Taxonomic and functional traits shape insect macronutrient regulation

Taxonomic and functional traits shape insect macronutrient regulation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 19 November 2025 V1 Latest version Share on Taxonomic and functional traits shape insect macronutrient regulation Authors : Christopher Brennan 0000-0003-2511-5720 and Spencer Behmer 0000-0002-5038-1046 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176357172.25769373/v1 244 views 128 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Nutrient regulation shapes fitness-related traits including growth, survival, and reproduction. Using the Nutritional Geometry Framework (NGF), we investigated how insects regulate intake of protein (P) and carbohydrates (C). To illustrate this approach, we used Schistocerca americana and found that nymphs regulated intake toward a balanced P:C target and exhibited reduced performance on imbalanced diets. We then synthesized data from 67 studies across six insect Orders to identify patterns. Most species regulated toward carbohydrate-biased or balanced intake targets, with protein-biased regulation rare. Intake strategies clustered by taxonomic Order and varied with functional traits. Herbivores selected more protein-biased targets than omnivores. Among herbivores, specialists showed narrower intake distributions than generalists, while among omnivores, scavengers were more carbohydrate-biased than predators. Immature insects favored more protein-rich diets than adults. Together, these findings highlight two key insights: performance depends on balancing both protein and carbohydrate intake, and evolutionary history and ecological traits shape nutrient regulation. Statement of Authorship: STB and CB conceived of the study idea. CB collected and analyzed the data. CB wrote the first draft. CB and STB edited subsequent drafts. Data accessibility statement: All supporting data and code is available from figshare DOI:10.6084/m9.figshare.29371910 Article Title: Taxonomic and functional traits shape insect macronutrient regulation Names and Affiliations: Christopher Brennan 1 and Spencer T. Behmer 1,2 1 Ecology and Evolutionary Biology PhD Program, Texas A&M University 2 Department of Entomology, Texas A&M University Running Title : Taxonomy and Traits Shape Insect Nutrition Keywords: Nutritional Geometry, Nutritional ecology, Nutrient balancing, Foraging behavior, Comparative analysis, Feeding Guilds Type of Article: Letters Word count (Abstract): 150 Word count (Manuscript): 5000 Number of references: 145 Number of figures: 5 Number of tables: 1 Name and address of correspondence: Christopher Brennan, [email protected] ABSTRACT Nutrient regulation shapes fitness-related traits including growth, survival, and reproduction. Using the Nutritional Geometry Framework (NGF), we investigated how insects regulate intake of protein (P) and carbohydrates (C). To illustrate this approach, we used Schistocerca americana and found that nymphs regulated intake toward a balanced P:C target and exhibited reduced performance on imbalanced diets. We then synthesized data from 67 studies across six insect Orders to identify patterns. Most species regulated toward carbohydrate-biased or balanced intake targets, with protein-biased regulation rare. Intake strategies clustered by taxonomic Order and varied with functional traits. Herbivores selected more protein-biased targets than omnivores. Among herbivores, specialists showed narrower intake distributions than generalists, while among omnivores, scavengers were more carbohydrate-biased than predators. Immature insects favored more protein-rich diets than adults. Together, these findings highlight two key insights: performance depends on balancing both protein and carbohydrate intake, and evolutionary history and ecological traits shape nutrient regulation. INTRODUCTION Animals require a suite of nutrients to support survival, growth, and reproduction (Karasov & del Rio 2007; Simpson & Raubenheimer 2012). These include both macronutrients (proteins, carbohydrates, and lipids) and micronutrients (vitamins and minerals) (Wu 2017; 2026). Among these, macronutrients are especially critical, as they provide the structural and energetic foundation of animal physiology. Consequently, research has focused