Harlequin ladybird (Harmonia axyridis (Pallas, 1773)) is an invasive species originating from Asia, posing a potential threat to the ecosystem and the wine industry in New Zealand due to wine taint, although it can also be a useful biocontrol agent. In this study, the response profiles of antennal olfactory receptor neurons (ORNs) to 32 plant volatiles were examined in male and female H. axyridis, using the single sensillum recording technique. Various types of ORNs were identified from four types of olfactory sensilla in both male and female H. axyridis, with no sexual dimorphism. The most abundant type of sensilla contained two ORNs exhibiting highly specialized responses to methyl benzoate and β-caryophyllene, respectively. Another type of sensilla also contained two specialized ORNs, one responsive to geranyl acetate and the other to some aromatic compounds such as 2-phenylethanol, benzyl acetate, methyl benzoate, and methyl phenylacetate. In contrast, two other types of sensilla contained broadly tuned ORNs, one containing ORN(s) responsive to six-carbon alcohols such as (Z)-3-hexen-1-ol, 1-hexanol, and isomers of 2-hexen-1-ol as well as some other non-alcohol green leaf volatiles, and the other containing ORN(s) exhibiting responses to β-myrcene, geraniol, linalool, nerol, benzyl acetate, and methyl phenylacetate. This study suggests that H. axyridis possesses a set of ORNs specialized for specific plant volatiles, providing insights into the olfactory communication system of this species and potential volatiles to be used for trapping this insect.

The ladybird beetles have been used as biological control agents against several pest species. The aim of this study is to evaluate the compatibility between ladybird beetles and an entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin AAD16. The virulence of AAD16 strain was compared with that of commercial GHA strain on three developmental stages of two coccinellids; Harmonia axyridis Pallas and Chilocorus stigma Say. The topical application method was used for both adult and larval stage while dipping method was used for egg stage. The LT50 and mycosis rate for all life stages of two ladybird beetles tested were not significant between AAD16 and GHA strain. In larval stage, the mycosis rates of H. axyridis and C. stigma were 36 and 40% and 63 and 60% in AAD16 and GHA strain. In adult stage, the mycosis rates of H. axyridis (male and female) and C. stigma (unsexed) were 20, 23, and 23% and 26, 30, and 30% in AAD16 and GHA strain, respectively. Therefore, the two predatory coccinellids could be compatible with these two fungus strains due to their relatively lower mycosis rate.

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.

Harmonia axyridis have become extremely popular through their extensive elytra and spot variation for many years. However, phenotypic polymorphism and striking visual phenotypes are not yet to be extensively studied. With growing attention to polymorphism of H. axyridis, it is imperative to study involved in genomic research in order to elucidate the molecular mechanisms underlying the diversity of elytra variation. To figure out suitable approach for analyzing genes of H. axyidis, we assessed RNAi as a tool for functional analysis. In order to demonstrate the utility of RNAi technique on H. axyridis, we conducted cDNA library screening to affirm silencing effect. We choose random genes from cDNA clones and the random genes were synthesized into dsRNA. After the injection of dsRNA into last instar larvae, we observed morphological defects or deaths. To investigate the effectiveness of knockdown in H. axyridis, we performed quantitative real time PCR on H. axyridis that appears the most distinctive features. These results provide that RNAi is highly applicable in H. axyridis and it will be useful for the gene functional analysis.

무당벌레(Harmonia axyridis)는 종내에서 초시색상패턴이 매우 다양하게 존재한다. 본 논문에서는 서로 다른 색상패턴의 무당벌레를 대상 으로 amplified fragment length polymorphism (AFLP)을 실시하여 무당벌레의 초시색상 패턴간 유전형질의 차이를 확인하고자 하였다. 총 28 개의 프라이머 조합으로 실험을 실시한 결과, 총 2,741 개의 밴드가 검출되었다. 그 중 20 개의 밴드(S1-S20)만이 특정 색상패턴에서 나타났다. 이들 가운데 9 개의 밴드를 색상에 연관된 AFLP 후보 지표로 선발하였다. 밴드 가운데 S1과 S2, S20은 Succinea 1, 2 변이형에 공통적으로 나타났으며, S3와 S5는 Conspicua 변이형에 특이적이었다. 또한 S13는 Spectabilis 변이형에, S15와 S18, S19는 Succinea 2 변이형에 특이적이었다. 특정 색상패턴에만 나타나는 9 개의 AFLP 지표들은 cloning을 통해 염기서열 분석을 실시하였고, GenBank를 이용해 다른 염기 서열과 비교를 해보았지만 아무런 상동성도 찾을 수가 없었다. 무당벌레 종 내 유전적 다양성을 평가한 결과, Spectabilis가 Conspicua보다 Succinea 변이형에 높은 유사성을 보였다. 색상에 연관된 AFLP 후보 지표를 기준으로 sequence characterized amplified region (SCAR) 지표로 변환하여 9 개의 AFLP 분자지표들 가운데에서 5 개만이 SCAR 지표로 전환될 수 있었으며, 이를 통해 AFLP 지표가 무당벌레의 색상과 연관되어 있는지 확인할 수 있었다.

