이중가닥 RNA (double-stranded RNA, dsRNA)는 표적 유전자의 발현을 억제하는 기능으로 해충방제에 응용되었다. 인테그린은 α와 β 단위체로 구성된 이량체 막 단백질이다. 진핵생명체에서 인테그린은 세포-세포 및 세포-세포외기질의 상호연결에 중요한 역할을 담당한다. 인 테그린 β 단위체 발현을 억제하는 특정한 dsRNA (= dsINT)는 해당 곤충에 뚜렷한 치사효과를 유발한다. 또한, dsINT를 발현시키는 형질전환 된 대장균도 해당 곤충에 뚜렷한 살충력을 가진다. 그러나 이 세균 살충제의 야외 적용을 위해서는 제형화 기술이 필요했다. 본 연구는 dsINT를 발현하는 재조합 세균을 동결 건조시켜 대상 곤충에 대해 살충효능을 검정하였다. 동결 건조된 세균은 파밤나방(Spodoptera exigua) 종령 유충에 높은 섭식독을 일으켰다. 파밤나방에 대해서 Bacillus thuringiensis 상용 살충제 처리는 불과 60%의 살충력을 보이는 반면, 동결 건조된 dsINT 발현 세균과 혼합 처리할 때 살충력은 크게 증가하였다. dsINT 발현 세균은 해당 인테그린 염기서열 유사성에 따라 차이를 보이는 해충 종에 선 택적 독성을 나타냈다. 이 결과는 인테그린에 특이적 dsRNA를 발현하는 세균이 동결 건조 제형화 조건하에서도 살충력을 유지한다는 것을 나타 냈다.

This study tested a hypothesis that the bacterial immunosuppresants enhance BtI susceptibility of two mosquitoes, the forest mosquito (Aedes albopictus) and the house mosquito (Culex pipiens pallens). Three symbiotic bacteria Xenorhabdus nematophila (Xn), X. hominickii (Xh), and Photorhabdus temperata subsp. temperata (Ptt) were isolated from their symbiotic nematodes and cultured in nutrient broth to allow them to produce the secondary metabolites. BtI gave significant toxicities to A. albopictus and C. pipiens pallens larvae: 50% of lethal concentration to be 2.9 × 105 spores/mL and 2.2 × 105 spores/mL at 16 h after treatment, respectively. Addition of each bacteria-cultured broth significantly enhanced BtI toxicity to the mosquito larvae by lowering LC50 values of BtI to A. albopictus larvae (1.5 × 105 to Xn, 1.7 × 105 to Xh, and 1.9 × 105 to Ptt, respectively) and to C. pipiens pallens larvae (1.2 × 105 to Xn, 1.3 × 105 to Xh, and 1.5 × 105 to Ptt, respectively). Based on these results, we developed a new mosquitocidal Bt formulation called ‘Dip-Kill’, which consisted of 80% Xn-cultured broth, 10% BtI (1010 spores/mL), and 10% preservative. Only 400 ppm of Dip-Kill showed 100% mortality to fourth instar larvae of A. albopictus and C. pipiens pallens 16 h after treatment.

A subtropical insect species, Maruca vitrata, is invasive to temperate zones. Supercooling capacity of M. vitrata was confirmed in all developmental stages, in which egg exhibited the lowest supercooling point (SCP) at –22.5oC. However, low temperature lethality was observed at much higher temperatures than SCPs in all developmental stages. In addition, this nonfreezing injury increased with incubation time at rearing condition. Uncontrolled phenoloxidase activation appeared to cause the nonfreezing injury. Preexposure to a cool temperature before lethal low temperature significantly increased survival of M. vitrata. This cold hardening accompanied with increase of glycerol content in hemolymph. Transcriptome of M. vitrata under cond-hardening was assessed.

Cotesia plutellae bracovirus (CpBV) is a bracoviral polydnavirus and a parasitic factor of an endoparasitioid wasp, C. plutellae, which parasitizes the diamondback moth (DBM) larvae. CpBV genome was reported to consist of 35 circular segments and encode 157 predicted open reading frames (ORFs). This study revisited and re-annotated the CpBV genome. Total ORFs consist of 15 groups of known gene or gene families as well as hypothetical (HP) group. All 74 HP genes are matched to other PDV genes. RNASeq analysis indicates that 147 genes out of 157 predicted ORFs are expressed in the parasitized DBM at the first day or 7 days after parasitization. At the early parasitic stage, CpBV genes annotated as ELP, BEN, E94K, DUFB, PTP, and HP are highly expressed compared to late parasitic stage. Interestingly, highly expressed genes at the late parasitic stages are HPs.

