from the abdominal segment of the ants collected in Incheon, and subjected to transcriptome sequencing. By using Illumina sequencer platform, 101 base pared-end sequencing generated 2 × 50,064,081 of raw reads to obtain 2 × 45.95 Gbase of quality filtered nucleotide sequences. The in silico cDNA library was constructed by Trinity de novo assembler followed by TransDecoder ORF finder and CD-HIT clustering program to streamline the library. The final version of cDNA library contains 20,442 contigs with protein coding capability. To survey the virome of this ant, these contigs were searched against the viral reference sequences from NCBI RefSeq database with BLASTN program. As a result, contigs which showed high sequence identities with several RNA viruses including previously reported SINV-2 were found from the fire ant. This virome information might give an idea of a shift of virological environment of this newly found ant isolate or population in Korea.

Origin of Red imported fire ant (RIFA : Solenopsis invicta) is Central America, a tropical climate region. It has settled in the invasive area, causing various problems. In recent, Solenopsis invicta were discovered in Busan in 2017 and then in 2018 at a construction site in Busan, Pyongtaek, Incheon and in Daegu. This study aims to confirm the origin of invasive colonies of Solenopsis invicta. We tried to test previously developed microsatellite markers so that we establish the tracing protocol for molecular epidemiology of RIFA. We justified 66 microsatellite markers already developed using DNA from the RIFA found in Incheon Harbor. As results, 30 markers were selected to facilitate amplification and fragment analysis.

Host defense against pathogen invasion highly relies on immune defense machinery that is controlled by the nuclear factor-κB (NF-κB) of transcription factors. The Toll pathway are well known as an insect innate immune mechanism to protect host itself from invaded pathogens. Basically, in the edible insect, Tenebrio molitor, the Toll pathway is primarily activated by polymeric Lys-type peptidoglycans (PGNs), and components of fungal cell walls, β-1,3-glucan. Based on the current studies, the tremendous study has been focused on recognition and subsequent activation of spätzle in haemolymph, hence, there is a grave gap for intracellular event. Herein, in order to understand intracellular event of Toll signaling pathway, the Dorsal gene were identified. Moreover, domain analyses of TmDorsal2 gene indicate that there are two major domains such as Rel homology domain (RHD), ig-like, plexins, and transcription factors (IPT) domains. Based on the achieved results, TmDorsal2 mRNA was highly expressed in 1-day old pupa. Furthermore, TmDorsal2 was highly expressed in Malpighian tubules and fat body in last instar larvae (LL), and likewise mainly expressed in Malpighian tubules during adult 5-day old period, also the lowest expression of TmDorsal2 was observed in gonads. Moreover, TmDorsal2 mRNA levels after infection with E. coli appreciably went up at 6 and 9h time points. To investigate the effects of TmDorsal2 RNAi on larval susceptibility against various pathogens namely E. coli, S. aureus or C.albicans, dsRNA of TmDorsal2 has been synthesized the larvae dissected after 24h. As a result, TmAttacin1a, 1b and 2, TmDefencine1 and 2, TmTenecin1, 2, 3 and 4, TmCecropin2, TmColeoptericin1 and 2, Thaumatin-like protein 1 and 2 markedly reduced in the gut after injecting all mentioned microbes. In contrast, TmTenecin 2, Thaumatin-like protein 1 and 2 strikingly increased after microbe injection in the fat body. Interestingly, the most AMPs gene expression in whole body experimental case were upregulated. On the horizon, we will investigate effects of TmDorsal1 RNAi on larval susceptibility against various pathogens. Taken together, our studies may aid to understand insect innate immunity.

It has been well known that IKK-β, -ε and –γ play a pivotal role in IMD pathway. In this study, TmIKK-ε was identified and their functions in countering pathogenic infections were investigated. We identified TmIKK-ε gene which including 2,196 bp nucleotides (encoding 731 amino acid residues). Domain analysis of TmIKK-ε indicates that there is one Serine/Threonine protein kinases catalytic domain. TmIKK-ε gene was highly expressed in 2 day-old pupal stage and the expression was gradually decreased until 1 day-old adults. Then the expression was slightly increased until 4 day-old adult stage. Tissue specific expression of TmIKK-ε mRNA was high in the gut, integuments and hemocytes in last instar larvae, and fat body, Malpighian tubules and testis in 5-daysold adult. In hemocytes, TmIKK-ε was drastically induced by E. coli injection after 3 h and by S. aureus at 3 and 12 h-post injection. In gut, expression level of TmIKK-ε was high at 6 h-post injection of microbial injection. Expression of TmIKK-ε in fat body was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-ε, gene specific RNAi and mortality assay were performed. TmIKK-ε RNAi caused increased larval mortality against E. coli, not S. aureus and C. albicans. Finally, to investigate the induction patterns of Tenebrio fourteen AMP genes in response TmIKK-ε RNAi, three microorganisms were treated into TmIKK-ε-silenced T. molitor larvae. Nine out of fourteen AMP genes were not induced by microbial challenge in TmIKK-β dsRNA-injected group. Taken together, our results indicate that TmIKK-ε may regulates nine antimicrobial peptide genes in response to microbial challenge in T. molitor fat body.