 ,  , The effects of gamma irradiation on the survival, development, fecundity, and sex ratio of the multicolored Asian ladybird beetle (Harmonia axyridis) were investigated. A dose of more than 50 Gy dose of irradiation on eggs completely inhibited hatch, and 1st and 3rd instar larva irradiated with over 40 Gy almost died. Adverse effects of gamma irradiation over 70 Gy were also observed in the survival and development of pupae and adults. tail indicating DNA damage was showed in survival individuals exposed to gamma irradiation.Although irradiated individuals usually survived, they had longer periods of larval and pupal development than untreated individuals. Slight female-biased sex ratios were observed in some treatments. However, sex ratio differences soon equilibrated. Generally, inherited harmful effects of gamma irradiation were observed in F1 and F2 generations. Comet tail indicating DNA damage was showed in survival individuals exposed to gamma irradiation.

Enterobacteria were isolated in the gut of the predacious multicolored Asian ladybird beetle, Harmonia axyridis, and their effects to the development of H axyridis were examined. Populations of H axyridis in this experiment were collected from Kimjae at Cheonbuk province (JK population), Geumsan at Chungnam province (CK population) and laboratory population at Laboratory of Insect Physiology in Chungnam National University, Daejeon. Thirty-four enterobacteria isolates were purified and isolated from the digestive tract of H. axyridis, and a total of 4 strains were classified into group by analysis of 16S rRNA gene sequences. About 70% of total isolates were phylogenetic groups of Bacillus genus and Staphylococcus genus, and they were commonly separated from the digestive tract of H. axyridis. After investigating their susceptibility against antibiotics with 18 representative enterobacteria isolates, ofloxacin and penicillin were selected for examination in this study of their ability to inhibit the growth of all of isolates. In order to remove the enterobacteria from the aphids, ofloxacin and penicillin were given to the green peach aphid, Myzus persicae, and the turnip aphid, Lipaphis erysimi. These aphids were provided to H. axyridis as prey. The weight of pupa, developmental periods of each larval instar, the number of eggs and their hatching ratio of H. axyridis with treatment aphids were lower compared with non-treatment aphids. Staphylococcus saprophyticus is a representative enterobacteria and commonly isolated from the digestive tract of H. axyridis. In the absence of S. saprophyticus, the developmental periods of each larval instar increased, however, the weights of pupa, the number of eggs, and their hatching ratio decreased.

곤충의 생태학적 역할에 있어서 공생미생물은 대부분의 곤충그룹에서 나타나 는 상호간 절대적 이익관계에서부터 해로운 기생적 관계까지 여러 다양성을 가지 고 있다. 곤충의 장내에서나 표면에서 많은 다른 미생물들과 서로 다양한 방법으 로 공존하면서 생활하고 있는데, 넓은 범위에서 공생관계가 있는가 하면 기주의 생리에 아주 밀접하면서 필수적인 관계만을 나타내는 것도 있다. 공생자는 대부분 곤충의 소화기관 부근에서 발견되어지고, 이것은 생장과 발육에 필수적인 아미노 산이나 비타민 등을 합성하는 것으로 밝혀지고 있다. 본 연구에서는 포식성 곤충 인 무당벌레(Harmonia axyridis)의 령기별, 먹이별로 장내에 존재하고 있는 세균 의 분리 및 동정을 통해, 무당벌레 장내미생물의 종류를 알아보고 무당벌레의 생 물학적 특성에 미치는 영향을 보고자하였다. 충남 금산의 월동개체군을 채집하여 무테두리진딧물로 실내에서 누대사육을 통해 확보한 자손세대 무당벌레 유충(1령~4령)과 진딧물, 설탕물, 닭간을 먹인 성 충의 소화기관을 분리하고 장내세균을 분리 동정한 결과, 총 31균주가 분리 동정 되었다. 설탕물을 먹인 성충을 제외하고 모든 령기와 성충에서 Staphylococcus saprophyticus가 분리 동정되어, 이 세균을 실험균주로 선발하여 S. saprophyticus 의 존재여부에 따른 기주곤충의 발육특성 및 생식특성에 미치는 영향을 조사하였 다. 대상균주의 부재로 인한 특성을 확인하기 위해 항생제인 ofloxacin을 처리한 처리구와, S. saprophyticus을 먹이인 진딧물에 처리하여 공급한 후, 무당벌레의 산란력, 부화율, 번데기 무게, 유충발육기간을 조사한 결과, 알의 부화율은 항생제 처리로 인한 실험 장내세균의 부재로 인해 감소하는 경향을 보였으나, 유충의 발 육기간이나 번데기 무게와 같은 발육특성에 미치는 영향은 확인되지 않았다.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