Integrin is a heterodimer protein that locates on cell membrane to interact with neighboring cells or extracellular matrix. A transcriptome analysis of the brassica leaf beetle, Phaedon brassicae, midgut identified both α and β subunits of integrin. RNA interference of β subunit genes significantly impaired survival of both larvae and adults of P. brassicae. A recombinant bacteria expressing double-stranded RNA specific to β integrin of P. brassicae were constructed and showed significant oral toxicities.

Polydnaviruses (PDVs) are a group of insect viruses and symbiotic to some endoparasitoid wasps. Genome analyses of different PDVs provide a number of genes putatively associated with alteration of host insect physiological processes. Especially, PDV gene products assist host wasp development by suppressing immune responses and delaying larval development of parasitized lepidopteran hosts. Thus, PDV genes can be applied to control insect pests by incorporating them into crop genomes. This talk illustrates two examples of PDV genes: CpBV-CST1 and CpBV-ELP1. CpBV-CST1 has been known to inhibit insect cysteine proteases to suppress immune and development, while CpBV-ELP1 exhibits a high cytotoxicity to insect cells. Transgenic tobaccos expressing CpBV-CST1 or CpBV-ELP1 were constructed by using Agrobacterium-mediated transgenic system and exhibited antixenosis against chewing and sucking insect pests on tobacco.

Entomopathogenic nematodes (EPNs) of the genus Steinernema are pathogenic to the insects and well known as ideal models for understanding parasite-host interaction. EPNs harbor a number of bacterial symbionts in their gut belonging to the noble genus Xenorhabdus which are capable of killing insects by themselves or by combination with nematodes by suppressing insect immune defense. Here, we report host range of Steinernema monticolum and its symbiont Xenorhabdus hominickii. S. monticolum has a diverse host range including lepidopteran and coleopteran insects although they showed higher pathogenicity to the lepidopteran insects. Especially, X. hominickii suppressed insect immune responses. A target insect, Spodoptera exigua, exhibited both cellular and humoral immune responses by expressing antimicrobial peptides and forming nodules in response to heat-killed X. hominickii. However, live bacteria significantly suppressed the immune responses. An addition of arachidonic acid to the bacterial infection significantly rescued the immune responses, suggesting eicosanoid biosynthetic pathway as a pathogenic target of X. hominickii.

Two Grapholita congener, G. molesta and G. dimorpha have difference in several biological characters such as flight time, emerging number/year, damage site, pupation site, and mating time although their host plants were similar. As a problem, cross-trapping was identified in each trap for monitoring. Effects on species-specific lure using minor sex pheromone components were observed in host plant orchards (apple, pear, peach, and plum) for continuative two years. Treatments of various ratios (0 to 10%) of Z8-12OH to G. molesta lure (Z8-12Ac/E8-12Ac = 95:5) allowed to increase the attraction of G. molesta, but not of G. dimorpha male. Other two minor components (14Ac and 12Ac) to G. dimorpha lure (Z8-12Ac/E8-12Ac = 85:15) were not showed species-specific responses. However, 10% treatment of Z8-14Ac to G. dimorpha lure was showed that G. molesta was decreased significantly although G. dimoprha was not affected. E8-14Ac treatment to new G. dimorpha lure (Z8-12Ac/E8-12Ac/Z8-14Ac = 85:15:10) not affected to attraction of two species. From these results, we suggest that optimum ratios for species-specific monitoring of G. molesta and G. dimorpha are Z8-12Ac/E8-12Ac/Z8-12OH = 95:55:5 and Z8-12Ac/E8-12Ac/Z8-14Ac = 85:15:10, respectively.