Autophagy is an important self-eating process to eliminate damaged or unused organelles. We identified nine autophagy-related genes (Atg) including AaAtg-1, -3, -4b, -4d, -5, -6, -8, -12 and -13 from the Asian tiger mosquito, Aedes albopictus. Developmental expression patterns indicate that mRNA levels of AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 were highly expressed in egg, whereas expression of AaAtg8 was high in 1stand3rdinstarlarvalstages. TissuespecificexpressionofthesegenesindicatesthatAaAtg1 was highly expressed in thorax and midgut in blood-fed adult female mosquitoes (BF), and head and thorax in sugar fed adult female mosquitoes (SF). Transcript level of AaAtg3 was high in thorax in BF, but head, thorax and Malpighian tubules in SF. AaAtg4b, -4d mRNA levels were significantly high in Malpighian tubules in BF, and head in SF, respectively. AaAtg-5 and -6 transcripts were highly expressed in head in BF, and expression of AaAtg-8 was high in Malpighian tubules in BF. Levels of AaAtg-12 and -13 mRNAs were significantly high in head and midgut in BF. Induction patterns of AaAtg genes against pathogens showed that AaAtg-1, -3, -4b, -8, -12 and -13 were strongly induced at 6 h-post injection of S. aureus, and mRNA levels of AaAtg-1, -3 and -13 were significantly induced by E. coli challenge after 3 h-post injection in SF abdominal carcass. In SF midgut, AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 transcripts were drastically induced at 9 h-injection of E. coli and S. aureus, while expression of AaAtg-8 was highly induced by S. aureus and C. albicans at 9 h-post injection. Each AaAtg gene was slightly induced by E. coli, S. aureus or C. albicans at different time points in abdominal carcass in BF. Interestingly, AaAtg-8 was not induced by microbial challenge. While eight other Atg genes except AaAtg-8 were highly influenced by S. aureus at 6 and 9 h-post injection, E. coli at 3 h-post-treatment, and 3, 6, and 9 h-post inoculation. In the future, we will characterize the functional roles of autophagy during mosquito-microbes interaction.

최근 기온이 높아지면서 강원도 대관령 감자 재배지로 날아오는 진딧물 발생이 늘어나고 있다. 그 중 복숭아혹진딧물(Myzus persicae)은 전 세계적으로 분포하고 경제적으로 중요한 해충으로, 감자, 고추 등 노지작물에 흡즙을 통한 바이러스 매개하고 생육저하를 일으키는 피해를 주고 있다. 본 연구는 복숭아혹진딧물의 지리적 차이에 따른 집단의 유전적 관계 규명을 통해 기주식물, 메타집단 등의 군집구조 및 발생패턴을 분석하고, 이러한 요인으로 인한 진딧물 집단의 분산 및 이주 관계를 추정하였다. 최종적으로 개체들 간의 집단유전학적 관계규명을 통해 이들의 확산과 비래 양상을 규명하고자 한다.

The genus Camponotus Mayr, 1861 distributes all over the world, even ranging from the African desserts to the Antarctic islands. Number of species in this genus is the largest: 1,488 taxa (species and subspecies) accounting for 10% ant species. Despite of it, number of available complete mitochondrial genomes was only one, Camponotus atrox (subgenus Camponotus). To decipher characteristics of mitochondrial genome of this genus, we completed mitochondrial genomes of Camponotus nipponensis Santschi, 1937, belonging to subgenera Myrmentoma using Illumina HiSeqX. The total length of mitochondrial genome of C. nipponensis is 15,532bp with 79.8% A + T content. It consists of 13 PCGs, 22 tRNA, and 2 rRNA genes and an A+T–rich control region. This mitochondrial genome will be a useful resource to understand features of Camponotus genus with adding more mitochondrial genomes of other subgenera, such as Paramyrmamblys.

