Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

Abstract

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first-or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JHindependent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broadinducing factor, i.e., a competence factor. juvenile hormone | metamorphosis | ecdysone | TALEN | Bombyx

Juvenile hormone signaling – a mini review

“…Moreover, null mutations of JH biosynthesis and JH receptor genes show precocious metamorphosis at the 3rd or 4th instar at the earliest. The authors suggest that the lack of competence for metamorphosis during the early larval instars may result from the absence of expression of the pupal specifier gene, Br (Daimon et al ., ; Daimon et al ., ). However, more recent experiments in B .…”

Resources and Recommendations for Using Transcriptomics to Address Grand Challenges in Comparative Biology

“…The goal is to develop N. denticulata into a tractable genetic model through transgenics and mutation screens. Methods used successfully in insects and amphipod crustaceans, such as CRISPR/Cas9, TALEN, and RNAi with tissue-specific expression constructs, may be applied to other crustaceans species (Gaj et al 2013; Daimon et al 2015; Enya et al 2015; Li et al 2015c; Martin et al 2016; Valzania et al 2016). Neocaridina denticulata has attributes that are essential for transgenic experiments: (1) they are freshwater animals that live in a wide range of temperatures and water chemistries; (2) relatively short intergenerational time (4–5 months); (3) small size for housing thousands of individuals in limited space; (4) fertilized eggs are relatively large and are brooded externally, facilitating the harvest of eggs for microinjection of gene constructs; (5) direct embryonic development; and (6) the cuticle is thin and transparent, which allows for observation of molt stages, gonad development, and tissue expression of reporter genes, such as red fluorescent protein, in live animals.…”

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

“…Daimon et al . () showed that embryonic growth and morphogenesis are largely independent of JHs, and precocious metamorphosis is induced at the earliest after the 2nd instar. They also suggested that an unidentified factor or signal is required to acquire the competence for metamorphosis.…”

“…However, this long-held paradigm was challenged when knockout B. mori with null mutations in JH biosynthesis or JH receptor genes were generated and characterized. Daimon et al (2015) showed that embryonic growth and morphogenesis are largely independent of JHs, and precocious metamorphosis is induced at the earliest after the 2nd instar. They also suggested that an unidentified factor or signal is required to acquire the competence for metamorphosis.…”