Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. To better understand the roles of Toll pathway in mealworm immune system, TmGPR and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGPR resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGPR. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

Recent research has suggested that the dietary protein:carbohydrate (P:C) balance is a critical determinant of fitness in insects. In this study, we examined the effects of dietary P:C balance on life-time reproductive success in the mealworm beetle, Tenebrio molitor. Both males and females lived the longest when fed on P:C 1:1 diet. Throughout their adult lives, females fed on P:C 1:1 diet laid significantly more eggs than those on nutritionally imbalanced diets (P:C 1:5 or 5:1). When given a choice, beetles regulated their intake of protein and carbohydrate to a ratio close to 1:1. Taken together, our results indicate the balanced intake of protein and carbohydrate maximizes life-time reproductive success in this species.

Distillers dried grain (DDG) and makgeolli spent grain (MSG) are agricultural by-product to produce alcoholic beverage. However, they are known to contain enough nutrients. Mealworm is a promising insect resource for an animal feed ingredient as well as alternative human food. With low cost, DDG and MSG were investigated as a feed ingredient for rearing high quality mealworms. DDG and MSG were mixed with wheat bran and compared to control feed (only wheat bran) for its effects on larval survivorship, larval weight, duration for larval development, pupation rate, and pupal weight. Adding DDG on wheat bran showed positive results for larval weight, duration for larval developmental period, and pupation rate. However, adding MSG made longer duration for larval development, but it also improved larval weight, pupal weight with more than 90% pupation rate. We confirmed that adding 30～50% of DDG or MSG to conventional wheat bran have a strong potential to replace the conventional wheat bran insect feed for quality insect production.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll signaling pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. TmGPR, antimicrobial peptide Tenecin 1 and Tenecin 2 were up-regulated after fungal infection. To better understand the roles of Toll signaling pathway in mealworm immune system, TmGRP and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 2 days post-dsRNA injection, and dramatically reduced at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGRP resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGRP. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

Entomopathogenic fungi are facultative microorganisms, dwelling in soil or infecting host insects, and some of the genera have been used as biological control agents worldwide. Collection of fungal isolates should be a platform for the development of highly effective resources, thus in this work we constructed a fungal library using a mealworm (Tenebrio molitor) pathogenicity-based fungal collection method and further characterized some isolates with high virulence. A phylogenetic three was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, spectrum of virulence, cultural characteristics, thermo-stability of fungi, production of biologically active materials, such as enzymes. This work reports an attractive entomopathogenic fungal library including the information of effective isolates in pest management.

This study was performed to develop and evaluate cookies prepared with various amounts (0 g, 12.5 g, 25 g and 37.5 g) of mealworm powder to serve as not only sports snacks but also for the general population as a new protein source. In the chromaticity of mealworm cookies, lightness and yellowness decreased whereas redness increased with more mealworm powder. The hardness of the mealworm cookies increased as more mealworm powder was added. In sensory evaluation, higher preference was shown with the measure of color, taste, and overall preference for mealworm cookies prepared with 50% mealworm powder (MP2). For the general composition of MP2, the moisture, carbohydrate, crude protein, crude fat and crude ash were higher compared with the control cookie. For the mineral contents of MP2, the contents of Ca (33.44 mg/100 g), P (225.13 mg/100 g), Mg (82.21 mg/100 g), Na (246.22 mg/100 g), and K (503.49 mg/100 g) were higher compared with the control cookie. The essential amino acids (valine, isoleucine, leucine, methionine, threonine, phenylalanine, and histidine) in MP2 were also higher compared with the control cookie. It was concluded that MP2 can be used as a new protein source for not only the maintenance of muscle but also for the prevention of muscle loss in old people.

Insect is an important player in the ecosystem as a prey for animals. Moreover, they are a valuable candidate food source for rearing animal. Tenebrio molitor (Coeloptera: Tenebrionidae) larvae are known as a good food source with high protein, unsaturated fatty acid, minerals. Therefore, it has strong potential to substitute the conventional meat consumption. To utilize T. molitor as a feed, the standard mass-rearing protocol is required. To make standard mass-rearing protocol, we tested different temperature(17.5, 20, 22.5, 25, 27.5 and 30°C) conditions for egg, larvae, pupae and adult T. molitor to identify the optimal rearing condition. Hatching was occurred within 15~32.5°C range. However, 17.5~27.5°C was required to get more than 70 % hatching rete. When the eggs were treated in 22.5~27.5°C, all eggs were hatched within 10 days. As larval development, shorter developmental period, higher pupation and eclosion rates were observed within 25~27.5°C temperature range. In addition, we compared the number of egg, oviposition duration and time required to start egg-laying. The minimum egg-laying(258.40±10.86) was observed at 17.5°C, but the maximum(749.10±7.45) was at 27.5°C. The maximum oviposition duration (137.00±12.73 day (mean±S.D.)) was achieved at 27.5°C, but the minimum (87.50±3.54 day (mean±S.D.)) was at 30°C. The time required to start egg-laying was less than 10 days at 17.5, 27.5, and 30°C. To consider all the factors, we concluded that the optimal temperature is 27.5 °C.