Toll and IMD pathways play an important role in producing antimicrobial peptides (AMPs) through NF-κB in insects. The functions of IκB kinase (IKK) complex regulating the NF-κB signaling cascade have not yet been investigated in Tenebrio model. Here, we identified TmIKK-β (or TmIrd5) which contains 2,112 bp encoding 703 amino acid residues. Domain analysis shows that TmIKK-β contains one Serine/Threonine protein kinases catalytic domain. Developmental expression patterns indicate that TmIKK- β gene was highly expressed in early pupal (P1) and adult (A5) stages. Tissue specific profiles show that TmIKK-β was highly expressed in the integuments in last instar larvae, and fat body and hemocytes in 5 day-old adults. TmIKK-β1 transcripts were strongly induced at 3 and 12 h-post injection of E. coli, and 3 h-post injection of S. aureus or C. albicans in hemocytes. In gut, TmIKK-β transcripts were slightly induced by E. coli (at 6, 9 and 24 h) and C. albicans (at 24 h), while it was not induced by S. aureus challenge. Moreover, it was highly induced at 6 h-post injection of E. coli and then it was gradually decreased in the fat body. To understand the immunological role of TmIKK-β, gene specific RNAi and mortality assay was performed. Depletion of TmIKK-β mRNA leads to increase microbial susceptibility of larvae against E. coli, S. aureus and C. albicans. In addition, induction patterns of fourteen AMP genes in response to microbial challenge was tissue specifically investigated in TmIKK-β–silenced T. molitor larvae. The results suggest that expression of ten AMP genes out of fourteen genes were drastically decreased by TmIKK-β RNAi in fat body, suggesting that TmIKK-β plays an important role in antimicrobial innate immune responses.

Incursions of red imported fire ant (RIFA), Solenopsis invicta into Korea have been increasing. After a first interception of a colony of S. invicta on Gamman pier, Pusan port while intensive surveillance by Animal & Plant Quarantine Agency (APQA) in September 2017, three more RIFA colonies have been found in sea port piers of Pyeongtek, Incheon and Pusan cities. The social forms and mitochondrial DNA haplotypes of the intercepted RIFA colonies were analysed by allelic discrimination assay (peptide nucleic acid probe based RT-PCR) of Gp-9 gene and mt-DNA fragment of 768 bp, which is part of the Cytochrome oxidaseⅠ gene. The colony on Gamman pier, intercepted in Sep. 2017 was previously reported as a haplotype 5 (H5) of mitochondrial DNA and a social form of polygyne. The colony on Hutchison pier of Pusan port, intercepted in June 2018 were confirmed as a H22 haplotype and a monogyne. Those different social forms show different origins of each colonies. Those on piers of Pyeongtek and Incheon ports, also found in 2018 were confirmed samely as H22 and monogyne. However, it could be putatively assumed that those two colonies were differently introduced via different container cargoes, considering those colonies were found in container yards of distantly located different sea ports. More genetic variation analyses using diverse sets of molecular markers such as microsatellites, genome-wide single nucleotide polymorphisms, etc. in nuclear gens are being proceeded for more exact introduction routes (origins).

Allomyrina dichotoma (order Coleoptera, family Scarabaeidae) is used for development of pharmaceuticals, pet or educational purposes and animal feedstuffs. The disease occurrence and distribution of Oryctes rhinoceros Nudivirus were investigated in Allomyrina dichotoma in Korea using PCR and analyzed the DNA seqeunces using BLAST(Basic Local Alignment Search Tool). The virus infected larvae were collected from 10 insect rearing farms in five different regions (Gyounggi, Chungbuk, Chungnam, Jeonnam, Daejeon). Frequency of OrNV virus infection appeared differently depending on the regions or rearing facilities (open field, vinyl house, indoor breeding system and etc.). The collected samples of Allomyrina dichotoma raised on open fields showed the highest possibilities of OrNV virus infection. The OrNV average infection rate of open fields rearing systems was 50.0%.

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce chitosan. In insects, this modification may contribute to the affinity and/or cross-linking of chitin/chitosan-like polysaccharides for a variety of structural proteins, which may lead to diverse mechanical properties of the cuticle. We previously reported the functional importance of Group I CDAs, TcCDA1 and TcCDA2, as well as the two alternative spliced isoforms of the latter, TcCDA2a and TcCDA2b from the red flour beetle, Tribolium castaneum in molting, morphology of cuticle and movement of legs. In this study, we further analyzed protein localization, ultrastructural defects of the cuticles and leg joints after RNAi of those genes. Both proteins are mainly present in the innermost procuticle region called the “assembly zone”. Loss of function of either TcCDA1 or TcCDA2 caused disorganized chitinous horizontal laminae and vertical pore canals in both the rigid and soft cuticles. RNAi of TcCDA2b affects cuticle integrity similar to that seen in RNAi of the two alternatively spliced forms of TcCDA2. In contrast, TcCDA2a-deficient adult, like that seen in the hypomorphic phenotype produced by RNAi of TcCDA1, exhibited ruptured tendons between femur and tibia, resulting in loss of locomotion ability. These results suggest that Group I CDAs play critical roles in molting, morphology, ultrastructure and mobility in T. castaneum. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).

