Dietary protein in sufficient quantity is required for post-embryonic development of insects, both molting and reproduction (Simpson et al., 2017). In contrast, the requirements for dietary protein quality (a balance of amino acids that supports growth) are less well understood. In Drosophila, a dietary amino acid composition similar to egg yolk protein speeds juvenile development and increases reproduction (Ma et al., 2020, Piper et al., 2017). Caterpillars (Spodoptera littoralis) slow development and reduce growth upon low-quality protein diets (mostly the corn protein zein) in comparison to high-quality protein diets (mostly the milk protein casein; Lee, 2007). Further, dietary restriction of methionine specifically can extend lifespan in Drosophila when fed low protein quantity diets (Lee et al., 2014).

We recently showed that a high-quality protein supplement, with amino acid composition similar to vitellogenin, increased reproductive output in lubber grasshoppers (Romalea microptera), while also increasing the levels of hexameric storage proteins (Hatle et al., 2022). Vitellogenin (Vg) is the precursor to egg yolk protein in grasshoppers, and it is the functional equivalent to fly yolk proteins. A weakness of our previous paper is that the high-quality diet was designed to match the protein required for reproduction, and the only developmental event we measured was reproduction. Because of this, the previous work does not show whether the diet can support a high-quality protein for a broad range of developmental events. To address this weakness, in the present paper we test whether molt development in juvenile males is supported by a high-quality protein diet that is based on the composition of vitellogenin. Because juvenile development in males does not directly require vitellogenin, we are testing whether this high-quality protein designed to match the composition of vitellogenin generally supports post-embryonic development.

Many developmental transitions (e.g., molting, initiation of reproduction) require attaining a threshold of feeding or storage before committing to the transition (e.g., tadpoles, Denver et al., 1998; dung beetles, Emlen and Nijhout, 1999, Shafiei et al., 2001; damselflies, Nilsson-Örtman & Rowe, 2020; mosquitoes, Telang et al., 2010; and tobacco hornworms, Truman et al., 2006, reviewed in Nijhout et al., 2014; but see an exception in solitary bees, Helm et al., 2017). Ecologically, attaining a threshold before initiating the developmental transition helps ensure that the animal is of sufficient size and condition to succeed during the next stage (e.g., Hill et al., 2018). Most demonstrations of thresholds have identified a critical size or amount of feeding. To our knowledge, no previous study has used amount of feeding on high-quality protein as a threshold. Another possible manifestation of a threshold is measuring current nutrient stores as a proxy for total feeding. A few studies have shown that the accumulation of hexameric storage protein (Hahn et al., 2008, Hatle et al., 2001, Hatle et al., 2022) can be associated with a developmental threshold.

Lubber grasshoppers are a good system for studying thresholds and storage in juvenile development, because they are easy to rear individually and large enough to allow collection of repeated hemolymph samples. For example, the middle instars showed the greatest plasticity in developmental time (Flanagin et al., 2000). Within the last juvenile instar, using abrupt switches in food availability (from ad libitum lettuce to low-quantity lettuce), 5th instar males attained a threshold by 8 days, before molting to adult at 15 days (Hatle et al., 2003b). During this 5th (last juvenile) instar, about half of the adult mass is gained.

Here, we test the effects of a range of dietary protein qualities based on vitellogenin on male development (i.e., duration of the 5th instar), mass gain during the entire instar, and accumulation of storage proteins. Our broad hypothesis is that diets with amino acid compositions matched to vitellogenin will support post-embryonic development in general (i.e., serve as a high-quality protein). Specifically, we predict that diets with an amino acid composition dissimilar to vitellogenin will slow development, reduce mass gained during the instar, and delay the accumulation of hemolymph storage proteins (i.e., serve as a low-quality protein). Next, because high methionine diets are known to be detrimental to health (e.g., Hine et al., 2015), we expect lowering methionine levels in otherwise low-quality diets will rescue developmental speed and mass gain. Last and most important, we predict that low-quality dietary protein will slow development and hexameric storage protein accumulation most strongly during the early part of the instar, indicating that dietary protein quality underlies a threshold for development.