Insect cuticle or exoskeleton is a complex extracellular matrix formed primarily from structural polysaccharide chitin and protein, and it plays a critical role in protecting them from various environmental stresses and pathogenic infection. Despite of limited composition, insect cuticle has remarkably diverse mechanical properties, ranging from soft and flexible to hard and rigid. My research has been focusing on functional importance of the genes involved in chitin metabolism and cuticle tanning (sclerotization and pigmentation) to comprehensively understand the genetic, enzymatic as well as molecular mechanism underlying differentiation, development and formation of insect cuticular extracellular matrices.

Insect cuticle consists of numerous structural proteins, which could interact with polysaccharide, chitin, and alter properly mechanical property of the cuticle. Cuticular Protein Analogous to Peritrophins (CPAPs) are characterized by presence of one (CPAP1s) or three (CPAP3s) chitin-binding domain belong to CBM14/ChtBD2 family. In this study, we investigated physiological functions of TcCPAP1-H and TcCPAP3-C in Tribolium castaneum. RNAi for either TcCPAP1-H or TcCPAP3-C at late instar larvae had no effect on larval-pupal molt nor pupal development. However, the resulting pharate adults failed to shed their old pupal cuticle and died entrapped in it without undergoing eclosion. TEM analysis, in addition, revealed disorganized chitinous horizontal laminae and/or vertical pore canals of rigid cuticle from TcCPAP1-H- and TcCPAP3-C-deficient adults. Desiccation-induced death produced by injection of dsTcCPAP1-H into young instar larvae is also discussed.

Insect cuticle tanning (pigmentation and sclerotization) is a complex and vital physiological process that begins with tyrosine and is responsible for production of both melanin- and quinoid-type pigments. In addition, these quinones undergo isomerization to quinone methides and cross-linking reactions with cuticular proteins for cuticle sclerotization. In this study, we studied the functions of TmDDC and TmY-y as well as TmNAT1, TmADC and Tmebony from Tenebrio molitor, which are involved in the tyrosine-derived melanin- and quinoid-type pigment productions, respectively. The temporal and spatial expression patterns of the genes were analyzed by real-time PCR. RNA interference was performed to understand the genetic regulation and molecular mechanism underlying the darkening and hardening of beetle cuticle.

The eggs of Asian tiger mosquito, Aedes albopictus, possess high desiccation resistance, which contribute the rapid spread of this mosquito across the world. Melanization of eggshell appear to play a role in the resistance to desiccation. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the eggshell. In this study, we demonstrated functional importance of two yellow genes, AalY-g and AalY-g2, in the chorion formation. Both genes were highly induced in the ovary at 48 h after blood meal. Injection of dsRNA for AalY-g or AalY-g2 into adult females had no effect on fecundity. However, the outermost colorless exochorion of the eggs obtained from both dsRNA-treated females was fragile and peeled off in places, and melanization of the endochorion was obviously delayed by several hours. In addition, unlike eggs from control females which acquired high desiccation resistance between 18 and 24 h after oviposition (HAO), 60-70% 24 HAO eggs from either AalY-g- or AalY-g2-deficient females were collapsed when they were moved to an air-dry condition, and the desiccation resistance was not increased in later stages of embryonic development analyzed. TEM analysis revealed that abnormal morphology and ultrastructure of the endochorion, particularly outer-endochorion, in the 24 HAO and older eggs from either AalY-g-and AalY-g2-deficient females. These results indicate that AalY-g and AalY-g2 are required for morphology and formation of the endochorion (outer-endochorion), a structure that appears to be critical for desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106)

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).

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 cuticle tanning (pigmentation and sclerotization) is a complex and vital process, which includes hydroxylation of initial amino acid, tyrosine, to DOPA and decarboxylation of DOPA to dopamine. In the pigmentation process, dopamine further undergoes two N-acylation reactions to yield N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD). In the former reaction, arylalkylamine N-acetyltransferase (AANAT1) converts dopamine to NADA, and in the later reaction, aspartate 1-decarboxylase (ADC) provides β-alanine, which is conjugated with dopamine catalyzed by NBAD synthase (Ebony) for production of NBAD. In this study, we performed functional genomics of TmAANAT1, TmADC and Tmebony to determine whether they are required for cuticle pigmentation in Tenebrio molitor adults. Loss of function of these genes by RNAi caused the significantly darker body color than that of control animals. Note that, although all phenotypes exhibited dark cuticle pigmentation, RNAi of either TmADC or Tmebony only altered brownish outer region of the cuticle to dark/black. In contrast, RNAi of TmAANAT1 had no effect on the brown hue of the outer cuticle layer, but less or no pigmented inner region of the cuticle became significantly darker than those of control adults. These results suggest that, like that seen in TcAANAT1- or TcADC-deficient Tribolium castaneum adults, NADA produced by a reaction by TmAANAT1 contributes the lighter inner cuticle layer(s), whereas NBAD appears to do the highly pigmented outer cuticle layer(s) of the cuticle of T. molitor adults. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).