Entomopathogenic fungi are facultative microorganisms, dwelling in soil or infecting host insects, and some of the genera have been used as biological control agents worldwide. Collection of fungal isolates should be a platform for the development of highly effective resources, thus in this work we constructed a fungal library using a mealworm pathogenecity-based fungal collection method and further characterized some isolates with high virulence. A phylogenetic three was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, spectrum of virulence, cultural characteristics, thermo-stability of fungi, production of biologically active materials, such as enzymes. This work reports an attractive entomopathogenic fungal library including the information of effective isolates in pest management.

Protein and carbohydrate are the two most important macronutrients that have profound impacts on fitness and demography in most insects. The purpose of this study is to investigate the effects of dietary protein:carbohydrate (P:C) balance and mating status on feeding behavior, longevity and fecundity in male and female mealworm beetles, Tenebrio molitor (Coleoptera: Tenebrionidae). In the first experiment, we measured the amount of protein and carbohydrate consumed by mated and unmated beetles of both sexes. Newly emerged beetles were allowed to mate for 24 h before they were simultaneously provided with two nutritional imbalanced but complementary synthetic diets (P:C =1:5 vs. 5:1) for 24 successive days. Mated females not only consumed significantly more nutrients but also exhibited a greater preference for protein than did mated males and unmated controls. In the second experiment, we determined longevity and fecundity from a total of 120 male and female beetles that were confined to feed on one of three no-choice foods differing in P:C balance (P:C=1:5,1:1 or5:1) throughout their entire lives. Fecundity was recorded as the total number of eggs laid by individual females until death. Both male and female beetles lived significantly longer and laid more eggs over the lifetime on a balanced diet (1:1) than on the two imbalance diets (1:5 and 5:1), suggesting that the Darwinian fitness was maximized when the diet was equally balanced in protein and carbohydrate. Mated male and female beetles had a shorter longevity compared to their unmated counterparts, indicating that there was a significant survival cost to mating in this insect.

본 연구는 갈색거저리(Tenebrio molitor)의 사료로 주로 이용되는 밀기울을 대체할 사료원료 개발을 목표로 수행되었다. 대체원료로서 열풍 건조시킨 귤껍질, 배추, 새송이버섯 및 팽이버섯 폐배지를 사용하였으며, 투여 후 유충의 생존율, 유충 무게, 유충의 발육기간, 용화율, 용무게를 측정하였다. 귤껍질과 배추를 첨가한 배지는 대체 먹이로 적합하지 않았다. 새송이버섯 폐배지의 모든 실험군에서 유충의 생존율은 대조군과 비 슷하였으나 유충의 무게는 대조군에 비해 다소 낮게 나타났고, 유충 발육기간은 폐배지의 함량이 높아질수록 길어짐이 관찰되었다. 팽이버섯 폐 배지 20~50% 첨가 조건에서 대조군과 비슷한 유충 및 용무게가 측정되었으며, 계대사육시 밀기울 사료에 40~50% 팽이버섯 폐배지를 첨가하 는 것이 용화율을 고려했을 때 가장 효과적일 것으로 분석되었다. 결론적으로, 팽이버섯 폐배지를 40%~50% 첨가한 조건에서 기존의 밀기울 사 육법과 비슷한 높은 효율성을 나타내기 때문에 팽이버섯 폐배지는 갈색거저리 유충의 대체사료로서 적합하다고 사료된다.

