To accommodate growth, insects must periodically replace their chitin/protein-rich cuticles in a process called “molting or ecdysis”. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. Here, we demonstrated a novel role for an endochitinase, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (Group III) that contain two catalytic domains, in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. The conservation of CHT7-like proteins among many insect and other arthropod species indicates a critical role for the Group III class of chitinases in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate exoskeletons.

Wood pellets are used by Biomass-solid refuse fuel (Bio-SRF) has been imported from many East-Asia countries especially,Vietnam in Korea. Wood pellets are made through grind and high-pressure process shows different physical characterizationlike high methyl bromide (MB) absorption unlike other wood materials. Furthermore, detected pests in wood pellets havesimilarity with detected pests in stored grain. In this study, we analyzed susceptibility on pupa and adult stages of redflour beetle, Tribolium castaneum in wood pellets against mixture of MB and phosphine (PH3). Mixture treatment ofMB and PH3 was more effective than single MB treatment compare with control. Pupal stage showed more tolerant thanadult stage on each fumigant and 100% mortality was determined at 27 mg/L of MB only at 20°C. However, PH3 increasedMB toxicity on pupae that 100% mortality was observed at 100:1 ratio of MB (5 mg/L) and PH3 (0.05 mg/L) at 20°C.Based on this study, mixture treatment of different type of fumigant will be applied to control of quarantine pests.

The red flour beetle, Tribolium castaneum, is one of economically important pests and can transmit several fungalspecies with their movement in stored grains. We collected T. castaneum samples from 34 rice processing complexes(RPC) nationwide during 2016-2017 and identified the contamination of fungal species on the surface of T. castaneum.Using sequence analysis of ITS region, 24 fungal genera were found. Among them, the genus Aspergillus spp. (28.8%)was the most frequently presented, followed by Cladosporium spp. (12.3%), Hyphopichia spp. (8.9%), Penicillium spp.(8.2%), and so on. In addition, as a known mycotoxin-producing fungal species, Aspergillus flavus (16 isolates) and Fusariumspp. (3 isolates) were also identified. Our study indicates that T. castaneum would play an important role in the contaminationof fungi and contribute potential production of mycotoxin in storage rice.

Tyrosine-mediated cuticle tanning (pigmentation and sclerotization) is a vital process for insect growth and development. In this metabolism, dopamine and N-β-alanyldopamine (NBAD) are major precursors to black melanin-like and yellowish quinonoid pigments, respectively. NBAD synthase (Ebony) catalyzes the synthesis NBAD by conjugation of dopamine and β-alanine, while NBAD hydrolase (Tan) dose a reverse reaction of NBAD synthesis catalyzed by Ebony. In this work, we cloned cDNAs of ebony and tan from two beetle species, Tribolium castaneum and Tenebrio molitor. Loss of function phenotypes produced by RNAi for these genes indicate that Ebony, but not Tan, is required for cuticle pigmentation in both beetles. This work was supported by NRFs (NRF-2015R1A2A2A01006614).

Insect chitinases (CHTs), an extracellular enzyme, belong to family 18 glycosyl hydrolases that hydrolyze chitin by an endo-type manner. In insect genomes, there are a large number of genes encoding CHT-like proteins, and they have been classified into eleven groups based on phylogenetic analysis. In this study, we have investigated functions of a group III chitinase (TcCHT7) in Tribolium castaneum. Although, unlike most insect CHTs, TcCHT7 contains a predicted transmembrane segment in N-terminal, immunohistochemical analysis reveals that it is localized in the newly forming procuticle, suggesting that TcCHT7 is released from the plasma membrane of underlying epidermal cells. RNAi for TcCHT7 does not affect on any types of molting. However the resulting pupae and adults fail to undergo wing-expansion and abdominal contraction. In addition, TcCHT7-deficient insects exhibit ultrastructural defects in both rigid (e.g. elytron) and soft (e.g. hindwing) cuticles. These results demonstrate that functional importance of TcCHT7 in the formation of the rigid and soft cuticles of the beetle.