곤충의 장내에는 여러 세균들이 서식하며 공생관계를 가지고 기주곤충의 소화, 발육, 생존, 번식 등에 중요한 역할을 하는 것으로 알려져 있다. 최근에 공생세균이 기주곤충의 생장과 발육에 필수적인 아미노산이나 비타민을 합성하는 것으로 밝 혀지고 있고, 이러한 공생세균은 곤충의 소화기관 부근에서 발견되어지는 것으로 보고되고 있다. 본 연구에서는 포식성 곤충인 무당벌레(Harmonia axyridis)의 장 내에 존재하고 있는 세균의 분리 및 동정을 통해, 무당벌레 장내미생물의 종류 및 이들 세균이 무당벌레의 생물학적 특성이나 생태학적 특성에 미치는 영향을 조사 하고자 하였다. 전북 김제와 충남 금산 월동개체군과 실내에서 누대사육한 무당벌레 유충과 성 충의 소화기관을 채취하여 장내세균을 분리하고 동정한 결과 Arthrobacter속 외 33균주가 확인되었으며, 분리된 균주를 여러 계통의 항생제가 첨가된 배지에 배양 하였다. 그 중 억제효과를 보이는 항생제인 ofloxacin, penicillin을 선발하여 무당 벌레의 먹이가 되는 복숭아혹진딧물에 직접 처리하여 사육하면서 번데기무게, 유 충기간, 성충의 산란력, 부화율 등의 생리적인 특성을 조사한 결과, 항생제 처리에 의한 장내세균의 부재시 무당벌레의 생물학적, 생태학적 특성에 있어 정상개체와 차이를 보이는 것으로 확인되었다. 특히나 실험에 이용한 세 지역의 무당벌레 개 체군의 장에서 분리된 Staphylococcus속 세균의 경우, 2령 유충단계에서부터 분리 됨에 따라 이 균의 존재여부에 대한 무당벌레의 유충기간, 번데기무게, 성충의 산 란력 및 부화율을 조사하여 장내세균의 역할을 구명하고자 하였다. 그 결과, Staphylococcus속 세균의 부재시 유충기간이 증가하였고, 번데기무게, 성충의 산 란력은 감소하였다.

무당벌레(Harmonia axyridis)는 동종 내에서의 색상패턴의 변이가 많은 종으로 잘 알려져 있으며, 100여 종류 이상의 다양한 초시무늬패턴이 보고되는 등 놀라운 무늬패턴 다형성을 가지고 있다. 현재까지 이러한 다양한 색상변이의 원인은 유충 시기의 먹이나, 번데기 시기의 온도와 습도조건 등 환경적인 요인과 교잡을 통해 유전적인 면의 일부를 확인하였으나 이러한 변이의 원인에 대해 확실히 밝혀진 것 은 없다. 따라서 본 연구에서는 저온조건이 무당벌레 초시의 멜라닌패턴 발현에 미치는 영향을 조사하기 위해, 멜라닌그룹의 conspicua과 비멜라닌그룹의 succinea1을 교잡하여 부화시킨 1령 유충을 사육온도(15와 25℃)를 달리하여 plant culture dish(D×H: 100×40mm)를 사육용기로 사용하고 진딧물을 공급하여 사육하여, 성충까지 사육하고 발육기간과 그들의 초시 무늬 패턴을 조사하였다. 멜라닌그룹(conspicua×conspicua) 사이의 우화한 성충의 초시패턴을 분석한 결 과, 멜라닌그룹의 발현비율은 저온에서는 71%, 상온에서는 31%로 사육온도간 차 이를 보였으며, 비멜라닌그룹(succinea1×succinea1)에서는 succinea2 패턴이 70.5%, 40%로 나타나 자손세대의 멜라닌색상패턴의 발현에 저온이 영향을 미치 는 것으로 여겨진다.

