Insect cuticular extracellular matrices (ECM) including the eggshell and exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous ECMs must be degraded at least in part during embryonic and post-embyonic molting periods to accommodate continuous growth all the way to the adult stage. In this study we investigated the functions of groups I and II chitinases, TcCHT5 and TcCHT10, in turnover of the eggshell and cuticle in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. However, although TcCHT5 is apparently involved in these vital physiological events, TcCHT10 could substitute for TcCHT5 except during the pupal-adult molting when both enzymes are indispensable to degrade chitin in the old pupal cuticle.

A new fumigant, carbonyl sulfide (COS), has potential for use as a replacement for methyl bromide, yet its mechanism of toxicity to insects remains poorly understood. In this study, transcriptome analysis was performed on Tribolium castaneum malpighian tubules and fat bodies, which are known to play an essential role in energy storage and utilization in insect species. In total, upon exposure to COS, 3,034 and 2,973 genes were differentially expressed in the T. castaneum malpighian tubules and fat body, respectively. These differentially expressed genes comprise a significant number of detoxification-related genes, including 105 P450s, 18 glutathione S-transferases (GSTs), 82 ABC transporters, 25 UDP-glucosyltransferases and 42 carboxylesterases and mitochondrial–related genes, including 9 complex Ⅰ genes, 2 complex Ⅱ genes, 1 complex Ⅲ gene, 9 complex IV genes, 8 complex V genes from both malpighian tubules and fat body tissues. Moreover, KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by ABC transporters and drug metabolism by other enzymes. We also investigated the role of carbonic anhydrases (CAs) in toxicity of COS using dsRNA treatment in T. castaneum. These results show that CA genes have a key role in toxicity of the COS. Furthermore, the results of transcriptomic analysis provide new insights into the insecticidal mechanism of COS fumigation against T. castaneum and eventually contribute to the management of this important stored grain pests.

Because of recent reports about phosphine resistance problem, development of effective fumigation method to control grain pests became very important. In this study, a chemical treatment, ethyl formate fumigant treatment, and a physical treatment, atmospheric control, were attempted as alternative solutions to this problem. In this study, for CA(Controlled atmosphere) treatment, 99.999% nitrogen was used to create a hypoxic condition with less than 5% oxygen, and for EF, the treatment concentration was 10 mg/L to 80 mg/L. As a result of the study, in CA single treatment, adult insects showed a mortality rate of less than 10% even after 2 weeks of treatment, and pupae and larvae showed a mortality rate 71% and 34%, but eggs showed a mortality rate of 100%. In EF single treatment, adults and larvae showed a 100% mortality rate at 80 mg/L, but eggs showed a 50% mortality rate and pupae were not affected. Considering the results, CA single treatment is not suitable for controlling Tribolium castaneum because of long treatment period, and in the case of EF single treatment, additional researches on longer treatment time is needed.

Insect eggshell and cuticle/exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous cuticular extracellular matrices must be degraded at least in part during embryo hatching and molting/ecdysis periods to accommodate continuous growth all the way to the adult stage. In this study, we investigated the functional importance of groups I and II chitinases, TcCHT5 and TcCHT10, in the turnover of chitinous cuticle during both embryonic and post-embryonic development in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. TcCHT10 appears to be able to substitute for TcCHT5 in all these vital physiological events except for the pupal-adult molting in which TcCHT5 is indispensable for complete digestion of chitin in the old pupal cuticle.

The fumigation toxicity of carbonyl sulfide to T. castaneum as a storage grain pest was evaluated. Carbonyl sulfide (COS) is registered in Australia for microorganism present in soil, root and fertilizer. the fumigation activity of carbonyl sulfide was investigated in 12 L desiccator for 24 h exposure to eggs, larvae, pupae, adults of T. castaneum. Eggs and pupae were showed 87.3% and 95.6% mortality for 25 mg/L of COS, respectively. Larvae and adults were investigated with 80.0% and 100.0% mortality at 15 mg/L treatment, respectively. Therefore, the eggs of T. castaneum showed the highest tolerance to COS.

The fumigation activity of phosphine (PH3) to T. castaneum as a storage grain pest was evaluated. The lethal concentration time (LCT) value of each developmental stage (egg, early larva, late larva, pupa and adult) of T. castaneum was analyzed in 12 L desiccator. At the T. castaneum larva stages, exposure for 4 h showed low LCT value, especially in early stage larvae (LCT99 = 0.32 mg·h/L) which is very high susceptibility to PH3. However, T. castaneum eggs were observed very high tolerance to PH3 at LCT99 77.47 mg·h/L. Therefore, the fumigant activity of PH3 against T. castaneum can be found to be significantly different depending on developmental stage.