on understanding how animals regulate their intake of macronutrients, particularly protein and digestible carbohydrates – the two primary nutrients underpinning growth and energetic needs (Simpson & Raubenheimer 1993; Behmer 2009; Raubenheimer et al. 2009). Because foods, especially plants, differ widely in their nutrients (Slansky et al. 1987; Sterner & Elser 2002; Schoonhoven et al. 2005; Karasov & del Rio 2007), animals must make complex foraging decisions to maintain nutritional balance and maximize fitness (Houston et al. 2011). Yet, do all animals regulate macronutrients in the same way? And if not, what factors explain variation in regulation strategies? Several approaches exist to study nutrient regulation (Raubenheimer & Simpson 2018; Demi et al. 2021; Deans & Behmer 2026), with the Nutritional Geometry Framework (NGF) being among the most widely used. This bottom-up method examines how animals regulate multiple nutrients simultaneously by offering them two nutritionally complementary diets (Raubenheimer & Simpson 1993; Raubenheimer & Simpson 1999; Behmer 2009). Through selective consumption of nutritionally complementary food, individuals can regulate intake to converge on an optimal nutrient balance – termed the intake target . The NGF can also be applied under constrained conditions, where animals are restricted to a single imbalanced diet. In such situations, individuals cannot reach their intake target and are forced to adopt a rule of compromise : they either overconsume one nutrient to obtain enough of another, or undereat one nutrient to avoid excess of the other. These compromises reveal how species prioritize certain nutrients over others, depending on which imbalance imposes the greater fitness cost (Simpson & Raubenheimer 1993; Behmer 2009). To date, most studies have focused on protein and digestible carbohydrates. Protein, which provides key amino acids, is essential for tissue growth, enzyme function, and reproduction (Wu et al. 2014), while carbohydrates are a primary source of metabolic energy required for movement, cellular function, and sustaining physiological processes (Chown & Nicolson 2004; Harrison et al. 2012). Though first applied to locusts – Locusta migratoria (Chambers et al. 1995) and Schistocerca gregaria (Simpson et al. 2002) – the NGF has since been applied to a wide range of organisms, including invertebrates, fish, birds, and mammals, including humans (Simpson & Raubenheimer 2020), demonstrating its broad applicability. While the NGF has been applied across diverse taxa, insects remain its primary focus (Cheng et al. 2008; Behmer 2009; Simpson et al. 2015). Most nutritional ecology research has focused on protein and carbohydrates, as these macronutrients are especially important for herbivorous insects (Behmer 2009; Deans et al. 2016, Tessnow et al. 2018). Although lipids can be equally important – particularly for predaceouse species or pollen-feeding insects – they have been studied less extensively, creating a research bias toward protein-carbohydrate regulation. The relative and total amounts of protein and carbohyrates can profoundly influence insect performance and fitness (Behmer & Joern 2008; Roeder & Behmer 2014; Deans et al. 2015; Simpson et al. 2015). To maintain nutritional balance, many insects feed on a variety of food sources that differ in protein-to-carbohydrate ratios, thereby regulating their intake through behavioral responses to nutrient availability (Bernays et al. 1994; Unsicker et al. 2008; Simpson & Raubenheimer 2012). Understanding nutrient regulation in insects is especially important given their taxonomic diversity and ecological roles (Scudder 2017). Their variation in nutrient niches, feeding behaviours, and physiologies (Behmer & Joern, 2008; Foriester et al. 2014; Machovshey-Capuska et al. 2016) also makes them excellent models for uncovering generalizable and occasionally counterintuitive trends in nutrient regulation. Insects can be broadly classified by taxonomy (e.g., Order), but nutrient regulation is likely shaped by finer-scale variation in functional traits (Chase & Leibold 2003; Grosdidier et al. 2025). Functional traits – defined as physiological, morphological, or phenological characteristics that influence an organism’s fitness or performance (Violle et al. 2007; Moretti