이산화염소는 병원미생물에 대한 소독제로 사용되고 있다. 최근 저곡해충에 대한 이산화염소의 산화적 스트레스에 의한 살충력이 확인되 었다. 그러나 이산화염소의 산화적 스트레스에 의한 대상 곤충의 체내 분자 종말점에 대해서는 알려지지 않았다. 본 연구는 화랑곡나방(Plodia interpunctella)의 아세틸콜린에스터레이즈가 이산화염소의 분자표적으로 가정하고 노출에 따른 이 효소의 활성을 분석하였다. 아세틸콜린에스 터레이즈 활성은 화랑곡나방 발육 시기에 따라 상이했다. 화랑곡나방 5령 유충에서 치사를 일으킬 수 있는 이산화염소 농도 처리는 아세틸콜린에 스터레이즈 활성을 뚜렷하게 증가시켰다. 또한 훈증제 형태의 이산화염소 처리에서도 아세틸콜린에스터레이즈 활성 증가가 유발되었다. 화랑곡 나방 5령 유충은 음성주광성을 보이는 데, 이산화염소 처리는 이 선천성 행동을 교란하였다. 이러한 결과는 아세틸콜린에스터레이즈가 이산화염 소 처리에 따른 산화적 스트레스의 분자표적 가운데 하나라는 것을 제시하고 있다.

기후변화는 국내 해충상의 변화에 영향을 주는 주요 요인 가운데 하나로 알려지고 있다. 특히 지구 온난화 모델에 따라 남방계 곤충의 서식 지 확대가 예상되고 있다. 휴면 기작을 가지고 있지 않은 파밤나방(Spodoptera exigua)과 배추좀나방(Plutella xylostella)은 시설재배지를 중심으 로 국내에서 월동이 가능한 것으로 알려지고 있다. 그러나 두 해충은 계절적 발생 양상에서 뚜렷한 차이를 보여주었다. 배추좀나방은 이른 봄철과 가을기간에 발생하고 여름 기간 중에는 발생하지 않았다. 반면에 파밤나방은 늦은 봄철에 나타나기 시작해 가을까지 지속적으로 발생하였다. 본 연구는 이러한 두 남방계 곤충이 계절적 발생 차이를 보이는 것이 이들이 갖는 고온에 대한 감수성 차이에 기인한 것으로 가정하였다. 이 가설을 증명하기 두 곤충의 내열성을 비교 분석하였다. 동일한 열처리(42℃)에서 배추좀나방 유충은 40 분 노출에 100% 사망률을 보인 반면, 파밤나방 은 대부분의 유충이 80 분의 노출에서도 생존하였다. 이러한 내열성은 두 곤충 모두 이들의 발육시기에 따라 상이했다. 배추좀나방은 4령 유충과 성충이 가장 높은 내열성을 보인 반면, 파밤나방은 1령 유충에서 가장 높게 나타냈다. 두 곤충 모두는 37℃에서 30 분간 전 처리 후 고온에 노출시 키면 생존율이 뚜렷하게 증가했다. 이러한 내열성유기는 두 곤충 모두 혈림프의 글리세롤 함량 증가와 관련성을 보였다. 또한 파밤나방의 경우는 열충격단백질의 발현도 증가하였다. 따라서 이상의 결과는 여름기간 배추좀나방의 발생이 없는 것은 이 곤충의 고온에 대한 높은 감수성에 기인 된 것으로 보이고, 반면에 파밤나방은 비교적 높은 내열성을 보유하여 여름 기간에도 발생을 지속시킨 것으로 해석되었다.

곤충 생리현상의 가소성은 후생유전적 변화와 밀접하게 관련을 지을 수 있다. 이 가설을 증명하기 위해 광식성인 파밤나방(Spodoptera exigua)을 대상으로 상이한 먹이 조건에 따라 이 곤충의 발육과 DNA 메틸화에 영향을 주는 지 분석하였다. 동일한 코호트로 부터 얻은 갓 부화한 유충을 최종령에 이르기까지 세 가지 다른 먹이(대파, 배추, 인공사료)로 섭식 처리하였다. 이 결과 상이한 먹이 조건에 따라 유충발육속도, 용화 율 및 우화율에서 뚜렷한 차이를 보였다. 인공사료로 사육된 유충이 가장 빠른 유충발육속도와 높은 용화율 및 우화율을 나타냈다. 반면에 두 자 연 기주 가운데는 대파가 배추에 비해 파밤나방 발육에 양호하였다. 이러한 먹이에 따른 변이는 혈림프 단백질 및 혈당에서도 차이가 나타났다. 또한 발육과 연계되었을 것으로 추정되는 인슐린유사펩타이드(SeILP1) 유전자의 발현 정도도 먹이조건에 따라 상이했다. 단일항체를 이용하여 파밤나방 게놈 DNA의 시토신 메틸화를 분석한 결과 이 부위에 DNA 메틸화가 검출되었으며, 메틸화 정도는 먹이 조건에 따라 상이했다. 이 결 과들은 동일 집단의 파밤나방이 상이한 먹이 조건에 따라 발육차이를 나타내고 또한 시토신 메틸화에 변이를 보여 이 곤충의 생리적 가소성에 후 생유전적 인자가 작용하고 있는 것을 제시한다.

