title: A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.
abstract: The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods.
authors.affiliation: Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile.
authors.forename: Constanza C
authors.initials: CC
authors.lastname: Macaya
authors.affiliation: Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile.
authors.forename: Patricio E
authors.initials: PE
authors.lastname: Saavedra
authors.affiliation: Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile.
authors.forename: Rodrigo E
authors.initials: RE
authors.lastname: Cepeda
authors.affiliation: Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile.
authors.forename: Viviana A
authors.initials: VA
authors.lastname: Nuñez
authors.affiliation: Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile. andres.sarrazin@pucv.cl.
authors.forename: Andres F
authors.initials: AF
authors.lastname: Sarrazin
mesh_terms: D000818:Animals; D046508:Culture Techniques; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000187:drug effects; D017403:In Situ Hybridization; D023421:Models, Animal; D015739:Nocodazole / Q000494:pharmacology; D014231:Tribolium / Q000187:drug effects / Q000254:growth & development*
chemical_list: D015739:Nocodazole; D004121:Dimethyl Sulfoxide
keywords: Arthropod development; Chemical genetics; Pharmacological manipulation; Tribolium castaneum; Whole embryo culture