et al. 2016; Brousseau et al. 2018; Wong et al. 2018; Junker et al. 2023; Schleuning et al. 2023) – include feeding guild, developmental strategy, and life stage. Among herbivores, some species are dietary specialists (monophagous or oligophagous), while others are generalists (polyphagous) (Schoonhoven et al. 2005). Similarly, omnivores can be categorized as facultative scavengers, which consume plant material and dead prey, or as predators, which feed on live plants and live prey (Price et al. 2011). Developmental strategy may also influence nutrient regulation. Hemimetabolous insects feed continuously across life stages and typically consume similar foods as both nymphs and adults. In contrast, holometabolous insects undergo complete metamorphosis, including a non-feeding pupal stage that separates the larval and adult phases (Nestel et al. 2016; Jindra 2019). As a result, larvae must accumulate sufficient nutrients to support not only growth but also the energy demands of metamorphosis and adult emergence. At the individual level, nutrient demands shift throughout development: protein is typically prioritized during juvenile stages to support tissue growth, while adults rely more on carbohydrates to fuel energetically demanding activities such as flight (Chapman 2012; Harrison et al. 2012). Although adults exhibit limited growth, protein remains important – particularly for somatic maintenance, immunity, and female reproduction (Schwenke et al. 2017; Macartney et al. 2022) Building on this framework of functional traits, our study takes a two-pronged approach to better understand insect nutrient regulation. First, as an empirical example, we applied the NGF to the generalist grasshopper Schistocerca americana to investigate how individuals regulate protein and carbohydrate intake under choice and no-choice conditions. The choice experiment allowed us to assess the extent to which this species actively regulates its nutrient intake when presented with two nutritionally suboptimal but complementary foods. In contrast, the no-choice experiment revealed how this species responds to: (1) varying protein-carbohydrate imbalances and (2) differences in total macronutrient content. It also demonstrated the physiological consequences of consuming diets with differing protein-carbohydrate ratios and concentrations. Second, we conducted a literature review to identify broad patterns of protein-carbohydrate regulation across insect taxa and functional groups. Analyzing 59 peer-reviewed studies that reported self-selected intake targets across seven insect Orders, we found that protein-biased intake targets were relatively uncommon. Instead, most species regulated toward balanced or carbohydrate-biased intake, with targets clustering within Orders but diverging across them. Intake targets also varied by functional traits. Herbivores selected more protein-biased intake targets than omnivores, and within each guild, foraging strategy (specialist vs. generalist, predaceous vs. scavenging) further shaped intake. Immature insects favored more protein-rich diets than adults. These patterns reveal how nutrient regulation strategies reflect both evolutionary history and ecologically relevant traits, with implications for insect nutrition, foraging behaviour, and the broader ecological interactions. MATERIALS AND METHODS (1) Applying the Nutritional Geometry Framework: Protein-Carbohydrate Regulation in a Generalist Grasshopper Insect Rearing Final-instar Schistocerca americana (Drury) were obtained from a colony reared on seedling wheat and wheat germ at Texas A&M University. Immediately upon molting nymphs were sexed, weighed, and placed in arenas (29-31°C) supplied with food dishes and water. Each treatment had 10 replicates. Synthetic Diets Seven artificial granular diets were prepared following Behmer et al. (2003) and varied in protein (p) and digestible carbohydrate (c) composition on a dry mass basis. Five diets (42% total macronutrient) were generated: (1) p7:c35, (2) p14:c28, (3) p21:c21, (4) p28:c14, and (5) p35:c7. Two additional diets – (6) p7:c7 and (7) p35:c35 differed in macronutrient concentration (14% and 70%, respectively). Cellulose acted as an inert bulk material and was adjusted inversely with total macronutrient content: (54% 82%, and 26% cellulose