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

Insect cuticle/exoskeleton covering the entire external surface of the body is essential for protecting insects from various environmental stresses. Tyrosine metabolism plays a major role in not only the darkening of cuticle but also its hardening. In this work, we have focused on the functional analysis of nine genes involved in tyrosine-mediated cuticle tanning (pigmentation and sclerotization) pathway in Tenebrio molitor, which has a unique adult cuticle coloration, dark/black dorsal thorax and elytron, and reddish ventral thorax and abdomen. The temporal and spatial expression patterns of the genes were analyzed by real-time PCR, and RNA interference (RNAi) was performed to study the functional importance of these genes in cuticle coloration and/or hardening in T. molitor. This work was supported by NRFs (NRF-2015R1A2A2A01006614 and NRF-2015R1A6A3A04060323).

We determined the effects of different water temperatures (15, 20, and 25℃) and photoperiod cycles (24L:0D, 12L:12D, and 0L:24D) on the oxygen consumption of the offspring of a cultured Japanese strain (JJ), a selected Korean strain (KK), and intraspecific hybrid strains (JK and KJ) of red seabream, Pagrus major, under starvation conditions. The different fish strains, water temperatures, and photoperiod cycles had effects on the mean oxygen consumption of fish. Oxygen consumption increased with increasing water temperatures for all photoperiod treatments (p <0.001). Fish held in continuous darkness (0L:24D) used consistently less oxygen than fish exposed to continuous light (p <0.05). The oxygen consumption of fish exposed to the light phase in a 12L:12D photoperiod was higher than that of fish in the dark phase of the 12L:12D cycle, and differences were significant in three of the strains: JJ (15℃), KK (15 and 20℃), and KJ (25℃). The oxygen consumption of the inbred (JJ and KK) and intraspecific hybrid (JK and KJ) strains varied with differing water temperatures and photoperiod cycles. The JK strain displayed significantly higher oxygen consumption than the other strains under all experimental conditions except 15℃ with a 0L:24D photoperiod. The JK and KJ strains usually showed the highest and lowest oxygen consumption values, respectively, whereas the inbred strains exhibited intermediate values. Oxygen consumption in the JJ and JK strains was usually higher than that of the KK and KJ strains. We propose that differences in the thermal sensitivity and photosensitization properties of the strains contribute to differences in their ability to adapt to changes in water temperature and photoperiod, thus resulting in differences in the amplitude of their metabolic rates.

Kisspeptin (Kiss) and its cognate receptor, kisspeptin receptor (KissR; G protein coupled receptor 54, GPR54), have recently been recognized as potent regulators of reproduction in teleosts. Additionally, leptin plays an important role in energy homeostasis and reproductive function in teleosts. The purpose of this study was to examine differences in the concentration of the hormones of the Kiss/KissR system and leptin and the expression of their underlying genes, all of which are involved in the sexual maturation of female goldfish, Carassius auratus, following treatment with Kiss. The expression levels of KissR increased after the Kiss injection. Furthermore, the peptide hormone leptin also increased after the injection (in vivo and in vitro). Additionally, the expression of GnRH and GTHs (GTHα, FSHβ, and LHβ) increased in the brain and pituitary (in vitro and in vitro). These results support the hypothesis that Kiss plays important roles in the direct regulation of the hypothalamus-pituitary-gonad axis and leptin in goldfish. Therefore, we suggest that Kiss system gene expression is correlated with energy balance and reproduction.

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).

Pigmentation or melanization is an important physiological event in insects and is involved in cuticle tanning, wound healing and encapsulation as a defensive response. Dopachrome-conversion enzyme (DCE, Yellow) significantly accelerates the melanization of the chorion in mosquito eggs. In this study, we demonstrated functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the Asian tiger mosquito, Aedes albopictus. The transcripts of both genes were detected in the ovary of adult females only 48-72 h after blood feeding. RNAi for AalY-g or AalY-g2 had no effect on fecundity. However, the outermost colorless exochorion of the eggs was fragile and partially peeled off, and initial melanization of the endochorion was significantly delayed. Eggs from control females exhibited high desiccation resistance, whereas those from dsAalY-g- or dsAalY-g2-treated females were collapsed (50-80%) under the air-dry condition. Ultrastructural analysis revealed abnormal morphology of the endochorion and vitelline membrane in the AalY-g and AalY-g2-deficient eggshell. These results indicate that AalY-g and AalY-g2 are critical for integrity and desiccation resistance of the Ae. albopictus eggs. This work was supported by NRFs (NRF-2015R1A6A3A04060323).