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce chitosan. Insect CDAs have been divided into five groups based on phylogenetic analysis. We previously reported the functional importance of group I CDAs, TcCDA1 and TcCDA2, from Tribolium castaneum in molting, morphology of cuticle as well as in movement of legs. However, ultrastructure in the cuticle after RNAi for these genes have not been investigated. In this study, we further analyzed precise localization of these proteins and ultrastructural changes/defects of the cuticles in TcCDA1- and TcCDA2-deficient insects. Loss of function of TcCDA1 and TcCDA2 causes disorganized horizontal laminae and vertical pore canals in both rigid (e.g. elytron and ventral body wall) and soft (e.g. hindwing and dorsal body wall) adult cuticles. These results indicate that TcCDA1 and TcCDA2 are critical for development and formation of the beetle cuticles

Insecticidal toxicities of the isolated constituent of Eucalyptus dives oil and its analogues were bioassayed. 3-Carvomenthenone was isolated by chromatographic techniques and determined by EI-MS, 13C-NMR,1H-NMR, 1H-1H COSY, and HMQC. In the fumigant bioassay against P. interpunctella, cyclohexenone exhibited the strongest insecticidal toxicity (LD50 against larvae and adults, 2.45 and 3.63 μg/cm3), followed by methylcyclohexenone, seudenone, and 3-carvomenthenone. In the structure-activity relationships between 3-carvomenthenone analogues and insecticidal toxicity, the mode of the insecticidal action of 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone was through the dermal organs of T. castaneum and P. interpunctella. This study indicates that 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone have potential capacity for the development as safety natural agents to control the stored grain insects.

Phototactic behavioral responses of Tribolium castaneum adults to light-emitting diodes (LEDs) of seven different wavelengths were determined under various conditions (light exposure times, light sources, and luminance intensities) and compared with those of a black light bulb (BLB) under laboratory conditions. Based on the attractive rate (%) of T. castaneum adults under optimal conditions (50 lx and an 48 h exposure time) in the dark, red LED (625±10 nm) exhibited the highest potential attractive rate (97.8%), followed by yellow (590±5 nm, 68.9%), green (520±5 nm, 55.6%), infrared (IR) (730 nm, 54.4%), white (450-620 nm, 41.1%), blue (470±10 nm, 34.4%), and ultraviolet (UV) (365 nm, 0.06%) LEDs. In comparison, red LED (97.8%) was approximately 3.4 times more attractive to T. castaneum adults than the BLB (28.9%). These results indicate that a red LED trap could be useful to control T. castaneum adults.

저장곡물의 곰팡이 발생과 저장곡물해충은 깊은 연관이 있는 것으로 알려져 있다(Miller, 1995). 저장곡물해충 중 거짓쌀도둑거저리(Tribolium castaneum Herbst)는 세계적으로 분포하며, 곡물가루를 가해하여 피해를 주는 주요해충 (Brich, 1945; Daniels, 1956)으로 곰팡이 포자를 옮긴다는 보고(Bosly, 2015)도 있어 농산물 안전에도 큰 위협이 되고 있다. 본 연구는 전국 21개 미곡종합처리장(RPC)에서 확보된 거짓쌀도둑거저리를 대상으로 습지배양을 실시하여 충체 외부에 붙어있는 곰팡이를 분리하였으며, 확보된 61균주에 대해 ITS 분석을 통하여 동정을 실시한 결과 Alternaria, Aspergillus, Penicillium, Cladosporium 등에 속하는 11종 및 속 수준 7속의 균주를 확인하였고, 이중 주요 독소생성 곰팡이로 알려진 Fusarium (zearalenone, fumonisin), Aspergillus flavus (aflatoxin), Penicillium (ochratoxin) 중 Aspergillus 및 Penicillium속에 속하는 균들이 동정되어 저장곡물에서 독소생성 곰팡이 발생이 우려되고 있다.

Insect chitinases (CHTs) belong to family 18 glycosylhydrolases and hydrolyze chitin by an endo-type manner. One of the functions of CHTs is in the turnover of chitin-containing extracellular matrices such as the cuticle and peritrophic matrix of the midgut. There are a large number of genes encoding CHT-like proteins in insects, and they have been classified into eleven groups based on phylogenetic analysis. We have investigated functions of a group III chitinase in Tribolium castaneum (TcCHT7) containing a predicted transmembrane segment in N-terminal region. Recombinant TcCHT7 exhibits chitinolytic activity against CM-Chitin-RBV. Immunohistochemical analysis shows that TcCHT7 is localized in newly formed procuticle in elytral cuticles, suggesting that TcCHT7 is released from the plasma membrane of underlying epidermal cells. TcCHT7-deficient pupae and adults fail to undergo wing-expansion and abdominal contraction. In addition, cuticular chitin accumulates in the inner region of the procuticle where disorganized horizontal laminae and pore canals are evident. These results demonstrate that TcCHT7 plays a critical role in the formation of the rigid and soft cuticles of the beetle. This work was supported by NRFs (NRF-2015 R1A2A2A01006614).