Toxicity of seven environment friendly agricultural materials (EFAM), which have been used in the domestic market were evaluated on honeybee (Apis mellifera) and asian multicolored ladybird beetle (Harmonia axyridis). Three EFAMs made from plant extract agents (Wangjoongwang Eco, Bogum Eco and Bestop Eco) and four EFAMs made from microbial utilizing agents (Worldstar Eco, Goodmorning, Bluechip and Cameleon) were investigated as EFAMs. In evaluation of toxicity on honeybee, the RT25 values of 3 EFAMs made from plant extract agents ranged from 1 to 3 days. Therefore, honeybee should be released 1-3 days after application of these EFAMs. Meanwhile, the four agricultural materials made from microbial utilizing agents did not show any mortality against honeybee. In evaluating the toxicity to adult and larva ladybird beetles, all seven EFAMs made from plant extract agents and microbial utilizing agents to show any mortality.

Gut symbiotic microorganisms of insects are known that play important roles in the digestion, development, survival, and reproduction of host insects, and it is widely accepted that symbionts synthesize essential amino acids or vitamins for host insect among these relationship. More recently, however, it has become increasingly apparent that symbiont's contribution to their host are not limited to nutrition. Actually, these symbionts are frequently observed near an insect's digestive tract. In this study, we isolated and identified gut bacteria of ladybird, Harmonia axyridis, which is a predator and used the factor for biological control of aphids, and was aimed to survey gut bacteria affecting to ladybird's biological or ecological characteristics. We separated the guts of larvae and adults ladybird and cultured them in 4 media, such as NA, skim-milk, CMC and CMA. About 33 isolates including genus Arthrobacter were identified in this study. These were cultured in NA media with several antibiotics. We selected ofloxacin and penicillin that inhibited these bacteria growth in media, and reared ladybirds as prey with aphids treated ofloxacin and penicillin 1000ug/1ml concentration. After that, weight of pupae, larval stage, emergence stage, fecundity and hatching rate were investigated. It was shown that, ofloxacin and penicillin treatment was caused by weightless of pupae, fecundity and delay of larval stage, and the affected hatching rate as well.

The multicolored Asian ladybird beetle, Harmonia axyridis, is a generalist predator of aphids also, shows a high level of phenotype polymorphism in color pattern of elytra. Although, it is not sure about genetic information of color polymorphism, it has been confirmed that this phenomenon comes from their genetic traits. The color of H. axyridis elytra is mainly composed of black and red pigment. Phenoloxidase (PO) plays an important role in many insect physiological functions, i.e. sclerotization and pigmentation of cuticle and melanization of parasites. Following activation, PO catalyses the hydroxylation of tyrosine and subsequent oxidation of phenolic substance into quinines, which are further converted to melanin. However, the molecular bases of H. axyridis color pattern formation are almost unknown but it may be that the different pro-POs have different expression. In this study, total RNA samples from four each color pattern individuals, for example, succinea 1, succinea 2, conspicua and spectabilis was extracted. A cDNA enconding pro-PO was molecular cloned from each color pattern of H. axyridis and its putative amino acid sequence shared homology with pro-PO of other insects. We are pursuing to elucidate that their pro-PO sequence will be similar with those other insect PPO sequence. There are also regions of high sequence similarity, including putative activation site and two copper binding sites.

There was different between two differential geographical and environmental condition areas on elytra color expression patterns of the multicolored Asian lady beetles (Harmonia axyridis). Especially, it was investigated that expression rates of melanic patterns (conspicua, spectabilis and axyridis) relatively increased in overwintering populations collected in highly mean temperature and longer cumulative daylength area. In addition, in the same collection site, the seasonal difference had influenced on color patterns of H. axyridis. Although these effects didn"t were not observed in the laboratory, environmental conditions such as temperature or cumulative daylength might be factors that gave an effect on color pattern formation.

To identify DNA markers linked to a elytra polymorphism, amplified fragment length polymorphism (AFLP) analysis was performed on DNA samples from four each colour pattern individuals (2 females and males), for example, succinea 1, succinea 2, conspicua, and spectabilis. As a result of performing AFLP analysis with the restriction endonuclease combination EcoRⅠ and Mse I, total of 2,269 AFLP fragments which were specific to succinea, conspicua and spectabilis was identified using 24 different AFLP primer combinations. Among these 2,269 fragments, 16 bands which were the most specific to one color patterns were isolated, cloned and sequenced. Subsequent UPGMA cluster analysis revealed that population of H. axyridis was divided four major group and these genetic tree showed that H. axyridis elytra colour diversity was affected by genetic polymorphism. It is considered that these genetic analyses may be facilitated the understanding of molecular genetic mechanism related with the wing colour pattern formation in this species.