for the 42%, 14%, and 70% P+ C diets, respectively). The remaining 4% was a mixture of salts, vitamins, and sterols. Experiment Protocol Two experiments were conducted. In choice assays, nymphs selected between paired suboptimal but complementary diets (p7:c35 vs p35:c7; p7:c35 vs p28:c14; p14:c28 vs p35:c7). In no-choice assays, individuals were restricted to one of seven diets (14-70% total macronutrient: p7:c7, p7:c35, p14:c28, p21:c21, p28:c14, p35:c7, or p35:c35. Food was replaced every 72 h, with intake measured gravimetrically after equilibration to ambient humidity. Feeding continued until a molt, when individuals were re-weighed to calculate performance metrics. (2) Review of Insect Protein-Carbohydrate Intake Targets Literature search and data extraction We searched Google Scholar, Wiley Online Library, and ScienceDirect for studies applying the NGF to insects using paired-food choice designs. To be included, studies had to (1) examine insects (Class Insecta), (2) measure simultaneous intake of protein and digestible carbohydrates, (3) use a paired-food choice design providing continuous access to two nutritionally complementary but individually suboptimal foods. Studies involving experimental manipulations condition – such as parasitism (Shik et al. 2018; Thompson and Redak 2005) or toxin exposure (Behmer et al. 2002; Deans et al. 2017) – were excluded. When intake targets were reported separately by sex, values were averaged. The final data set included 69 insect species from 67 studies (Supplementary Table 1). If intake targets were tabulated, reported means were used; when presented graphically, protein and carbohydrate coordinates were extracted digitally. For species with data across instars, cumulative consumption was analyzed. All intake targets were expressed as log-transformed P:C ratios to standardize comparisons. Exploratory Variables We analyzed P:C intake targets across four categorical variables, with the number of species in each category shown in parentheses: (i) Taxonomy – Blattodea (7), Coleoptera (5), Diptera (9), Hymenoptera (8), Lepidoptera (13), and Orthoptera (23). Orthoptera was further divided into the suborders Ensifera (crickets and katydids; 9) and Caelifera (grasshoppers and locusts; 14); (ii) Consumer type – Herbivores (34) vs. omnivores (31) . Herbivores were further divided into specialist (feeding on one or a few host plants; 6) and generalist (feeding on diverse plant taxa; 28) while omnivores were subdivided into predaceous (feeding on live prey; 14) and scavenging (consuming detritus, carrion, or decaying organic matter; 17). If a species could be classified into multiple categories, it was assigned based on its primary feeding behavior; (iii) Life stage - Immature (37) vs. adult (28); (iv) Metamorphosis type – hemimetabolous (33) vs holometabolous (32). (3) Data Analysis For the S. americana choice experiment, paired t-tests assessed feeding preferences between foods (null hypothesis: equal consumption), and MANOVA tested consistency of P:C targets among diet pairings. Additional ANOVAs evaluated differences among treatments in total consumption, stadium duration, and mass gain. For no-choice experiment, total consumption was first compared across all seven diets using ANCOVA. We then conducted three complementary analyses to examine nutrient regulation and performance. First, the rule of compromise (Simpson & Raubenheimer 1993; Behmer 2009) was assessed by comparing Euclidean distances from the intake target across the five 42% macronutrient diets differing in P:C ratio using ANCOVA. Second, we evaluated whether grasshoppers achieved the same P:C intake target across the three 1:1 diets (p7:c7, p21:c21, p35:c35), which varied in total macronutrient concentration, also using ANCOVA on Euclidean distances to the intake target. Third, efficiency of conversion of ingested food (E.C.I. = [mass gain / food intake] x 100; Waldbauer 1968) was analyzed sequentially across: (a) the 42% macronutrient diets varying in P:C ratio, (b) the three 1:1 diets (p7:c7, p21:c21, p35:c35) differing in total macronutrient concentration, and (c) the two 35%-protein diets differing in carbohydrate content. Starting wet mass was included as a covariate to control for sex-related size differences. When treatment effects with