Chitin deacetylases (CDAs) are chitin-modifying enzymes that deacetylate chitin to form 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 that may lead to diverse mechanical properties of the cuticle. DmCDA1 (serpentine) and DmCDA2 (vermiform) from Drosophila melanogaster play roles in development and morphology of embryonic tracheal tubes. We previously reported the functional importance of TcCDA1 and TcCDA2 orthologs to DmCDA1 and DmCDA2 from Tribolium castaneum, in molting, morphology of cuticle, and movement of legs. In this study, we further analyze ultrastructural defects of the cuticles and leg joints in TcCDA1- and TcCDA2-deficient insects. Loss of function of TcCDA1 and TcCDA2 causes disorganized chitinous horizontal laminae and vertical pore canals of rigid adult cuticle (e.g. elytron). Both proteins are also required for laminal organization in soft cuticle (e.g. hindwing). Morphological analysis of TcCDA1- and TcCDA2A-deficient adult revealed that ruptured tendons between femur and tibia cause the defects in movement of the leg joint. This work was supported by NRF (NRF-2015R1A2A2A01006614).

Temperature as a major environmental factor affects on organisms on various levels including molecular, physiological, behavioral and ecological levels. Transient receptor potential channels (TRPs) are a cation channel family. Among them, thermo-TRPs are known as a thermosensor. The potential role of thermo-TRPs have been identified in the fruit fly, Drosophila melanogaster, in thermotaxis and in thermal acclimation. With RNA interference (RNAi) technique, the role of thermo-TRPs in the red flour beetle, Tribolium castaneum, was identified by measuring thermal avoidance behavior in a behavioral assay. RNAi of trpA1 reduced high temperature avoidance, 39 and 42 °C. Moreover, the effects of RNAi of thermo-TRPs on the heat-induced knockout and the death after short exposure to high temperature was measured after one minute exposure at 52 °C, either with or without a 42 °C 10-minute thermal acclimation period. Even though it was relatively short time exposure to high temperature, it was enough to induce high temperature thermal acclimation. RNAi of trpA1 made faster knockout at 52 °C. With RNAi of painless, the recovery rates from heat-induced knockout after thermal acclimation. RNAi of pyrexia reduced long-term total survivorship without thermal acclimation.

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.

We divided the sample into four groups by temperature regimes and comparing the Lethal effect after exposure to high room temperatures for 50~58 days. After inoculating Cnidium officinale Makino with Tribolium castaneum Herbst, the storage insects of medicinal herbs, was 20 respectively. The results of treating cut Cninium officinale Makino are listed below. Survival rate of group A by exposure of 5 times at 35~36.5℃ is less than 7.5%. Survival rate of group C by exposure of 5 times at 35.5~39.5℃ and group D by exposure of 23day at 37~44℃ is less than 2.5%. Especially in the case of group D, we found that complete eradication of the insect is difficult despite exposure high temperature for a long time. Also most of the grinded Cninium officinale Makino has the same patten but group B by relatively treated low temperature has a significant difference in mortality. Insect mortality in cut Cninium officinale Makino by exposure at 35℃ and 39.5℃ is 7.5% and the insect mortality in grinded Cninium officinale Makino by exposure at 39. 5℃ is only 75%. The reason is estimated that heat conduction of grinded Cninium officinale Makino is slow and it was less shocked by heat because The final core temperature of medicinal herbs is relatively low about 0.5~2℃. This means that storage insect(Tribolium castaneum Herbst) can be suppressed at mid-high temperature if it is to be treated more than 50 days without problem of quality deterioration of medicinal herbs that can be caused by high temperature.