EMS(ethyl methansulphonate)는 alkylalting 물질에 속하는 화학적 돌연변이원으로 초파리, 꼬마선충, 애기장대, 모기 등 다양한 개체에 single-site mutation을 일으키는 것으로 잘 알려져 있다. 또한 자외선은 곤충이 자외선에 노출됐을 때 치사율이 낮고, 노출량을 쉽고 정확하게 측정할 수 있어 돌연변이유발에 빈번하게 사용된다. 본 연구에서는 다양한 색상패턴을 나타내는 무당벌레의 알과 번데기에 EMS와 자외선이 미치는 영향을 조사하기 위해, 0.1%, 1% 농도의 EMS를 처리하였고, 장파선 자외선인 UV-A(360nm)에 각각 1시간, 12시간 동안 노출시킨 후, 이들의 발육기간, 성비, 색상패턴의 발현비율 등을 조사하였다. 그 결과, 각 농도의 EMS와 UV-A를 알에 처리했을 때, 발육기간은 무처리구와 비교했을 때 큰 차이는 없었으나, 성비의 경우에 있어서는 EMS(무처리는 39.3:60.7, 0.1%처리는 52.9:47.1)와 UV-A(무처리는 39.7:60.3, 0.1%처리는 66.5:33.4) 처리구에서 암컷의 비율이 감소해 성비의 불균형이 확인되었다. 또한 EMS와 UV-A를 처리한 번데기의 우화율은 무처리구와 비교했을 때 큰 차이가 없었으나, 우화한 성충의 성비를 조사했을 경우에는 EMS를 처리했을 때 암컷의 비율이 증가하는 것을 확인할 수 있었다. 또한 유충시기의 다리와 우화한 성충에서 기형 개체를 확인할 수 있어 EMS와 UV-A가 무당벌레에 돌연변이원으로 작용함을 확인할 수 있었다.

무당벌레(Harmonia axyridis)는 동종 내에서의 색상패턴의 변이가 심한 종으 로 잘 알려져 있으며, 현재까지 무당벌레 개체군 안에서 이러한 색상변이의 주요 원인은 유전적 차이와 물리적 환경요인들에 의한 것으로 암시되고 있으 나, 정확하게 구명된 것은 없는 상태이다. 본 연구에서는 환경적 요인에 의해 무당벌레의 초시다형화 현상이 나타나는지를 조사하기 위해 사육온도와 먹이, 광주기 등의 조건을 달리하여, 초시의 반점의 색상이나 무늬패턴을 확인하여 모세대와의 초시표현형을 비교하고자 하였다. 2007년 12월 중 충남 금산군에서 채집한 월동개체군에서 3개의 주요 표현 형 패턴(succinea, conspicua 그리고 spectabilis)을 암수 1쌍씩 선발하여 목화진 딧물을 공급하며 25±2℃와 50±10% RH, 광주기 L:D=16:8의 실험실 조건에서 Plant culture dish(D×H=100×40mm,SPL Life Science,Korea)를 사육용기로 사용하 여 사육하였으며, 교잡 후 산란한 알을 모두 수거하여 부화시켰다. 부화한 1 령 유충은 실내에서 먹이(복숭아혹진딧물, 목화진딧물, 무테두리진딧물), 광주 기(16:8, 12:12, 8:16), 온도(20℃, 25℃, 30℃)를 달리하여 성충까지 사육하고 우화한 성충의 초시패턴을 분석한 결과, conspicua패턴의 경우, 사육된 광주기 별 모세대의 발현비율이 각각 85.9, 83.1, 88.1%(F=2.95, P=0.061)로 광주기는 자손세대의 초시발현비율에 영향을 미치지 않는 것으로 확인되었다. 먹이에 따른 발현비율은 각각 85.7, 83.3, 88.5%(F=3.51, P=0.038)로 먹이간 차이를 보 여, 자손세대의 초시패턴의 발현에 먹이가 영향을 미치는 것으로 여겨진다

The surface of most coccinellids (particularly the elytra) has characteristic colour patterns, which show great variability within many species. Individuals of a species of ladybird often differ from one another in colour and pattern. In case of Harmonia axyridis, environmental factors, such as temperature or food, can probably influence colour and pattern. However, virtually no work has been carried out in the influence of such factor. Therefore, in this study, according to rearing temperature, photoperiod and diet, variation of elytra color patterns were investigated. First instar larva on each color pattern were reared in an incubator at one of two temperatures; 25 and 30℃, under the following photoperiod; L16: D8, L12: D12 and L8: D16 and were provided three species of aphids. And their color pattern of adults were estimated.