Juvenile hormone alone is insufficient in developmental programing for supernumerary larva under crowded condition in the beetle species Zophobas atratus and Tribolium freemani

A number of tenebrionid beetles undergo continuous larval-larval molting in crowded conditions (LLC). This unique developmental programming, observed in Giant mealworm, Zophobas atratus, and in the flour beetle, Tribolium freemani, delays development into immobile pupal stage which often becomes the victim of cannibalism in crowded condition. In isolated rearing condition, Z. atratus and T. freemani pupate at the end of the 12^th instar or 8^th - 9^th instar, respectively, whereas they continue larval molting in crowded condition.  Juvenile hormone (JH) and its chemical analogue methoprene are each sufficient to induce supernumerary larva in isolated conditions, therefore an increased titer of JH has been proposed as the mechanism in LLC (Nakakita, 1990; Quennedey et al., 1994). We tested this proposal by examining whether the opposite was true; that suppression of JH in crowded conditions allows metamorphosis to pupal stage.  We performed RNA interference (RNAi) to knockdown methyltransferase 3 gene (MT3) which is a crucial enzyme for JH biosynthesis in T. castaneum (Minakuchi et al., 2008). MT3 RNAi induced precocious metamorphosis to pupa in isolated conditions in both Z. atratus and T. freemani. However, in larvae of both species under crowded conditions, MT3 RNAi resulted in larval death instead of pupal molting. We conclude that increased JH is not sufficient, or perhaps not necessary, for LLC.  We suggest that other or additional factor(s) are required for continued larval molting in the crowded condition.