significant, differences among groups were identified using Tukey’s post-hoc tests followed significant effects. Outliers (defined as over 2 standard deviations from the mean log-transformed P:C) were excluded (n=2). Analytical procedures followed standard approaches used in NGF studies (Simpson & Raubenheimer 1993; Behmer 2009; Raubenheimer & Simpson 2018). In the comparative literature analysis, ANOVAs tested for differences in log-transformed P:C ratios among taxonomic Orders and functional traits, with Tukey’s post-hoc tests for pairwise contrasts. Distributional variability was assessed using bootstrap resampling of kurtosis (1,000 iterations with replacement), followed by Wilcoxon rank-sum tests to compare variance among groups. All analyses were conducted in R (version 4.2.0). Outliers (> 2 SD from Order means) and the single Hemipteran data point were excluded from the final dataset. Three species were removed as outliers: Conocephalus spartinae (Orthoptera), Spodoptera litura (Lepidoptera), and Apis mellifera scutellate (Hymenoptera). (1) Applying the Nutritional Geometry Framework: Protein-Carbohydrate Regulation in a Generalist Grasshopper We used two distinct methods to assess whether 6 th -instar S. americana nymphs regulate their protein-carbohydrate (P:C) intake. First, we tested for non-random feeding. When given a choice between two nutritionally suboptimal but complementary foods, nymphs exhibited non-random feeding in two of the three food-pairing treatments (14:28 with 35:7: t (9) =-2.35, P=0.043 ; 7:35 with 28:14: t (8) =2.93, P=0.019 ; 7:35 with 35:7: t (8) =-0.86, P=0.416 ). Second, we examined whether they defended a specific intake target. As shown in Figure 1A, nymphs regulated their protein-carbohydrate intake, maintaining a 1:1 ratio across all food-pairings (MANOVA: F 2,25 =0.790, P= 0.537). Consistent with this tight regulation, there were no statistical differences in total consumption, dry mass gain, or stadium duration among treatments (Table 1; Figure 1A). Figure 1B shows a bicoordinate plot of protein and carbohydrate consumption by nymphs offered diets with varying P:C ratios. Unlike the choice experiment, total consumption was significantly affected by diet composition (ANCOVA: F 6,54 =50.74, P< 0.001). Nymphs restricted to p7:c7 consumed significantly more food than those in all other treatments (Figure 1B). Among the 42% macronutrient diets, total intake followed a linear trend. Although P:C intake on 1:1 diets (p7:c7, p21:c21, and p35:c35) can overlap as they share a nutrient rail, locusts on each diet regulated to a distinct point in nutrient space (ANCOVA: F 2,22 =42.53, P<0.001 ). We also examined the efficiency with which ingested food was converted into body mass (E.C.I.) across diets. When nutrient density was consistent (p+c=42%) but P:C ratio varied (p7:c35, p14:c28, p21:c21, p28:c14, p35:c7), E.C.I. differed significantly among treatments (ANCOVA: F 4,39 =13.54, P< 0.001). Efficiency was highest when dietary P:C matched or closely bracketed the self-selected intake target (Figure 2A), and lowest on the most imbalanced diets (p7:c35 and p35:c7), which did not differ significantly despite contrasting protein-carbohydrate content. When foods had the same P:C ratio but different nutrient densities (14%, 42%, 70%), E.C.I increased with nutrient density (ANCOVA: F 2,22 =193.88, P< 0.001; Figure 2A). Finally, among treatments with equivalent protein content (35%) but varying carbohydrate levels, E.C.I. differed significantly (ANCOVA: F 1,12 =80.35, P <0.001), indicating carbohydrate availability also shaped growth efficiency (Figure 2C). Together, these results highlight the importance of regulating both macronutrients for optimal performance. (2) Review of Insect Protein-Carbohydrate Intake Targets Taxonomy Insect taxonomy, defined here at the insect Order level, had a significant effect on the self-selected P:C intake target (ANOVA: F 5,59 =32.74, P< 0.001; Figure 3). On average, lepidopterans (1.2:1) and coleopterans (1.1:1) selected slightly protein-biased diets and did not differ significantly. In contrast, orthopterans (1:1.4), hymenopterans (1:1.5), blattodeans (1:3.2), and dipterans (1:4.1) selected carbohydrate-biased intake targets. The P:C ratios of coleopterans, orthopterans, and hymenopterans were