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).

Stored grain pests can cause reduction of grain quantity, quality, commercial value and germination rate. Susceptibility of three fumigants, methyl bromide, ethyl formate and phosphine, were assessed on Tribolium castaneum, which is an important stored grain pest. On susceptible insects, LCT50 of phosphine was 0.654mg h/L for egg, 0.127mg h/L for late larvae, 0.105mg h/L for pupae and 0.048mg h/L for adult stage, respectively. LCT50 of methyl bromide was 33.193mg h/L for egg, 14.585mg h/L for late larvae, 8.616mg h/L for pupae and 11.967mg h/L for adult stage, respectively. LCT50 of ethyl formate were 25.165mg h/L for egg, 80.912mg h/L for late larvae, 176.326mg h/L for pupae and 68.578mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine were 82.325mg h/L for egg, 33.315mg h/L for late larvae, 73.546mg h/L for pupae and 55.707mg h/L for adult stage, respectively. LCT50 of methyl bromide were 19.250mg h/L for egg, 43.413mg h/L for late larvae, 76.842mg h/L for pupae and 19.387mg h/L for adult stage, respectively. LCT50 of ethyl formate were 87.552mg h/L for egg, 113.457mg h/L for late larvae, 200.122mg h/L for pupae and 85.394mg h/L for adult stage, respectively.

Insect cuticle or exoskeleton is an extracellular matrix consisting of three major morphologically distinct layers, the water-proofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. To accommodate growth, insects must periodically replace their cuticles in a process called “molting or ecdysis”. During each molt cycle a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. In this study, we show a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, belonging to a subfamily (group III) of insect chitinases in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to maintain the integrity and the mechanical strength of a light-weight, yet rigid, adult cuticle. Because group III chitinases are highly conserved among insect and other arthropod species, these enzymes have a critical role in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate cuticular extracellular matrices. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2018R1A2B6005106)

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.