Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum.
Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA. Electronic address: sujata.chaudhari@unmc.edu.
Sujata S
SS
Chaudhari
Department of Applied Biology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Mi Young
MY
Noh
Interfaculty Institute for Cell Biology, Animal Genetics, University of Tübingen, Tübingen, Germany.
Bernard
B
Moussian
Department of Medicine, University of Massachusetts, Worcester, MA 01605, USA.
Charles A
CA
Specht
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Karl J
KJ
Kramer
Department of Entomology, Kansas State University, 123 W. Waters Hall, Manhattan, KS 66506, USA.
Richard W
RW
Beeman
Department of Applied Biology, Chonnam National University, Gwangju 500-757, Republic of Korea. Electronic address: arakane@chonnam.ac.kr.
Yasuyuki
Y
Arakane
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Subbaratnam
S
Muthukrishnan
D000818:Animals; D005260:Female; D019476:Insect Proteins / Q000378:metabolism*; D034622:RNA Interference; D014231:Tribolium / Q000196:embryology* / Q000378:metabolism
D019476:Insect Proteins
Chitin; Chitin synthase; Knickkopf; RNA interference (RNAi); Retroactive; Serosal cuticle