title: Neverland regulates embryonic moltings through the regulation of ecdysteroid synthesis in the water flea Daphnia magna, and may thus act as a target for chemical disruption of molting.
abstract: Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Basic Biology, Faculty of Life Science, SOKENDAI, (Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.|Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Sumiya
authors.affiliation: Department of Basic Biology, Faculty of Life Science, SOKENDAI, (Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.|Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Ogino
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Toyota
authors.affiliation: Center for Bioscience Research and Education, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi, 321-8505, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Miyakawa
authors.affiliation: Department of Basic Biology, Faculty of Life Science, SOKENDAI, (Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.|Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Miyagawa
authors.affiliation: Department of Basic Biology, Faculty of Life Science, SOKENDAI, (Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. taisen@hotmail.co.jp.|Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. taisen@hotmail.co.jp.
authors.forename: Taisen
authors.initials: T
authors.lastname: Iguchi
mesh_terms: D000818:Animals; D003621:Daphnia / Q000187:drug effects / Q000254:growth & development*; D026461:Ecdysteroids / Q000096:biosynthesis* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics*; D018978:Molting / Q000187:drug effects / Q000235:genetics*
chemical_list: D026461:Ecdysteroids; D019476:Insect Proteins
keywords: Daphnia magna; Neverland; ecdysteroid synthesis; ecdysteroids; embryogenesis