similar. However, blattodeans and dipterans selected significantly more carbohydrate-biased diets than the other Orders, with no statistical difference between them. Among the six insect Orders, orthopterans exhibited the widest range in self-selected P:C intake targets. This variation may reflect differences between its two sub-orders: Ensifera (crickets, katydids, and allies), which are omnivorous, and Caelifera (grasshoppers and allies), which are primarily herbivorous. When Orthoptera was subdivided accordingly, we found a significant difference in their P:C intake targets (ANOVA: F 1,21 =10.01, P= 0.005). Caeliferans regulated to a near-balanced intake (1:1.1) whereas ensiferans selected a more carbohydrate-biased intake (1:1.8). Consumer Type Insects span a wide range of feeding guilds, playing key ecological roles across ecosystems (Scutter 2017; Cornwallis et al. 2023). To explore how feeding strategy influences nutrient regulation, we compared the P:C intake targets of herbivorous and omnivorous insects (Figure 4A). On average, herbivores selected more protein-biased diets than omnivores (ANOVA: F 1,63 =13.51, P< 0.001). Further subdivision of these feeding categories revealed additional patterns. Among herbivores, specialists (1:1.1) and generalists (1:1.2) had similar P:C intake targets (ANOVA: F 1,32 =0.11, P= 0.744). However, kurtosis analysis showed that specialists exhibited a significantly narrower range of P:C ratios than generalists (Wilcoxen rank-sum test: W=1380, P <0.001; Figure 4B). Among omnivores, predatory and scavenging omnivores differed significantly, with the latter showing a stronger carbohydrate bias (ANOVA: F 1,29 =5.97, P=0.021 ; Figure 4C). Additionally, predatory omnivores showed less variation in P:C ratios than scavengers (Wilcoxen rank-sum test: W=8197, P <0.001; Figure 4C). Life Stage and Metamorphosis Type We found a significant difference in the self-selected P:C intake targets between immature and adult insects (ANOVA: F 1,63 =26.42, P< 0.001). On average, adults selected more carbohydrate-biased diets (1:2.3) compared to immatures (1:1.2; Figure 5A). In contrast, metamorphosis type had no effect as hemimetabolous (1:1.6) and holometabolous (1:1.5) insects did not differ significantly (ANOVA: F 1,63 =0.07, P= 0.800; Figure 5B). DISCUSSION This study combined experimental and comparative approaches to examine how insects regulate their intake of protein and carbohydrate – two macronutrients essential for growth, survival, and reproduction. As an illustrative example, we used final-instar Schistocerca americana to demonstrate how the Nutritional Geometry Framework (NGF) can be applied to understand nutrient regulation and its consequences for performance across nutritional environments. Through both choice and no-choice experiments, we showed that S. americana nymphs actively defend a specific intake target (Figure 1A) and experience reduced performance when forced onto imbalanced diets (Figure 2). These results underscore the physiological costs of deviating from an optimal nutrient balance, even when protein is abundant. This application of the NGF highlights how insect feeding strategies and performance outcomes can be mapped under variable nutritional conditions. Expanding beyond S. americana , our synthesis of 67 studies revealed two key findings: 1) most insects regulated towards carbohydrate-biased or balanced diets, and 2) intake targets showed consistent, taxon-specific patterns. These targets clustered within insect Order but diverged significantly among them (Figure 3), suggesting that evolutionary history strongly shapes nutrient regulation strategies. Functional traits also played a role. Herbivores tended to regulate towards balanced P:C ratios, with generalists exhibiting broader intake ranges than specialists (Figure 4). Among omnivores, predaceous species maintained near-balanced targets, while scavengers were carbohydrate-biased despite having access to protein-rich diets. Development stage further influenced intake preferences. Immatures selected more protein-rich diets, while adults prioritized carbohydrates (Figure 5). Together, these patterns highlight how evolutionary lineage and functional traits contribute to insect nutritional strategies. To complement the comparative patterns observed across