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.

A Tenebrio molitor larva that is called mealworm is widely used as feed for raising a pet. Also, it is the insect that would be produced as the edible food for human. For these reasons, many researchers are revealing about physiologic characteristic of Tenebrio molitor to make good use of it. However, morphological characteristic of different larval stage is not specifically indicated and researchers have different opinions about the average number of Tenebrio molitor’s instar until now. This study, therefore, was carried out to examine the physiological and morphological characteristics of Tenebrio molitor larva in different larval stages, and the average number of Tenebrio molitor’s instar. As a result, all of the Tenebrio molitor larvae had 7~8 days during their incubation period and 3~4 days during the 1st instar period. After the 1st instar, there were comparatively large differences in the number of days in each instar. Before emergence, most of the larvae comparatively showed 15th~17th instars, particularly, the largest rate of pupae (28.83%) was observed in the 17th instar. In terms of the body length of each Tenebrio molitor larval stage, the body length was gradually increased by the 17th instar. Beyond the 17th instar, however, the body length was smaller than 17th instar. In the 1st instar, the larva had white color, and the color gradually changed into brown from the 2nd instar. Through this study, we could identify the accurate incubation period, 1st instar period, the average number of instar, body length and the color change in different larval stages. These results would be used as a fundamental data for further study about the physiological and morphological characteristic of each instar period.

As demand for Tenebrio molitor increases in animal feed market, improvement of mass-rearing indoor system became more crucial. This study is aimed at establishing the technology for conserving genetic resource of Tenebrio molitor, and increasing the technology utilization. This year, in advance, this experiment was designed for examining the developmental characteristics of eggs and larvae. To research the developmental characteristics under different temperatures, eggs, larvae and papae were treated at 17.5, 20, 22.5, 25, 27.5 and 30°C. At 25°C, larvae were exposed to various photoperiod conditions, LD 14:10, 12:12, and 10:14. The results show that the temperature range which affected hatching was 15~32.5°C. To achieve 70 percent or higher hatching rate 17.5~27.5°C was required. Furthermore, hatching occurred within 10 days, when the eggs were treated in 22.5~27.5°C. In regard of larval development, shorter developmental period, and higher pupation and eclosion rates were observed in 25~27.5°C. The shortest larval period was recorded at 14L:10D. At 10L:14D, in contrast, pupae showed the lowest chance of eclosion. Overall, larval development was optimal in long-day condition.

As a part of a study on insect food, the nutritional and harmful components in the mealworm (Tenebrio molitor) were analyzed. In addition, due to a recent introduction of live Chinese mealworm in the Korea market, those components between the Korean and Chinese mealworms were compared. Analysis of the composition of the general components (moisture, crude protein, crude fat, crude ash, crude fiber, and carbohydrates) showed that abundant crude protein (50.32– 52.79%) was present in both Korean and Chinese mealworm powders, and the protein content in the Chinese mealworm powder was higher than that in the Korean mealworm powder by 2.67%. The amino acid compositions were similar, but the fatty acid composition differed with respect to each component in the Korean and Chinese mealworm powders. The unsaturated fatty acid contents were 76.80-80.55% of the total fatty acid content in the mealworms. The linoleic acid contents in the Korean and Chinese mealworms were 20.8±1.1% and 34.69±1.9%; the linolenic acid contents, 0.47% and 1.31%; and the oleic acid contents, 51.40±0.9% and 40.20±1.5%, respectively. With respect to harmful components such as heavy metals and bacteria that cause food poisoning, bacteria such as Escherichia coli O157:H7 and Salmonella spp. were not detected in both Korean and Chinese mealworms, and the mercury content was below the standard value for common food (Korea, 0.03 mg/kg; China, 0.08 mg/kg).

Many insects adjust feeding behavior to meet their optimal requirement for multiple nutrients. In the present study, we investigated the behavioral regulatory responses of male and female adults of an omnivorous beetle, Tenebrio molitor (Coleoptera: Tenebrionidae), using choice and no-choice feeding experimental designs. In the choice experiment, newly eclosed adults of both sexes received one of four food-pairing treatments and so were allowed to self-compose their preferred nutrient intake by selecting between two nutritionally-complementary diets (protein-rich vs. carbohydrate-rich diet; 35:7 vs. 7:35, 35:7 vs. 5.6:28, 28:5.6 vs. 7:35, or 28:5.6 vs. 5.6:28, P%:C% by dry mass). Our data showed that both sexes independently regulated their intake of protein and carbohydrate to a 1:1 ratio, indicating that they are capable of adjusting their feeding behavior to optimize their nutrient preference. In the no-choice experiment, adults were forced to eat one of seven single diets that varied in P:C ratio (0:42, 7:35, 14:28, 21:21, 28:14, 35:7, or 42:0). Results showed that both sexes ingested similar amounts of nutrients when the dietary P:C ratio was moderately balanced (14:28, 21:21 and 28:14), but males ingested significantly more than females on those diets that were extremely imbalanced with respect to their P:C ratio (0:42, 7:35, 35:7 and 42:0). This indicates that there exists a sex-specific difference in nutrient intake when the nutritional balance of diets deviates away from the optimal level under restricted feeding condition.