배추좀나방(Plutella xylostella)은 시설재배지를 중심으로 국내 자연 상태에서 월동한다. 안동지역에서 이른 봄부터 배추좀나방 성페로몬트 랩을 이용하여 배추좀나방의 성충 발생 시기를 주기적으로 조사한 결과 연중 4 회의 성충 발생 피크를 보였다. 월동 집단을 대상으로 서로 다른 지 역 집단 간 생물적 특성을 조사한 결과 내한성, 약제감수성 및 발육속도에서 뚜렷한 집단 특성을 나타냈다. 분자마커로 집단변이를 분석한 결과 월동세대의 높은 집단변이는 계절이 진행됨에 따라 낮아지는 양상을 나타냈다. 이 결과는 배추좀나방의 국내 월동 집단 사이의 생물적 특성 차이 를 나타냈고, 이들의 높은 유전적 분화는 계절이 진행됨에 따라 감소하여 이들 집단 사이의 개체들의 이동에 따른 유전적 교환이 이뤄졌다는 것을 제시했다.

Heat treatment has been widely used to control some stored insect pests. The Indian meal moth, Plodia interpunctella, gives massive economic loss to most stored grains. Heat tolerance of P. interpunctella was assessed in most developmental stages in this study. Heat treatment at 44oC for 1 h was lethal to P. interpunctella. Compared to other developmental stages, late larval instar larvae were more tolerant to the heat treatment. Some of the fifth instar larvae could survive under 44oC for 80 min, but completely died at exposure to 45oC for 80 min. However, the survival rates of the larvae significantly increased by pre-exposure to sublethal temperature at 37oC. The rapid induction of heat tolerance was accompanied with increases in gene expressions of two heat shock proteins, Hsp70 and Hsp90, but not in that of Hsp25. Interestingly, hemolymph trehalose levels also increased from 39.2 mM to 51.3 mM by pre-exposure to 37oC for 30 min. These results suggest that P. interpunctella induces a rapid heat tolerance by up-regulation of heat shock protein levels and hemolymph trehalose levels.

Trehalose is a major blood sugar in insects. It functions as carbohydrate nutrient source to all tissues with a relatively constant level and also serves as a cryoprotectant at high levels in some insects. Thus, its hemolymph levels need to be controlled according to physiological conditions. An insulin-like peptide, SeILP1, was identified and showed to mediate down-regulation of the hemolymph trehalose levels of Spodoptera exigua larvae. This study showed that hemolymph trehalose levels of S. exigua significantly increased upon immune-challenge and under starvation in a time-dependent manner. During up-regulation of the hemolymph trehalose, the gene expression of trehalose phosphate synthase significantly increased, but that of trehalase significantly decreased. Injection of biogenic monoamines, such as octopamine and serotonin, did not increase the hemolymph trehalose levels. Either treatment of arachidonic acid or eicosanoid biosynthesis inhibitor did not change the hemolymph trehalose levels. However, injection of adipokinetic hormone (AKH) of Manduca sexta significantly increased the trehalose levels. Interrogation of Spodobase identified an AKH-like gene of S. exigua. Its expression increased with starvation in the larvae. Its RNA interference significantly prevented the up-regulation of the hemolymph trehalose levels in S. exigua.

