Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose

Abstract

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

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

“…Relative mRNA expression levels normalized against rp49 are shown. As shown in a previous study (6), the Kr-h1 expression levels were lower in the mod strain than in the control. Note the very low levels of Kr-h1 mRNA in JHAMT, JHAMT mod, Met1, and Met2 Met1 mutants.…”

“…This clear difference between L2 and L3 larvae may reflect a physiological difference between the two instars. In this study, we obtained definitive evidence supporting the hypothesis that there are two phases in the life of both holometabolous and hemimetabolous larvae (6)(7)(8): (i) a JH-independent phase (L1 and L2) in which JH does not have an important function and (ii) a JH-dependent phase (L3 and thereafter) in which the antimetamorphic action of JH is required to prolong the larval stage until the attainment of appropriate size for metamorphosis.…”

“…This analysis provides evidence that larval status is maintained independently of JH signaling during L1 and L2. As proposed by Smykal et al (6), our results may suggest that the competence for metamorphosis is acquired gradually during larval development, given that L3 tissues were much more sensitive than L2 tissues to the absence of JH signaling (Fig. S5).…”

“…It is widely considered that this "status quo" action of JH is required to maintain the larval status throughout the larval stage. Interestingly, however, several attempts to deplete JHs using classic and modern techniques have failed to induce precocious metamorphosis during the very early larval instars (6)(7)(8)(9)(10)(11)(12).…”

“…In addition, the dimolting (mod) mutant larvae of Bombyx with a null mutation in a JH biosynthetic enzyme, JH epoxidase CYP15C1, metamorphosed into miniature pupae after the third or fourth instar (7). Similarly, RNAi of a JH receptor Methoprene tolerant (Met) or Krüppel-homolog 1 (Kr-h1), a repressor of pupal metamorphosis, in the early instars of a hemimetabolous Pyrrhocoris bug did not cause precocious metamorphosis until after the third instar (6). In addition, in the fruit fly Drosophila melanogaster, deprivation of the CA or double mutation of two paralogous JH receptor genes, Met and germ cell expressed (gce), did not reduce the number of molts (9,10).…”