Insect cuticle or exoskeleton is an extracellular matrix consisting of three major morphologically distinct layers, the water-proofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. To accommodate growth, insects must periodically replace their cuticles in a process called “molting or ecdysis”. During each molt cycle a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. In this study, we show a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (group III) of insect chitinases in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to maintain the integrity and the mechanical strength of a light-weight, yet rigid, adult cuticle. Because group III chitinases are highly conserved among insect and other arthropod species, these enzymes have a critical role in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate cuticular extracellular matrices. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2018R1A2B6005106)

The eggs of Asian tiger mosquito, Aedes albopictus, possess high desiccation resistance, which contribute the rapid spread of this mosquito across the world. Melanization of eggshell appear to play a role in the resistance to desiccation. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the eggshell. In this study, we demonstrated functional importance of two yellow genes, AalY-g and AalY-g2, in the chorion formation. Both genes were highly induced in the ovary at 48 h after blood meal. Injection of dsRNA for AalY-g or AalY-g2 into adult females had no effect on fecundity. However, the outermost colorless exochorion of the eggs obtained from both dsRNA-treated females was fragile and peeled off in places, and melanization of the endochorion was obviously delayed by several hours. In addition, unlike eggs from control females which acquired high desiccation resistance between 18 and 24 h after oviposition (HAO), 60-70% 24 HAO eggs from either AalY-g- or AalY-g2-deficient females were collapsed when they were moved to an air-dry condition, and the desiccation resistance was not increased in later stages of embryonic development analyzed. TEM analysis revealed that abnormal morphology and ultrastructure of the endochorion, particularly outer-endochorion, in the 24 HAO and older eggs from either AalY-g-and AalY-g2-deficient females. These results indicate that AalY-g and AalY-g2 are required for morphology and formation of the endochorion (outer-endochorion), a structure that appears to be critical for desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106)

Korean horn beetles, Allomyrina dichotoma (Linnaeus, 1771) reared in many local farms are suffering from a fatal viral disease as reported in 2015, and recent sequencing analysis revealed that the virus is very closely related to Oryctes rhinoceros nudivirus. In a nationwide investigation, it was concluded that the virus can transmit vertically from an infected adult to the offspring by indirect manner, and 70 to 77% of young larvae dead in early stage were diagnosed as this virus positive. Here, we report for the establishment of on-site diagnosis method for the viral disease using loop-mediated isothermal amplification (LAMP) assay. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting OrNV_gp102 gene. To evaluate the LAMP specificity, two bacterial pathogens, Bacillus thuringiensis and Serratia marcescens, causing disease in A. dichotoma were tested along with OrNV. Only from the OrNV-infected larvae the reaction resulted positive. Also, to avoid DNA extraction process, the LAMP assay used diluted hemolymph directly and 50-fold dilution was set for diagnosis standardization and convenient on-site application for infected larvae from local farms. The LAMP diagnosis is fast and convenient for nontechnical user in the field and expected to stop this foreign virus spread all over the country.

The multicolored Asian ladybeetle Harmonia axyridis is characterized by polymorphism of the elytral pattern. Melanization in Harmonia axyridis is crucial for their elytral coloration, but the molecular mechanisms are not fully understood in this species. Tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), two key enzymes in the melanin pathway, convert tyrosine and dopa into dopa and dopamine, respectively. In this study, it was to determine the role of TH and DDC of Harmonia axyridis (HaTH and HaDDC) in body and wing pigmentaion produced via melanin pathway. The cDNA sequences of HaTH and HaDDC were cloned to perform RNAi-based functional analysis. Injected dsRNA to the 4th larvae caused knockdown of target genes, and it was verified by quantative realtime PCR. Both TH and DDC RNAi adult show loss of black pigmentation in their body and wing pigmentaion. These results is expected to be helpful to investigate polymorphism by melanin pigment in Harmonia axyridis.

Promalactis established by Meyrick in 1908, is one of the highest species richness genera of the family Oecophoridae. Over 240 described species are mainly distributed from Oriental and Palaearctic regions. They are usually a tiny-sized and exhibit subtle morphological differences which make problems on morphological-based identification. In this study, we performed DNA barcoding study for the Promalactis by using mitochondrial cytochrome c oxidase subunit I (COI). From analyzing 155 COI sequences, we observed the usefulness of the COI in species identification with intraspecific genetic variation (range 0.0-2.1%) and interspecific genetic divergence (range 2.8-15.4%).

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.