Abstract
The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.
Highlights
Two endocrine hormones, ecdysteroid and sesquiterpenoid, play important roles in arthropod physiology, development, and phenotypic plasticity [1,2,3,4,5]
We performed a TBLASTN search using the amino-acid sequences of Spo and Juvenile hormone acid o-methyltransferase (Jhamt) obtained from several insect species against the D. magna genome database
Because sesquiterpenoids are known as “status quo” hormones in insects, we examined the timing of molting after the end of embryogenesis. siRNA-Jhamt-injected embryos showed a significant reduction during the embryonic period compared with the control (Fig 3A; siJhamt), suggesting that sesquiterpenoid biosynthesis represses the precocious transition of embryos into the juvenile stage
Summary
Ecdysteroid and sesquiterpenoid, play important roles in arthropod physiology, development, and phenotypic plasticity [1,2,3,4,5]. The conversion of cholesterol to active ecdysteroids (ecdysteroidogenesis) requires a series of hydroxylation reactions involving multiple enzymes, mostly of the P450 families, including Noppera-bo (Nobo), Neverland (Nvd), Non-molting glossy/shroud (Sro), Spook (Spo), Spookier (Spok), Cyp6t3, Phantom (Phm), Disembodied (Dib), Shadow (Sad), and Shade (Shd) [13]. Genes encoding these enzymes (except Nvd, Cyp6t3, and Spok) are collectively termed as the Halloween genes [13,14,15]. Mutants of these genes exhibit common phenotypes that result from abnormally low ecdysteroid titers, including a poorly differentiated embryonic cuticle, dorsal closure failure, defective midgut morphogenesis, and eventually embryonic lethality [8, 16, 17]
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