The changes of stresses and ecdysteroid biosynthesis gene expression levels in Kynurenine 3-monooxygenase mutant Bombyx mori

( Chan Young Jeong ),( Chang Hoon Lee ),( Su Bae Kim ),( Sang Kuk Kang ),( Wan-taek Ju ),( Seong-wan Kim ),( Nam-suk Kim ),( Kee Young Kim ),( Jong Woo Park ) 한국잠사학회 2021 International Journal of Industrial Entomology Vol.43 No.1

Silkworms have long been bred with human history to produce silk. It has been with humans for longer than other industrial insects, and the silkworm variety has been continuously improved. Silkworms have been developed into the optimal form for producing high quality silk and pupae. Recently, the production of transgenic silkworms has further expanded the possibility of industrial value of silkworms. Kynurenine 3-monooxygenase (KMO), which is a flavin enzyme, is known for its involvement in ommochrome pigment synthesis. In the field of mammals, including humans, previous studies have revealed the function and role of KMO, which is an important enzyme for various immune responses and cell protection. However, in the case of insects, the function of KMO has only been studied to be involved in the formation of pigment, and accordingly, KMO is used exclusively on screening for generation of transgenic insects as a marker. In this study, using KMO-edited silkworms, it was intended to discover the novel functions and roles of KMO in silkworms by identifying changes in the expression of various genes associated with stress and growth. The changes were observed in expressions of genes regulating on stresses to survive and those on ecdysteroid hormone between wild-type (WT) silkworms and kmo mutant silkworms. The loss of KMO, in particular, decreased the expression of the shadow (sad) gene, one of the Halloween genes in the synthesis of ecdysteroid. In conclusion, these results suggest that silkworm KMO is responsible for potential functions regarding stress response and ecdysteroid synthesis.

밤바구미(Curculio dentipes) 월동 중 휴면 유충의 Ecdysteroid의 농도 변화

유진수,이충언,이경로 建國大學校基礎科學硏究所 1996 理學論集 Vol.21 No.-

월동 중인 밤바구미(Curculio dentipes) 휴면 유충의 발생에 따른 ecdysone과 20-hydroxyecdysone의 농도를 high performance liquid chromatography(HPLC)를 이용하여 측정하였다. Ecdysone 농도는 pre-diapausing larvae(19.81ng/g)에서 최저치를 나타내었고, diapausing larvae(29.97ng/g)에서 최대를 보였으며, post-diapausing larvae(25.71ng/g)에서 약간 감소하였다. 그러나 20-hydroxyecdysone은 pre-diapausing larvae(29.29ng/g)에서 가장 높은 농도를 보인 후, diapausing larvae(5.05ng/g)에서 급격히 감소하였고, post-diapausing larvae(12.53ng/g)에서 다시 증가하는 경향을 보여 생리기능상 역관계를 나타내었다. The concentration of ecdysteroid was measured in the pre-diapausing, diapausing, and post-diapausing larvae of the chestnut weevil. Curculio dentipes. Ecdysteroid titers were measured by high performance liquid chromatography(HPLC) during developmental stages. The concentration of ecdysone was the minimum(19.81ng/g) at pre-diapausing larvae and the maximum(29.97ng/g) at diapausing larvae, and it decreased to 25.71ng/g at post-diapausing larvae, and then kept relatively at high level. But the amount of 20-hydroxyecdysone was the highest value at pre-diapausing larvae(29.29ng/g) and minimum(5.05ng/g) at diapausing larvae, but it increased to the 12.53ng/g at post-diapausing larvae. In conclusion, the relationship between ecdysone and 20-hydroxyecdysone titer was shown to be inverse.