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs with high mortality. PEDV is belong to Coronavirus, enveloped, single-stranded, positive-sense RNA virus. PEDV particles were composed of four structure proteins such as a glycosylated peplomer (spike, S) protein, envelope (E), glycosylated membrane (M) protein, and unglycosylated RNA-binding nucleocapsid (N) protein. Many of previous studies talk about this four structure proteins have a great potential to diagnosis and prevent PEDV. In this study we investigated expression of these structure proteins using the bacterial and baculovirus expression system. In bacterial expression system, our results showed that structure proteins fused polyhedrin and intein gene were expressed higher than non-fusion structure proteins. The expressed fusion proteins were used to immune mice for generating a polyclonal antibodies. In baculovirus expression system, co-infection of insect cells with these four recombinant baculoviruses led to self-assembly of virus-like particles as demonstrated by Transmission electron microscopy. They were confirmed by western blot analysis using pre-made polyclonal antibodies. Finding in this study may provide important information for vaccine and diagnostic development.

Insects are among the most diverse groups of animals on the planet, representing more than half of all known living organisms. These insects are found in nearly every environment. Although humans regard certain insects as pests and attempt to control them using insecticides, most insects perform complex ecological functions, and provide either direct or indirect economic benefits to humans. Recently, the importance of insects used as food sources or as pets has increased in many countries, including Korea. In addition, several insects have a strong influence on people's emotion. Insect-mediated mental healthcare program is designed to help people who have disorders with physical, behavior and development. Children who have mental disorder, the experimental group that was provided with an insect-mediated mental healthcare program over a total of 8 sections, one section per week, 60 minutes per section, followed by pre-test and post-test. They responded to therapeutic effect after the completion of the program. Further research on the basis of this study is expected to help children with emotional therapy in other areas.

The population of managed honey bees has been dramatically declining the recent past in worldwide. The one of most common disease of bees is nosemosis, the nosemosis is caused by microsporidia in the genus Nosema. Nosema apis and N. ceranae have been described as honeybee pathogens. These microsporidia are highly evoloved fungi with an obligately intracellular parasitic lifstyle. The disease causes significant detriment to honey production and results in economic losses. In our knowledge, Fumagillin is the only antibiotic approved for control of nosemosis in honey bees, however this antibiotic may have unintended effects on the honey bee host, ultimately contributing to increased prevalence and pathogenicity of Nosema. Therefore, we screened anti-Nosema substances from entomopathogenic fungal culture filtrates using in vitro polar tube germination assay. These fungal metabolites are employed as antibiotic agents. As results, Total 3 samples (23% of 13 total samples) showing the germinating inhibition against N. ceranae. This screening method may be useful for the detection of anti-Nosema substances from various samples and selected samples in this study may be a good feature to be used in the development of a new biocontrol method of nosemosis.

We study the physical and chemical properties of the molecular clump hosting a young stellar cluster, IRAS 20160+3636, which is believed to have formed via the “collect and collapse” process. Physical parameters of the UC Hii region associated with the embedded cluster are measured from the radio continuum observations. This source is found to be a typical Galactic UC Hii region, with a B0.5 type exciting star, if it is ionized by a single star. We derive a CN/HCN abundance ratio larger than 1 over this region, which may suggest that this clump is being affected by the UV radiation from the Hii region.

Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a non-enveloped virus and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2 is the main target for neutralizing antibodies in PPV. When VP2 was expressed in large amounts, it assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. In this study, we generated the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) to express the VP2 protein. Expression of the VP2 protein was analyzed by SDS-PAGE and Western blot. The recombinant VP2 protein of approximately 64 kDa was detected by both analyses. The formation of VLP by recombinant VP2 was confirmed through transmission electron microscopy examination. The purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm.

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 first physical barrier to protect their body from multifarious environments. Cuticle tanning (sclerotization and pigmentation) 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 cuticular proteins (CPs) resulting in protein cross-linking. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to NADA whose covalent-linkage of CPs is correlated with colorless cuticle (β-sclerotization). In this study, we analyzed functions of TcNAT1 on cuticle tanning of adult Tribolium castaneum by RNAi. Injection of dsRNA for TcNAT1 (dsTcNAT1) had no affect on animal development and growth. 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 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). On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles located at the intervein regions, suggesting that NADA mediated β-sclerotization is occurred in these regions. Similarly, darker pigment was evident at veins of the hindwings of TcNAT1-deficient adults. These results suggest that TcNAT1 plays important roles in cuticle tanning of T. castaneum adult. To characterize enzymatic properties of TcNAT1, furthermore, recombinant TcNAT1 protein expressed in E. coli was purified by utilizing Ni-NTA affinity column chromatography. This work was supported by NRF (NRF-2012R1A2A1A01006467).