갈색거저리는 소맥피와 가축사료에서 2일간 평균 44.3~55.3개를 산란하였고 발육기간은 80.0~83.7일, 번데기크기는 18.1~18.7mm로 유사하였다. 갈색거저리 번데기의 평균 무게에 따라 0.12g, 0.15g, 0.18g의 세 중량으로 구분하여 우화 후 성충의 일생동안 평균 산란수를 조사한 결과 각각 172.3, 191.2, 215.0개로 중량에 비례하여 증가하였다. 산란경향은 우화 2주차까지 51.8%를 산란하여 가장 왕성하였으며 그 후 급격히 감소하였다. 번데기 무게에 따른 성충의 수명은 각각 27.1±6.9, 30.1±7.8, 27.4±8.5일로 차이 없는 것으로 조사되었다. 갈색거저리 유충사육용기(16×21.5×7cm)에 유충 사육밀도를 100, 300, 500, 700마리로 사육한 결과 용화 시작 후 3주 이내에 62.7%가 용화되었으며 단위 공간당 사육 밀도가 증가함에 따라 번데기의 중량은 용화 시작 후 3주까지 각각 146.9±6.5, 142.1±8.3, 122.0±9.6, 118.5±5.2mg으로 크게 감소하였으나 3주 이후에는 사육밀도에 따라 각각 134.9±48.9, 148.8±18.1, 149.0±8.6, 140.4±8.2mg으로 차이 없이 조사되었다.

The developmental time of larvae of mealworm beetle, Tenebrio molitor was studied at six temperatures ranging from 17 to 30℃ with 60~70% RH, and a photoperiod of 14L:10D. Mortality of 1st~13th larva was very low at 17 and 20℃ but did not die over 22℃. Developmental time of larva decreased with increasing temperature. The total developmental time was longest at 17℃ (244.3 days) and shortest at 30℃ (110.8 days), suggesting that the higher temperature, the faster development period. The lower developmental threshold temperature and effective accumulative temperatures for the total larval stages were 6.0℃ and 2564.1 day-degrees. The relationship between developmental rate and temperature fitted a linear model and nonlinear model by Logan-6 (r2=0.95). The distribution of completion of each development stage was well described by the 3-parameter Weibull function (r2=0.89).

일반적으로 곤충의 영양성분은 단백질과 지방, 광물질 및 비타민을 풍부히 함유하고 있고, 아미노산 조성은 대부분의 케이스에서 곡물이나 콩류에 비해 우수하고 단쇄불포화지방산이 풍부하다. 이에 곤충을 이용한 사료는 매우 안전하고 저단가의 고품질 단백질 사료의 생산을 가능하게 할 수 있기에 사료용 곤충 갈색거저리 특성을 파악하고자 하였다. 갈색거저리는 성충이 된 후 빠르게는 5일 이후부터 산란하기 시작하여 성충 전기간동안 산란하는 특성이 있으나, 주로 성충 초기･중기 기간에 집중적으로 산란하는 것을 확인하였다. 수명 및 산란성을 조사한 결과, 수명(암컷/수컷) 152.4/150.7일, 산란전기간 7.8일, 산란기간 평균 114.1일, 산란수 364개이었다. 또한 성충은 먹이에 알을 붙여서 산란하는 특성이 있으며, 사육상자 내 곡물류, 채소류 등 함께 사용 시 밀기울이 가장 산란선호성이 높았으나, 각 재료를 단독으로 사용 시 밀기울, 옥수수분말이 선호도가 높았다. 알의 색깔은 유백색, 크기는 장경 1.93㎜, 단경 1.00㎜이었다. 알 보호온도별 부화율은 17.5~27.5℃일 경우 80%이상이었으나, 15℃이하에서는 거의 부화되지 않았다.

Large amounts of genetically modified (GM) grains, including maize, cotton and soybean, have been imported to Korea for food, feed and processing (FFP). To evaluatethe environmental impacts, particularly on non-target insects, of FFP GM grains of unknown source, it is a prerequisite to identify Cry protein types in the test GM grains and to establish proper risk assessment protocols. Imported GM maize grains were randomly obtained and their Cry toxins were analyzed by ELISA using Cry1A, Cry1F, and Cry3A antibodies. Since all tested GM maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as a non-target insect species. A domestic maize strain was used as a non-GM control, which did not show any differences in major nutritional composition from the GM maize grain. Slightly increased survival rate and head capsule width of T. molitor larvae were observed when reared on GM maize powder, demonstrating no sub-chronic adverse effects of GM maize on T. molitor larvae. Head capsule width of T. molitor neonate increased steadily from hatch to 70-day-old, regardless of being fed Bt or non-Bt maize. ELISA test using Cry1A-antibody revealed that concentration of Cry1A protein slowly increased in the whole body of T. molitor from 0 to 50 post-feeding days when the insects were fed GM maize but rapidly decreased within 5 days when Bt maize-fed larvae were transferred to non-Bt maize, showing that the Cry toxin is not accumulated inside the body of T. molitor once the exposure source is removed. In addition, no Cry protein was detected in the hemolymph of the larvae reared on Bt maize, suggesting little possibility of Cry toxin exposure to higher tropic level. Taken together, the imported GM-maize grains is not likely to cause any side impacts on non-target insect T. molitor.