이산화염소(ClO2) 훈증제는 살충효과를 나타낸다. 그러나 일부 곤충은 이산화염소에 대해 회피행동을 보여, 이 훈증제에 대한 방제효율을 크게 떨어뜨리고 있다. 본 연구는 이를 해결하기 위해 이산화염소 처리에 열처리를 추가하여 곤충의 이산화염소에 대한 회피행동을 줄여 살충효과를 극대화하는 전략을 세웠다. 이산화염소 훈증 처리는 거짓쌀도둑거저리(Tribolium castaneum)에 대해 살충효과를 주었으며, 시험 곤충이 노출된 조건에서 12 시간 처리할 때 유충에 대해서 383.67 ppm (153.63 - 955.78 ppm: 95% 신뢰구간), 성충에 대해서 397.75 ppm (354.46 - 446.13 ppm: 95% 신뢰구간)의 반수치사농도를 나타냈다. 그러나 먹이인 밀가루를 충분히 제공한 상태에서 이산화염소를 처리하면, 처리 약제에 반응하여 시험 곤충이 먹이 속으로 들어가는 회피행동을 보이면서 방제효과는 크게 낮아졌다. Y 튜브를 이용한 이 곤충의 먹이 선호성 행동을 분석한 결과 거짓쌀도둑거저리 성충은 이산화염소가 처리된 먹이를 회피하는 행동을 보였다. 그러나 촉각을 제거한 경우 이러한 회피행동은 둔화되었다. 거짓쌀도둑거저리에 6 시간 동안 46℃ 열처리를 하면 살충효과는 10% 이하로 낮지만, 처리된 성충들이 먹이 밖으로 나와 있는 것을 관찰하였다. 반면 400 ppm의 이산화염소를 단독으로 6 시간 처리한 결과 회피행동에 따라 전혀 살충효과를 보이지 않았다. 그러나 46℃ 열처리와 400 ppm의 이산화염소를 병행하여 6 시간 처리한 결과 살충효과는 95%로 크게 증가하였다. 따라서 열처리는 거짓쌀도둑거저리의 이산화염소에 대한 회피행동을 억제하여 살충효과를 증가시켰다.

비티 포자와 Xenorhabdus nematophila (Xn)의 배양액을 혼합하여 비티플러스가 개발되었다. 높은 살충력에도 불구하고 비티플러스는 다 양한 해충에 대한 넓은 살충범위를 보이지 않는 한계가 있다. 본 연구에서는 Xn 대사물질 첨가를 통한 파밤나방과 같은 비감수성 해충에 대한 비 티플러스의 살충력 향상에 초점을 맞추었다. Xn의 주요 대사물질인 oxindole (OI)과 benzylideneacetone (BZA)는 비티의 살충력을 향상시킨 다고 보고되었다. 본 연구에서 OI 또는 BZA의 첨가는 비티플러스의 살충력을 향상시켰다. 그러나 동결건조된 Xn 배양액의 첨가는 보다 낮은 농 도의 OI 또는 BZA로도 충분히 비피플러스의 살충력을 상승시켰다. HPLC 분석에서 Xn 배양액에 최소 12개의 대사물질이 포함되어 있는 것을 확인하였다. 이러한 결과는 OI와 BZA 외에도 Xn 대사물질에 생리활성물질이 존재하는 것을 제시한다.

Histone H4 is a protein subunit of nucleosomes in eukaryotes and play crucial roles in DNA package and in regulation of gene expression by covalent modification. A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV). The viral H4 (CpBV-H4) is highly homologous with other H4 proteins except 38 extended residues in N terminus. Its expression alters insect gene expression and suppresses immune and development. In this study, CpBV-H4 was expressed in a natural host, Plutella xylostella, and its suppressive activity on host gene expression was detected by suppressive subtractive hybridization (SSH) technique. SSH targets, of which expressions were down-regulated by CpBV-H4, were read by 454 pyrosequencing and annotated using the published P. xylostella whole genome. Resulting targets were assigned to most GO functional categories. Two chromatin remodeling factors were included in the SSH targets. Lysine demethylase (Px-KDM) of P. xylostella was highly expressed during entire larval period in all tested tissues. However, the suppression of Px-KDM expression by a specific RNA interference (RNAi) did not affect immune response, but significantly impaired the larval development. SWI/SNF of P. xylostella (Px-SWI/SNF) was expressed in all developmental stages. Its RNAi did not affect larval development, but led to significant alteration in adult metamorphosis. CpBV-H4 suppressed expressions of both Px-KDM and Px-SWI/SNF, but its truncated mutant lacking in the extended N-terminal tail did not. These results suggest that the developmental alteration in P. xylostella parasitized by C. plutellae can be caused by an epigenetic control of CpBV-H4 against chromatin remodeling factors.

