28Although the mechanisms that control growth are now well understood, the mechanism by which 29 animals assess their body size remains one of the great puzzles in biology. The final larval instar 30 of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a 31 threshold size. We investigated the mechanism of threshold size assessment in the tobacco 32 hornworm, Manduca sexta. The threshold size was found to change depending on the amount of 33 exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower 34 threshold size. Under these various conditions, the mass of the muscles plus integuments was 35 correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at 36 the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. 37 castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage 38 permanently even after passing the threshold size. We propose that increasing levels of Myo 39 produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis 40 at the threshold size. 41 42 43 44 Keywords 45 Myoglianin; threshold size; body size; muscle growth; Manduca sexta 46 BACKGROUND 47

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