Ecdysteroid Stimulates Virus Transmission in Larvae Infected with Bombyx mori Nucleopolyhedrovirus

Kang,Kyung-Don,Lee,Eun-Jung,Shizuo Geroge Kamita,Susumu Maeda,Seong,Su-Il The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.1

Most baculoviruses have an ecdysteroid UDP-glucosyltransferase (egt) gene, whose product inactivates ecdysteroid within the infected host. Bombyx mori larvae infected with BmEGTZ, a mutant B. mori nucleopolyhedrovirus (BmNPV) in which the egt gene has been inactivated, die more rapidly compared to larvae infected with wild-type BmNPV. In this study, the profile of hemolymph proteins, and progression of virus infection in BmEGTZ- and BmNPV-infected B. mori larvae, was analyzed by SDS-PAGE and histochemically. These analyses showed that virus-encoded and virus-induced proteins were expressed quicker in BmEGTZ-infected larvae than in BmNPV-infected larvae. This suggests that the decrease in time to death, following BmEGTZ-infection, results from the stimulation of virus-specific protein expression. In order to examine the effect of ecdysteroid on virus transmission, the profile of hemolymph proteins, and progression of virus infection, were analyzed following an ecdysteroid injection of BmEGTZ- or BmNPV-infected larvae. In the BmNPV-infected larvae, ecdysteroid treatment had no apparent effect on hemolymph protein expression. This suggests that the injected ecdysteroid was inactivated by the BmNPV-expressed ecdysteroid UDP-glucosyltransferase. An Ecdysteroid injection into BmEGTZ-infected larvae increased the speed of virus-specific protein expression and virus transmission. These results suggest that ecdysteroid stimulates protein expression, which in turn results in the stimulation of virus transmission.

Ecdysteroid Stimulates Virus Transmission in Larvae Infected with Bombyx mori Nucleopolyhedrovirus

Kang, Kyung-Don,Lee, Eun-Jung,Kamita, Shizuo George,Maeda, Susumu,Seong, Su-Il Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.1

Most baculoviruses have an ecdysteroid UDP-glucosyltransferase (egt) gene, whose product inactivates ecdysteroid within the infected host. Bomhyx mori larvae infected with BmEGTZ, a mutant B. mori nucleopolyhedrovirus (BmNPV) in which the egt gene has been inactivated, die more rapidly compared to larvae infected with wild-type BmNPV. In this study, the profile of hemolymph proteins, and progression of virus infection in BmEGTZ- and BmNPV-infected B. mori larvae, was analyzed by SDS-PAGE and histochemically. These analyses showed that virus-encoded and virus-induced proteins were expressed quicker in BmEGTZ-infected larvae than in BmNPV-infected larvae. This suggests that the decrease in time to death, following BmEGTZ infection, results from the stimulation of virus-specific protein expression. In order to examine the effect of ecdysteroid on virus transmission, the profile of hemolymph proteins, and progression of virus infection, were analyzed following an ecdysteroid injection of BmEGTZ- or BmNPV-infected larvae. In the BmNPV-infected larvae, ecdysteroid treatment had no apparent effect on hemolymph protein expression. This suggests that the injected ecdysteroid was inactivated by the BmNPV-expressed ecdysteroid UDP-glucosyltransferase. An Ecdysteroid injection into BmEGTZ-infected larvae increased the speed of virus-specific protein expression and virus transmission. These results suggest that ecdysteroid stimulates protein expression, which in tum results in the stimulation of virus transmission.