농가 선호도가 높은 분홍색 비모란 선인장 ‘Yeonbit’는 분홍색 품종 ‘Yeonsi’을 모본으로, 분홍색 ‘Yeonsil’ 품종을 부본으로 하여 2011년에 교배하여 육성된 교배종이다. 종자를 70% C2H5OH와 2% NaOCl로 멸균처리한 후, 100mL 삼각플라스크 0.1% 활성탄이 첨가된 Kyoto 배지에 파종하였다. 발아된 유묘는 기저부를 잘라 기배계대 배양된 어린 삼각주에 접목한 후 동일한 배지가 첨가된 시험관에 치상하였다. 6개월 재배 후 온실로 옮겨서 순화과정을 거쳤으며, 기외에서 매년 접목을 하였으며 총 4번을 접목하여 2013년부터 2015년까지 총 3회에 걸쳐 생육특성을 조사하였다. ‘Yeonbit’ 품종은 편원형 모양과 분홍색 구색을 가지고 있다. 그리고 7~9개, 평균 8.0개의 능(rip), 갈색의 직립형 짧은 가시, 그리고 혹(tubercle)이 돌출된 형태로, 모구가 구성되어 있다. 정식 10개월 후 ‘Yeonbit’ 품종의 구직경은 34.5mm였으며 자구는 모구와 같은 분홍색으로 평균 15.4개가 생성되었다. 2015년 육성계통 평가회에서 ‘Yeonbit’ 품종은 기호도 점수 3.7을 받았다. 2017년 6월 12일에 이 품종은 국립종자원에 등록되었으며 종자산업법에 의해 품종보호(등록번호 6752)를 받게 되었다.

Pesticide application in agriculture provides significant benefits such as protection from disease, prevention of harmful insects, and increased crop yields. However, accurate toxicological tests and risk assessments are necessary because of many related adverse effects associated with pesticide use. In this review, we discuss and analyze residual pesticides contained in livestock feed in Korea. A pesticide residue tolerance standard for livestock feed has not been precisely established; so, risk assessments are required to ensure safety. Standards and approaches for animal criteria and appropriate methods for evaluating residual pesticides are discussed and analyzed based on technology related to animal product safety in Korea. The safety of livestock feed containing pesticides is assessed to establish maximum residue limits relative to pesticides. Analysis of residual pesticides in milk, muscle, brain, and fat was performed with a livestock residue test and safety evaluation of the detected pesticide was performed. Efficacy of organic solvent extraction and clean-up of feed was verified, and suitability of the instrument was examined to establish if they are effective, rapid, and safe. This review discussed extensively how pesticide residue tolerance in livestock feed and hazard evaluation may be applied in future studies.

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig (GalT-MCP/-MCP). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain GalT-MCP/-MCP/CD39/CD73 pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from GalT-MCP/-MCP pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate GalT-MCP/-MCP/CD39/CD73 pig expressing CD39 and CD73 at endothelial cells.

In Korea, concrete pavements were first applied to highways in 1981 and as a result of continued increase in length over the past years, 2,592 km of concrete pavement network is currently in service, of which 1,399 km(54%) of concrete pavements is 10 years or older, and 233km(9%) is 20 years or older. The length of concrete pavement sections nationwide has been steadily on the rise every year (EXTRI, 2017). Approximately 54% of current concrete pavement highway network will reach the service life limit in 2025 which means around 660 billion won is needed for future pavement repair project (EXTRI, 2017). Given that concrete pavements beyond design life still have a remaining service life, it is economically advantageous to repair them before reconstruction. Asphalt overlays are a major repair method for older concrete pavements. Depending on the concrete pavement condition, thickness and mixture of asphalt overlays are determined. Service life of asphalt overlays varies by the presence, time and size of cracks in existing concrete pavements and reflecting crack at joints. Temperature change of concrete pavement is among the major reaction parameters of reflecting crack. Reflecting crack develops when asphalt bottom-up cracking by longitudinal shrinkage and expansion due to temperature change of the concrete base layer, top-down cracking by temperature difference between top and bottom of concrete, and shear stress by traffic loading are combined (Baek, 2010). Crack and joint behaviors of concrete pavement vary between the base layer and the concrete surface of composite pavement system, and different conductivity by mixture and thickness of asphalt overlay leads to temperature change of concrete base course. This study measured temperatures of each layer of diverse composite pavements in place on site and analyzed differences in temperature change of concrete base layer depending on mixture and thickness of asphalt overlays. Overlay thickness parameters were 5cm and 10cm, two values most widely used, while mixture parameters were SMA and porous asphalt. Based on temperature change of concrete surface, this study also evaluated the difference of temperature change in concrete base layer with an asphalt overlay on top. Findings from this study are expected to be utilized for studies on mechanism and modeling of reflecting crack in old concrete pavements with asphalt overlays.

The objectives of the present study were to examine the antioxidant activity of fractions with different isoelectric points from salmon enzymatic hydrolysates and obtain peptide fractions of sufficient amounts with higher antioxidant peptide fraction, which could be applied to the food and animal model systems. The salmon enzymatic hydrolysates were fractionated on the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing without toxic solvents and reagents, which is termed autofocusing. Acidic and basic fractions showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than the other fractions. The basic fractions showed higher hydroxyl (OH) radical scavenging activity. The weak acidic and weak basic fractions showed higher oxygen radical absorbance capacity (ORAC) values than the acidic and basic fractions. The acidic fraction showed higher metal chelating activity than other fractions. The acidic fraction suppressed lipid oxidation in the cooked patties to a greater extent than other fractions. These results suggest that peptides fractions from salmon enzymatic hydrolysate are effective antioxidant, and that autofocusing could be useful to increase antioxidant activity for application to food and animal model systems.

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.

Eggs from the mosquito genus Aedes exhibit high desiccation resistance that likely facilitate spreading some of them as a vector of human disease throughout the world. However, molecular mechanism underlying the embryonic resistant to desiccation has not well understood. In this study, we performed functional study of two ovary-specific yellow genes, AalY-g and AalY-g2, in the Asian tiger mosquito, Aedes albopictus. The eggs obtained from AalY-g or AalY-g2 RNAi females showed poor desiccation resistance. TEM analysis revealed that, unlike that seen in the dsEGFP-control eggs, no high electron-dense outer-endochorion was evident in the eggs from AalY-g- or AalY-g2-deficient females. These results suggest that both yellow genes at least play roles in the chorion formation, which appears to be critical for integrity and desiccation resistance in Ae. albopictus eggs.