In insect exoskeleton/cuticle, structural cuticular proteins (CPs) and the polysaccharide chitin are the major components of the procuticle. CPs are cross-linked by quinones or quinone methides produced by the laccase2 (Lac2)- mediated oxidation of N-acylcatechols. We reported that two major CPs, TcCPR27 and TcCPR18, belong to the CPR family that contain the RR-2 consensus motif (Rebers & Riddiford), are essential for formation and stabilization of the rigid cuticle of Tribolium castaneum adults. In this study, we characterized and investigated functions of the third most abundant protein, TcCP30, in extracts of elytra. TcCP30 cDNA encodes a protein with 171 amino acid residues containing a putative signal peptide. Unlike TcCPR27 and TcCPR18, TcCP30 mature protein lacks an RR motif, with a very unique amino composition, 36% Glu, 21% His, 20% Arg and 16% Gly. TcCP30 gene is highly expressed right before and after eclosion (in 5 d-old pupae and 0 d-old adults). Immunohistochemical studies revealed that TcCP30 protein was present in rigid cuticle such as elytra and ventral abdomen but not soft cuticle such as hindwings and dorsal abdomen of adult T. castaneum. Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. However, the subsequent pupal-adult molt, more than 50% adults were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly because probably due to the malformed elytra. These results indicate that TcCP30 is critical for formation of rigid adult cuticle as well as development and growth of T. castaneum.

Insect cuticle is a complex biocomposite material consisting of three major morphologically distinct layers, the waterproofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. Structural cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers that comprise the procuticle. During cuticle tanning (sclerotization and pigmentation), CPs are cross-linked by quinones derived from the oxidation of catechols, resulting in hardening of the exoskeleton. However, the factors that lead to synthesis and assembly of cuticular regions with differing mechanical properties are not well understood. To gain a better understanding of the development and differentiation of rigid cuticle, we performed transmission electron microscopic (TEM) analysis of elytral cuticle (highly sclerotized and pigmented forewing) from 2 d-old pupae to 9 d-old adults of the red flour beetle, Tribolium castaneum. In 2-3 d-old pupae, pupal cuticle separated from the underlining epidermal cells (apolysis), and outermost envelope and protein-rich epicuticle begun to form. A numerous horizontal chitinous laminae and vertical pore canals were evident in the procuticle of 4-5 d-old pupae. By one day after adult eclosion, less-compact horizontal chitinous laminae were deposited, followed by block-type cuticular layers with no horizontal laminae were formed by 9 days. These results will lead to a) a better understanding of insect cuticle formation, structure and mechanics, b) the potential for development of novel insect control agents that target cuticle physiology, and c) the production of biomimetic materials with physical properties like those of the insect exoskeleton for use in biomedical or other technological devices. This work was supported by NRF (NRF-2012R1A2A1A01006467).

To accommodate growth, insects must periodically replace their exoskeletons. The cuticle or exoskeleton consists of multiple functional layers including the waterproof envelope (cuticulin layer), the protein-rich epicuticle (exocuticle) and the chitinous procuticle (endocuticle). After shedding the old cuticle, the newly formed soft and transparent cuticle must harden and tan. During tanning, cross-links form between adjacent polypeptide chains, causing progressive hardening, dehydration, and close packing of the polymers. This cross-linking occurs as a result of oxidative and nucleophilic reactions between highly reactive tanning agents derived from catechols and nucleophilic side chain groups of cuticular proteins (CPs). The initial steps of tanning in most cuticles involve formation of quinones and quinone methides derived from N-acylcatecholamines, followed by their oxidative conjugation with CPs, leading to changes in mechanical properties and pigmentation. This vital physiological step occurs during each stage of development and is required to stabilize and harden the exoskeleton. The mechanism of the insect sclerotization, however, is poorly understood, and the factors that lead to synthesis of cuticular structures with differing physical properties that are unique to each type of cuticle (e.g. elytron, hindwing, pronotum, dorsal and ventral body wall) are not well defined. In this study, we investigated development and differentiation of rigid cuticle using the red flour beetle, Tribolium castaneum adult, as a model insect. Tribolium as a beetle is superior model for studying rigid cuticle formation because they have a highly modified (sclerotized and pigment) forewing (elytron) which can be separated from other tissues easily and cleanly. We analyzed ultrastructure of elytral cuticle during development (from 3 d-old pupae to 3 d-old adults) by transmission electron microscopy (TEM). In 3 d-old pupae, pupal cuticle separated from the epidermal cells (apolysis), and the outermost envelop of adult cuticle was being formed. Protein-rich epicuticle and procuticle composed numbers of horizontal laminae and vertical canals were formed at 4 and 5 d-old pupal stages. After adult eclosion, additional thick horizontal laminae were evident and apical membrane of the epidermal cells became undulae like-structure at 1 d-old adult, and then final three layers with no horizontal laminae were formed by 3days after adult molting. Furthermore, protein localization of several high abundant adult CPs is also discussed. These results will contribute understanding cuticle formation and differentiation in insect during post-embryonic development.

Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers or procuticle. CPs contain a conserved sequence known as the Rebers & Riddiford (R&R) motif, which may function as a chitin-binding domain that helps to coordinate the interaction between chitin fibers and the protein network. We identified two highly abundant RR-2 CPs, TcCPR18 and TcCPR27, in protein samples extracted from elytra (rigid cuticle) of Tribolium castaneum adults and determined that these two CPs are required for rigid cuticle morphology. In this study, we identified the third most abundant protein (TcCP30) extracted from the elytra, and cloned a full-length cDNA. It encodes a very unusual 171 amino acid residue protein of which 36% of the residues of the mature protein are Glu, 21% are His, 19% are Arg, and 16% are Gly, organized in a regular pattern but not R&R consensus motif. TcCPR18 and TcCPR27 genes are expressed at 4 d-old pupae, while TcCP30 is highly expressed at 5 d-old pupae (last pupal stage) and 0 d-old adults. Immunohistochemical studies revealed the presence of TcCP30 in rigid adult cuticle (e.g. elytron, pronotum and ventral abdomen) but not soft cuticle (e.g. hindwing and dorsal abdomen). Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. The subsequent pupal-adult molt, however, more than 50% adults were unable to shed their exuvium and died. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly. These results indicate that TcCP30 may play critical roles in rigid adult cuticle formation, development and insect growth and survival. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect cuticle/exoskeleton is a first physical barrier to protect their body from multifarious environments such as desiccation, natural enemies and entomopathogenic microorganisms. Cuticle tanning (sclerotization and pigmentation) is a vital procedure for generating suitable cuticle depending on body region by sclerotization and pigmentation in insects. Insect cuticle tanning is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticle protein (CP) side chains resulting in protein cross-linking. One type of pigmentation (quinone tanning) is associated with the covalent linkage of CPs to the ring component of NBAD. In contrast, linkage of CPs to the side chain of NADA (b-sclerotization) is correlated with colorless cuticle. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to N-acetyl dopamine (NADA) in cuticle tanning pathway. In this study, we studied function of TcNAT1 on adult cuticle tanning by double stranded-RNA (dsRNA) mediated gene silencing. Injection of dsTcNAT1 had no affect on animal development, growth and molting such as larva to larva, larva to pupa and pupa to adult. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults (older than 3 days) was darker than that of control dsTcVer treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment) and dopamine quinone-mediated protein crosslinking. On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles that are located in the intervein regions, suggesting that NADA mediated b-sclerotization is occurred at these regions. Similarly, darker pigment was evident at veins of the hindwings of the dsTcNAT1-mature adults. These results suggest that TcNAT1 have important roles in sclerotization and pigmentation of adult body and wings (elytron and hindwing). This work was supported by NRF (NRF-2012R1A2A1A01006467).

A nuclear receptor, Met, mediates juvenile hormone (JH) action to control gene expressions associated with metamorphosis in many insects. In this study, we showed that RNA interference (RNAi) of the Met or Kruffel homolog 1 (Kr-h1) induced the precocious metamorphosis of Tribolium castaneum larvae. JH significantly inhibited cellular immune response of T. castaneum hemocyte by suppressing hemocyte-spreading behaviour and nodule formation in response to bacterial injection. However, either RNAi of Met or Kr-h1 expression did not prevent the JH-inhibitory effect on hemocyte behaviors. However, several inhibitors specific to JH membrane action significantly inhibit the JH action hemocytes. These results suggest that JH responsiveness of hemocyte is not mediated by the nuclear receptor.