Polydnavirus (PDV) is a group of double-stranded DNA insect viruses. PDV is mutualistic with some ichneumonid and braconid wasps to parasitize specific lepidopteran hosts. The viral genome is located on the wasp chromosome(s) as a proviral form and replicates only at the female reproductive organ during late pupal stage. The viral particles are accumulated in the oviduct lumen and delivered to the parasitized host along with wasp eggs during parasitization. The viral particles enter target tissues in the parasitized larvae and alter host physiological processes for the wasp development by suppressing immune responses and extending larval period. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. The viral particles of CpBV encode 157 open reading frames classified into different gene families. CpBV-PTP family is the largest and comprises of at least 40 gene members. CpBV-BEN, CpBV-ELP, and CpBV-IkB families also share common motifs in each gene group. In addition, two homologous genes of CpBV15α and CpBV15β are encoded in a viral genome segment. To apply these viral genes to enhance an alpha-baculovirus in insecticidal activity, they were recombined with AcNPV under a PDV promoter. As a control, under the same promoter, the recombinant baculovirus expressed an enhanced green fluorescence protein (EGFP). Upon injection or oral feeding tests, three different recombinant baculoviruses (AcNPV-ELP1, AcNPV-CpBV15α, AcNPPV-CpBV15β) enhanced the insecticidal activity compared to a control recombinant (AcNPV-EGFP). However, there was a variation in the up-regulation of the insecticidal activities among the recombinants. AcNPV-ELP1 showed the greatest potency in the insecticidal activity against the beet armyworm, Spodoptera exigua, larvae. AcNPV-ELP1 exhibited a significant variation in insecticidal activity among different larval stages of S. exigua. In the fifth instar, 1.435x107 PIB treatment of AcNPV-ELP1 showed a median lethal time at 112.7 h. ELP1 protein was detected in the hemolymph at 24 h after the viral treatment. Foliar spray of AcNPV-ELP1 was performed in pot assay and resulted in 88% control efficacy against S. exigua, while control efficacies of AcNPV-EGFP and bifenthrin (a pyrethroid insecticide) resulted in 65% and 96%, respectively. These results suggest that a PDV gene, ELP1, may be applied to develop a novel control agent by ameliorating commercial microbial insecticides or by generating transgenic crops.

An oxidative fumigant is potent to kill insect pests infesting stored grains. Its oxidative activity generates reactive oxygen species (ROS), which has been considered to be a main insecticidal factor. Furthermore, the oxidative fumigant has cytotoxic effect to insect cell lines, but the cytotoxicity is abrogated by antioxidant treatment. This study aimed to extend the usefulness of the oxidative fumigant in terms of medical purpose against cancer cells. Five cancer cell lines HCT 116 (human colorectal), Lovo (human colorectal), SW480 (human colorectal), MDA-MB-231 (human breast), and MCF-7 (human breast) were tested to determine their susceptibility to the oxidative fumigant with reference to two insect cell lines (Sf9 and Hi-Five). All cancer cell lines were highly susceptible to the oxidative fumigant, compared to the insect cell lines. Interestingly, basal ROS levels of the cancer cell lines were much higher than the insect cell lines. Furthermore, the oxidative fumigant significantly increased the ROS levels in the cancer cells. Treatment of vitamin E as an antioxidant mitigated the cytotoxicity of the oxidative fumigant. Thus, the high susceptibility of cancer cells to the oxidative fumigant may be induced by their high inducible ROS production. This study also investigated the antiviral activity of the oxidative fumigant against insect and plant viruses. The oxidative fumigant significantly inactivated a baculovirus (dsDNA virus) by inhibiting polyhedral production in Sf9 cells. It also inactivated tobacco mosaic virus (ssRNA virus) by suppressing phytopathogenicity. These results support a broad effect of the oxidative fumigant, which can be applied to agricultural and medical purposes.

Cabbage is one of the major vegetable crops especially in Korea. Various insect pests intimidate the cabbage production. Moreover, an excessive spray of chemical insecticides leads to development of insecticide resistance as well adverse effects on environment and humans. The brassica leaf beetle, Phaedon brassicae, is a coleopteran pest. Both larval and adult stages feed cabbage. This study aimed to develop an effective microbial biopesticide against P. brassicae by adding immunosuppressive agent to Bacillus thurigiensis (Bt). To determine an optimal immunosuppressive agent, two bacterial species of Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila were compared in their insecticidal activity against P. brassicae, and Ptt was selected. Among four subspecies of Bt, B. thuringiensis subsp. tenebrionis (Btt) was selected. Ptt bacterial culture broth possessed immunosuppressive agent(s) because the bacterial culture broth significantly increased other bacterial growth in P. brassicae hemolymph. When the Ptt broth was added to Btt, it significantly increased the insecticidal activity of Btt. Bt-Plus was prepared by mixing Btt and Ptt bacterial culture and sprayed the cabbage infested by P. brassicae. The Bt-Plus was much more effective than Btt treatment. This study reports a novel Bt insecticide to control P. brassicae.