검정금파리의 변태기에 따른 엑디스테로이드와 난세포성숙에 미치는 엑디스테로이드의 효과

이종진 한국응용곤충학회 1992 한국응용곤충학회지 Vol.31 No.4

검정금파리의 변태에 따른 엑디스테로이드를 Radioimmunoassay법으로 측정하고, 난세포성숙에 미치는 엑디스테로이드의 효과를 조사하여 얻을 결과는 다음과 같다. 산락직후 존재하였던 난내 엑디스테로이드는 발생과정 중 감소하다가 부화 직전 다시 증가하였으며, 유충기와 용기의 성장 변태시 엑디스테로이드함량의 변화를 보면 유충-유충과 유충-용으로의 탈피시에 일시적인 증가현상을 나타냈다. 특히 용화 후 48시간에 높은 엑디스테로이드의 농도를 보였는데 이는 큐티클분비와 경화작용과 밀접한 관계가 있는 것으로 생각된다. 성충기에서는 수컷의 경우 엑디스테로이드가 거의 검출 되지 않은 반면, 암컷에서는 단백질원 섭식 후 96시간에 최고의 함량을 나타내어 난성숙도와 일치하는 변화를 보였다. 엑디스테로이드 처리와 난성숙도와의 관계를 보면, 1g 처리군은 대조군에서와 같은 성숙도를 나타내 차이를 보이지 않았으나, 5g처리군에서는 대조군에서 보다 12시간 빠르게 난세포성숙이 완료되어, 엑디스테로이드 처리시 임계농도 이상에서는 난세포조기성숙에 직접적인 영향을 미치는 것으로 나타났다. The ecdysteroid titers of representive developmental stages of the blackblow fly, Phormia regina, were determined by radioimmunoassay and the effect of ecdysteroid on the oocyte maturation was investigated. Prior to every molts ecdysteroid levels began to increase sharply, suggesting ecdysteroid was the major component for egg-larval, larval-larval, and larval-pupal transformation. A difference in the levels of ecdysteroid between male and female was observed during adult life span. Following the protein meal, ecdysteroid in the females increased rapidly to a maximum at 96 hr of age when terminal oocyte fully matured. Effect of ecdysteroid on oocyte development was determined for control and ecdysone-treated female flies after the liver-feeding. The growth of oocyte in the flies treated by g of ecdysone, along with the control flies, was not facilitated. When the flies treated by 5 g of ecdysone, however, duration of oocyte maturation was shorter than those of other two groups. This can be suggested that oocyte development in P. regina is due to the critical level of ecdysone.

Ecdysteroid 화합물들의 화학구조 특성에 대한 비교연구

황갑수,Hwang, Gab-Soo 한국환경보건학회 2012 한국환경보건학회지 Vol.38 No.4

Objectives: This study was conducted in order to investigate the specific correlation between physicochemical properties and bioactivity in ecdysteroids found in living organisms. Methods: The examined steroidal compounds were classified into three groups according to their relevance to ecdysone activity. Each compound molecule was completely drawn to automatically calculate its physicochemical parameters and docked against 20-hydroxyecdysone to calculate the total distance. Electronic charge distribution was also observed for each molecule. All procedures were conducted using a computational chemistry program. Results: Ecdysone agonists showed different ranges of parameter values, such as log P, hydrophilic-lipophilic balance (HLB), solubility parameter (SP), hydrophilic surface (HPS), hydrogen bond (HB) and Kappa 2, when compared with antagonists and steroids without ecdysone activity. They also showed a similar electronic charge distribution that is significantly different from the electron charge distribution of antagonists and steroids without ecdysone activity. The total distance values of agonists, estimated by docking them with 20-hydroxyecdysone, were relatively small but showed no correlation with binding affinity with receptor ligand. Conclusions: These results suggest that physicochemical properties such as steric and electronic effects, hydrophobicity and hydrogen bonding may operate in combination to determine the binding activity of ecdysteroids to the receptor protein.

Physiology and Biochemistry Ecdysteroids Stimulate Corpora Allata Activity and Specific Allatotropin mRNA-3 Expression in Early Fifth Instar Larvae of the Tobacco Hornworm, Manduca sexta

Lee, Kyeong-Yeoll Korean Society of Applied Entomology 2003 Journal of Asia-Pacific Entomology Vol.6 No.1

Effects of ecdysteroids on two components of the juvenile hormone (JH) biosynthetic pathway were determined during the early stage of fifth instar larvae of tobacco hornworm, Manduca sexta. When ecdysed fifth instars were fed a diet containing nonsteroidal ecdysteroid agonist, RH-5992, the normal decrease of the JH biosynthetic activity in corpora allata (CA) was prevented until day 2 larvae. In addition,20-hydroxyecdysone (20E) directly stimulated CA activity in a long-term culture for 24 hours, and its effect was not influenced by the simultaneous treatment with M. sexta allatotropin (Manse-AT). The Manse-AT mRNA-3, which is one alternatively spliced mRNA among 3 isoforms of the Manse-AT gene, was exclusively present in the terminal abdominal ganglion (TAG) of the fifth instar larvae(Lee and Horodyski, 2002). The level of Manse-AT RNA-3 was increased in the TAG of the larvae which ingested RH-5992. These results suggest that the levels of hemolymph ecdysteroids exert a tropic activity in CA and the TAG and could be associated with the control mechanism of internal physiological changes, such as the nutrientrestricted condition.

