Phosphine (PH3) fumigation has been widely used for controlling storedgrain insect pests, causing the development of resistance of stored-grain insect pests to phosphine. PH3 resistance in Sitophilus oryzae has been reported in Korea. However, PH3 resistance in Tribolium castaneum has not been reported yet. This study was conducted to determine susceptibilities of T. castaneum collected from five different domestic locations to PH3. The susceptibility to PH3 was investigated using the FAO fumigation method. All domestic T. castaneum individuals were controlled by PH3 at 0.04 g m-3. At 0.01 g m-3, T. castaneum collected from two domestic locations did not exhibit 100% mortality. A P45S point mutation in dihydrolipoamide dehydrogenase (dld) gene was found in a PH3-resistant strain of T. castaneum (Aus07), but not in five domestic stains or a PH3-susceptible strain (Aus10). No significant difference was found in dld or cyt-b5-r gene expression across all tested strains. However, the Gyeongju-collected strain of T. castaneum showed more than a 1.7-fold increase in cyt-b5-r expression compared to the Aus07 strain. cDNA sequence analysis revealed that P45S (C133T) in the dld gene was only present in Aus07. A characteristic single nucleotide polymorphism in the cyt-b5-r gene sequence was identified in the five domestic strains. This study suggests that it is necessary to continuously monitor PH3-susceptibility of T. castaneum in Korea to quickly identify resistant individuals and prevent the spread of PH3 resistance through rapid control.

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.

Structural cuticular proteins (CPs) and the liner polysaccharide, chitin, are the primary components of insect cuticle or exoskeleton. A large number of insect CP family proteins are divided into several distinct subfamilies defined by the presence of specific amino acid sequence motifs. One of these subfamilies is composed of Cuticular Protein Analogous to Peritrophins (CPAPs), containing one (CPAP1s) or three (CPAP3s) type-2 chitin-binding domains. In this study, we report a novel function of TcCPAP1-C from Tribolium castaneum in movement of legs. RNAi for TcCPAP1-C at larval stage has no effect on insect molting, growth and development. However, the resulting adults exhibit impaired leg movement, in which internal tendon cuticles are ruptured near the femur-tibia joint. The exoskeletal cuticle, hemiadherens junctions, microtubule array, myotendinous junctions and muscle fibers exhibit normal morphology before the tendon breakage. These results indicate functional specialization of TcCPAP1-C in structural integrity of the internal tendon cuticle, and loss of function of TcCPAP1-C caused breakage of the tendon cuticle, resulting in defective limb movement and locomotion.

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 effect of fumigation on the phosphine-susceptible and -resistant strains in the T. castaneum was evaluated using phosphine, ethyl formate, and combination with phosphine and ethyl formate (phosphine+ethyl formate). The Lethal Concentration Time (LCT)50 analysis of susceptible strains, late larva showed that phosphine (0.13 mg·h / L), ethyl formate (80.91 mg·h / L), and phosphine + ethyl formate (19.36 mg·h / L). The LCT50 of adult was 0.05 mg·h / L, 68.58 mg·h / L and 17.84 mg·h / L when treated with phosphine, ethyl formate, and phosphine + ethyl formate. The LCT50 of resistant strains, late larva was found to 33.32 mg·h / L of phosphine, 113.46 mg·h / L of ethyl formate and 129.85 mg·h / L of phosphine + ethyl formate, and the LCT50 of adult was 55.71 mg·h / L of phosphine, 85.39 mg·h / L, phosphine + ethyl formate 85.83 mg·h / L. The treatment of three fumigants (phosphine, ehtyl formate, and phosphine+ethyl formate) showed the possibility of controlling against T. castaneum of phosphine-susceptible and –resistant strains.

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.

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce 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, which may lead to diverse mechanical properties of the cuticle. We previously reported the functional importance of Group I CDAs, TcCDA1 and TcCDA2, as well as the two alternative spliced isoforms of the latter, TcCDA2a and TcCDA2b from the red flour beetle, Tribolium castaneum in molting, morphology of cuticle and movement of legs. In this study, we further analyzed protein localization, ultrastructural defects of the cuticles and leg joints after RNAi of those genes. Both proteins are mainly present in the innermost procuticle region called the “assembly zone”. Loss of function of either TcCDA1 or TcCDA2 caused disorganized chitinous horizontal laminae and vertical pore canals in both the rigid and soft cuticles. RNAi of TcCDA2b affects cuticle integrity similar to that seen in RNAi of the two alternatively spliced forms of TcCDA2. In contrast, TcCDA2a-deficient adult, like that seen in the hypomorphic phenotype produced by RNAi of TcCDA1, exhibited ruptured tendons between femur and tibia, resulting in loss of locomotion ability. These results suggest that Group I CDAs play critical roles in molting, morphology, ultrastructure and mobility in T. castaneum. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).