Insect structural cuticular proteins (CPs) play a major role in determining the diverse physical properties of the cuticle as a result of interactions/cross-linking among themselves and with chitin. CP genes compose a large gene family and have been classified more than ten distinct families based on the presence of unique amino acid sequence motifs. In this study, we performed RNAi-based functional analysis of eleven genes (TcCPLCP1-11) in Tribolium castaneum, which belong to CPLCP (Cuticular Proteins of Low Complexity, Proline rich) cuticular protein family. RNAi for TcCPLCP7-11 caused lethal pupal-adult molting defects and/or abnormal cuticle morphology in the resulting adults. Ultrastructural defects of the cuticles from TcCPLCP7-11-deficient insects by TEM are also discussed.

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.

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.

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).

Insects have a protective exoskeleton consisted with cuticle to adapt various environments and pathogens. Insect cuticle mainly composed of the polysaccharide chitin and numerous of cuticular proteins (CPs). CPs are important for insect cuticle formation, development, and growth because it produces proper combination of mechanical and physical properties of cuticle depend on the regions of an exoskeleton. The largest family of CPs contains a 28-residue motif known as the Rebers-Riddiford (R&R) consensus sequence. When sequences containing the R&R consensus are aligned, they fall into three groups based on sequence similarity, and these groups tend to correlate with the type of cuticle (soft or hard) from which the proteins are derived. Proteins with the RR-1 motif have been found primarily in soft cuticle, whereas many proteins from rigid cuticle have an extended region of similarity called RR-2. We recently reportedthat two major CPs, TcCPR18 and TcCPR27 belong to RR-2, are essential for formation of highly sclerotized modified-forewings (elytra) of a beetle. In this study, we performed functional genomics of TcCPR4, which encodes RR-1 motif. The transcript levels of TcCPR4 drastically increased in 3 d-old pupae at when adult cuticle synthesis appears to be begun. Immunohistochemical studies revealed that TcCPR4 protein was detected in the rigid cuticle of elyton and ventral abdomen but not in the flexible cuticle of hindwing and dorsal abdomen of T. castaneum adult. Furthermore, TcCPR4 protein was specifically present at basal side of the procuticle (near the epidermal cells) and vertical canals, whereas TcCPR27 protein was found entire procuticle. Injection of double-stranded RNA of TcCPR4 (dsTcCPR4) into late instar larvae had no effect on development and any types of molting such as larval-larval, larval-pupal or pupal-adult. Interestingly, depletion of both TcCPR4 and TcCPR27 transcripts could rescue the elytral cuticle defect and mortality produced by injection of dsTcCPR27 alone. Transmission electron microscopy analysis revealed that depletion of TcCPR4 had abnormal vertical canals in rigid adult cuticle while dsTcCPR27 injection showed less electron-dense-horizontal laminae and vertical canals. Surprisingly, co-injection of dsRNA for TcCPR4 and TcCPR27 exhibited more severe cuticle defect with thinner elytral cuticle and abnormal vertical canals and chtin laminae compared to those from insects treated with dsRNA for each gene. These results suggest that TcCPR4 as a RR-1 is essential structural component in the rigid cuticle of T. castaneum adult.

Phospholipase A2 (PLA2) is the committed catalytic step of eicosanoid biosynthesis, which has been a common molecular target of several entomopathogens to induce insect immunosuppression. Despite critical importance of PLA2 in insect immunity, its gene structure was not known. This study identified insect PLA2 gene associated with immune reactions in the red flour beetle, Tribolium castaneum. Based on a previous study that an immune-associated PLA2 in insect is secretory type of PLA2 (sPLA2), five highly matched cDNA sequences were obtained from T. castaneum genome database using an sPLA2 sequence probe encoded in Drosophila melanogaster. The expressions of these five putative PLA2 were confirmed by reverse transcriptase-polymerase chain reaction. Out of five genes, one PLA2 gene called TcPLA2B was chosen because it showed specific expression in hemocyte and fat body. TcPLA2B was cloned and expressed in Escherichia coli and its protein was purified. The purified TcPLA2B showed PLA2enzyme activity, which was specifically inhibited by bromophenacyl bromide (a specific sPLA2inhibitor) and dithiothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 6.0) and reaction temperature (optimum at 10-30°C), and calcium dependency. An immunofluorescence assay indicated that TcPLA2B was localized near to cellular membrane of the cytosol in the hemocytes of T. castaneum at immune chanlenge. Double-stranded RNA (dsRNA) of TcPLA2B-treated larvae showed knockdown of its mRNA expression and did not form hemocyte nodule formation, while control larvae could exhibit time- and bacterial dose-dependent nodule formation in response to bacterial challenge. Addition of arachidonic acid (the catalytic product of PLA2) to the dsRNA-treated larvae rescued the inhibition of nodule formation. These results suggest that TcPLA2B gene is associated with insect immune reaction.