Relationship between Ecdysteroids and Sex Pheromone, 2,6-DCP, of Female Haemaphysalis longicornis (Acari: Ixodidae)

Liu, Jing-Ze,Lee, Hai-Poong,Song, Da-Xiang 한국곤충학회 1999 Entomological Research Vol.29 No.1

The relationship between ecdysteroids (ECs) and the attractant sex pheromone, 2,6-dichlorophenol (2, 6-DCP), in female Haemaphysalis longicornis was investigated by GC and RIA separately. 2, 6-DCP firstly appeared in female foveal gland on d5 after emerged from nymph, then it increased and deposited in foveal gland and reached a peak (10.71 ng/tick) on the day just when capable of feeding. After feeding, 2, 6-DCP declined on d3 and maintained at a relatively stable level from d3 to d5. After mating, 2, 6-DCP decreased significantly and could not be detected on the day of engorgement. A peak of ecdysteroids in female whole body extracts was found on d3 after emerged from nymph. Inoculation of 20-hydroxyecdysone (20-E) on d1 newly emerged female could enhance the level of 2, 6-DCP. The above results suggested that female 2,6-DCP synthesis was regulated by ecdysteroids in this species.

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera)

( Bok Ri Park ),( Yong Gyun Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.6

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site (``AGCTTT``) from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication. [BMB reports 2011; 44(6): 393-398]

17β-estradiol이 기수산 물벼룩의 Ecdysteroid 경로에 미치는 영향

인소연,유제원,조하영,이영미 (사)한국해양생명과학회 2020 한국해양생명과학회지 Vol.5 No.2

17β-estradiol (E2)는 난소로부터 방출되는 호르몬으로 가정 및 축산 오폐수에 포함되어 환경으로 지속적으로 유출된다. E2는 높은 에스트로겐 활성을 가지고 있어 갑각류의 발달과 생식에 영향을 미치는 내분비계교란물질로 알려져 있다. 갑각류의 발달은 탈피호르몬(ecdysteroid)의 신호 전달 과정에 의해 이루어지지만 E2가 소형 갑각류의 탈피호르몬 경로 유전자를 어떻게 조절하는지에 대한 연구는 매우 미흡하다. 본 연구에서는 기수산 물벼룩 Diaphanosoma celebensis에서 E2에 대한 급성 독성 시험을 통해 24-h LCx 값을 도출하였고, E2 노출에 따른 탈피호르몬 경로에 관여하는 7개의 유전자(CYP314a1, EcRA, EcRB, USP, ERR, Vtg, VtgR)의 시간별 발현 변화를 quantitative real time polymerase chain reaction (qRT-PCR)을 이용하여 분석하였다. D.celebensis의 24-h LC50 값은 9.581 mg/l (95% C.I.: 7.697~11.927 mg/l), 24 h-LC10 값은 4.842 mg/l (95% C.I.: 3.683~6.366 mg/l)로 나타났다. CYP314a1, EcRA, USP, VtgR 유전자의 발현이 12시간 또는 24시간에 유의하게 증가하는 양상을 보였다. 이러한 결과는 E2가 D. celebensis의 탈피호르몬 경로에 관련하는 유전자의 발현을 조절함으로써 탈피와 생식에 영향을 미칠 수 있을 것임을 시사한다. 본 연구는 소형 갑각류에서 내분비계교란물질이 탈피 경로에 미치는 영향에 대한 분자 기전을 이해하는데 도움이 될 것이다.