갈색거저리의 온도에 따른 유충 발육시험을 15, 17, 20, 22, 25, 28 및 30℃의 7개 항온조건, 광주기 14L:10D, 상대습도 60~70% 조건에서 수행하였다. 유충은 13령까지 경과하였고 항온 조건에서 사망률은 17, 20℃에서 극소수 개체만이 발견되었고, 22℃ 이상의 항온조건에서는 발견되지 않았다. 유충의 발육기간은 17℃에서 244.3일로 가장 길었고, 30℃에서 110.8일로 가장 짧았다. 15℃는 부화되지 않아 유충 발육 조사가 불가능하였다. 온도와 발육율과의 관계를 알아보기 위하여 선형모형과 비선형모형(Logan 6)을 이용하였으며, 선형모형을 이용하여 추정한 전체유충의 발육영점온도는 6.0℃, 발육 유효적산온도는 2564.1DD 였으며 선형, 비선형 모두 결정계수값(r2) 이 0.95로 높은 값을 보였다. 전체유충의 발육완료분포는 2-parameter Weibull 함수를 사용하였으며 전체 유충의 결정계수 값은 0.8502~0.9390의 양호한 모형 적합성을 보였다.

Peptidoglycan recognition proteins (PGRPs) are family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (IMD) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts followed by a challenge with L. monocytogenes showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infections in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

Tenebrio 모델은 Tribolium과 비교하여 실험실에서 배양 및 조작하기가 쉬운 생물 종이다. 하지만 현재까지는 해독되어진 유전체 정보가 그리 많지 않다. Mycoplasma genus는 세포벽이 없으며 사람에서 pneumonia 나 다른 호흡기 질환을 일으키는 박 테리아로 본 연구에서는 갈색거저리 유충을 이용하여 Mycoplasma genus와 유사 한 acholeplasma lysate를 처리하여 접종 전과 후의 비교 전사체학적 방법을 통하여 선천성 면역 관련 유전자들을 동정하고 기능 분석을 위한 기초 데이터를 확보 하고 자 하였다. 갈색거저리 유충에 acholeplasma lysate를 처리하기 전과 후의 각 샘플 들로부터 cDNA library를 구축한 후 random sequencing 을 통해 해독 되어진 염기 서열들을 비교 분석 하였다. 얻어진 서열들로부터 NCBI nr 데이터베이스에 Blastx 분석을 하여 획득한 서열들을 생물정보학적 방법을 이용하여 비교한 결과, 곤충의 선천성면역(innate Immunity)에 중요한 것으로 알려진 serpin 관련 유전자들이 약 5종류 동정되었다. Serpin (XP_974209.1) 의 경우 acholeplasma에 처리하여 전과 후 비교하였을 때, 약 3배 정도 증가를 나타내었고, serpin 6 (XP_972660.1)의 경우 에는 acholeplasma를 처리한 샘플에서만 동정되었다. 또한 serpin 3a (XP_969874.1), serpin 1(BAI59109.1), serpin 40(BAI59106.1) 등의 유전자들도 동정되었다. 추후 이러한 유전자들을 대상으로 한 기능연구를 통하여 보다 구체적인 면역기능을 구 명하고자 한다.

Serine protease는 병원체의 표면 melanization, hemolymph coagulation, antimicrobial peptide synthesis 등을 통해 여러 무척추동물의 방어기작을 조절하는것으로 알려 져 있다. 곤충의 경우 Tribolium을 대상으로 이와 같은 연구가 이루어져 왔지만, 혈 액의 량이 그리 많지 않아 연구자들은 최근 갈색거저리(Tenebrio)를 이용하기 시작 하였다. 하지만 아직 유전체(자) 서열정보가 충분하지 않은 상황이다. 본 연구에서 는 이러한 갈색거저리 유충을 이용하여 세포벽이 없으며 사람에서 pneumonia나 다른 호흡기 질환을 일으키는 mycoplasma 와 유사한 acholeplasma lysate를 처리 한 후 접종 전과 후의 전사체의 비교를 통하여 무척추 동물에서의 선천성 면역 관련 유전자들을 동정하고자 하였다. Acholeplasma lysate를 처리하기 전과 후의 각 샘 플들로부터 cDNA library를 구축한 후 random sequencing 을 통해 염기서열을 분 석하였고, 얻어진 서열들로부터 NCBI nr 데이터베이스에 Blastx 분석을 하여 획득 한 서열들을 comparative transcriptomic 방법을 이용하여 분석한 결과, 여러 종류 의 Serine protease 관련 유전자들이 동정되었다. Serine protease (XP_970766.1)의 경우에는 acholeplasma를 처리한 샘플에서 2배 정도 발현이 증가하였고, serine protease P66 (EFA09207.1)의 경우 증감의 변화는 보이지 않았다. 또한 serine protease P146 (EFA04636.1), serine protease H1 (EEZ99180.1) 등의 유전자들도 동정되어 연구하고 있다.

CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, T.molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type β-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, L. monocytogenes suggesting putative function in innate immunity.