taxa, we used S . americana to illustrate how NGF experiments can uncover the nutrient regulation strategies of individual species and the performance consequences of nutritional imbalance. Our choice experiments revealed that final-instar nymphs maintain tight nutritional homeostasis, regulating both the total quantity and relative balance of protein and carbohydrate intake. Despite variation in nutrient availability across diet pairs, individuals converged on a consistent intake target (P:C ~ 1:1.1), resulting in similar performance across treatments in terms of growth, development time, and conversion efficiency. These findings are consistent with previous studies on orthopterans (Simpson et al 2002; Behmer 2009; Le Gall & Behmer 2014; Cease 2024) and highlights the NGF’s value for quantifying intake targets under ecologically relevant conditions (Raubenheimer & Simpson 2003; Raubenheimer et al. 2009; Kearney et al. 2010; Le Gall et al. 2019). When S. americana nymphs were restricted to a single diet – when self-selection was not possible – clear rules of compromise emerged. As seen in other insects, performance declined on nutritionally suboptimal diets, either due to imbalanced P:C ratios or low total macronutrient content (Raubenheimer & Simpson 2003; Lee 2007; Deans et al. 2022). The distribution of intakes for the 42% macronutrient diets was approximately linear indicating that locusts are nutrient maximizers, following the equal distance rule (Simpson & Raubenheimer 2012), a strategy shared by many generalist feeders (Raubenheimer & Simpson 1997; Simpson & Raubenheimer 1999; Lee et al. 2002, Merx-Jacques et al. 2008; Krabbe et al. 2019). Although total consumption remained similar across most treatments, nutrient utilization efficiency (E.C.I.) varied substantially. Grasshoppers achieved the highest conversion efficiency on diets closest to their self-selected intake target, with reduced efficiency observed on the most imbalanced diets – highlighting the physiological importance of aligning intake with nutritional demands (Simpson et al. 2015). When fed dilute diets (e.g., p7:c7), nymphs increased consumption to compensate, but this strategy proved ineffective as they failed to achieve a similar macronutrient intake. Despite optimal P:C proportions, nutrient dilution led to reduced mass gain and lower E.C.I., a pattern also documented in caterpillars (Simpson & Simpson 1990; Lee et al. 2004; Lee 2007; Couture et al. 2016). Conversely, individuals on high-concentration diets (e.g., p35:c35) exhibited elevated conversion efficiency, underscoring the importance of both nutrient ratio and total nutrient availability. Beyond patterns of intake regulation and compromise, our results offer key insights into which macronutrients constrain performance. Although protein is essential for growth, our findings indicate it is not the sole limiting nutrient for insects, especially herbivores (Mattson & Scriber 1987; White 1993; Fagan et al. 2002; Behmer & Joern 2012; Hansen et al. 2020). When protein levels were held constant (e.g., at 35%) but carbohydrate increased (from 7% to 35%) conversion efficiency improved substantially – indicating that energy from digestible carbohydrates was critical to realizing the growth potential supported by protein. This directly supports the emerging view that insects must regulate both protein and carbohydrates to achieve optimal performance (Lihoreau et al. 2018; Raubenheimer & Simpson 2018), rather than prioritizing protein alone. While the S. americana experiments provide detailed insights into nutrient regulation at the species level, our literature synthesis places these findings in broader taxonomic and functional context. Among the 65 species examined, we observed two broad trends. First, nearly all insect species regulated toward a carbohydrate-biased or balanced intake target, with protein-biased regulation being exceptionally rare. This widespread pattern suggests that energy acquisition through carbohydrates may be a widespread driver of nutrient regulation. Second, taxonomic identity was a strong predictor of nutrient intake. Across our 6 insect Orders, mean intake targets ranged between 1.2:1 and 1:4.1. However, only two Orders exhibited a slight protein preference: Lepidoptera (1.2:1) and Coleoptera (1.1:1). Lepidoptera are unique because larval and adult feeding niches differ: larvae consume plant tissues, while adults rely primarily on nectar (Krenn 2010). Consequently, Lepidoptera follow a largely capital-breeding strategy in which most adult nutritional needs are acquired during the larval stage (Colasurdo et al. 2009). Although plant tissues are generally low in protein relative to carbohydrates, larvae obtain sufficient protein through selective feeding and high intake rates, supporting rapid growth – shortening vulnerable larval stages, which also reduces predation risk (Merkx-Jacques et al. 2008; Thaler et al. 2012) – and, in females, fuelling egg production and development (Roeder & Behmer 2014; Barragan-Fonseca et al. 2019). In contrast, Coleoptera generally maintain feeding activity in both larval and adult stages and exhibited intake targets centered on balanced P:C ratios, which may help buffer performance under fluctuating nutrient environments (Choi & Lee 2022). However, Coleoptera remain underrepresented in nutritional studies, limiting our ability to fully evaluate how feeding guild diversity shapes their regulatory strategies. Orthoptera (1:1.4) and Hymenoptera (1:1.5) showed slightly carbohydrate-biased targets. Within Orthoptera, divergence emerged between the suborders Caelifera (grasshoppers and locusts) and Ensifera (crickets and katydids) approximately 350 MYA (Song et al. 2020). The Caelifera showed a more balanced intake, while Ensifera leaned more carbohydrate-biased. These differences likely reflect the generalist, diet-mixing strategies of grasshoppers (Bernays & Bright 1993; Behmer and Joern 2012) – unlike other herbivorous Orders which are dominated by specialists (Forister et al. 2014). In contrast, katydids (plants and animals) and crickets (detritus) are omnivores. Our data suggest that the ensiferan lifestyle entails substantial energetic demands. Frequent locomotion associated with navigating patchy environments adds to the cumulative costs of mobility, and these demands are further amplified by energetically costly acoustic signalling during mating (Hawkes et al. 2022). Hymenoptera also leaned toward carbohydrate-biased targets. Many studies have focused on ants, where colony-level regulation depends on feedback to adjust adult foraging and stockpiling behavior (Cannon & Fell 2002; Cook et al. 2010). Colonies with larvae showed slightly more protein-biased targets than those without (Dussutour and Simpson 2009), though both fell within the overall range for Hymenoptera. Bees are less represented in P:C studies because most pollinator nutritional research examines protein-lipid dynamics (Stabler et al. 2021; Lau et al. 2025). Nonetheless, existing data suggest bees may be even more carbohydrate-biased than ants because of the nature of floral resources (Altaye et al. 2010). The most carbohydrate-biased orders were Blattodea (1:3.2) and Diptera (1:4.1). Such extreme carbohydrate intake can impose physiological costs, including excessive lipid accumulation from carbohydrate conversion, which can produce obesity-like effects and impair growth and immune function (Warbrick-Smith et al. 2006; Graham et al. 2014). However, both groups have evolved nutritional strategies that facilitate efficient resource use in niches defined by low-protein food availability. Blattodea, as opportunistic scavengers, consume a wide range of food resources and recycle nitrogen by ingesting exuviae, frass, and secretions (McPherson et al. 2021). They also rely on endosymbionts to utilize nitrogen sources that would be otherwise unavailable (Schapheer et al. 2024). Similarly, many Diptera feed on nutrient-poor substrates such as rotting fruit, carrion, and other decaying organic material, and remain confined to these resources throughout larval development. Like blattodeans, many dipterans harbor microbiomes that supply essential nutrients, particularly amino acids that are scarce in their diets (Sontowski & van Dam 2020). These microbial symbionts likely enable Diptera to exploit low-protein substrates that are inaccessible to insects lacking such associations. For example, larvae feeding on rotting fruits benefit from fungal colonization; fungi enrich the substrate and serve as a direct food resource, supplying protein and other key nutrients needed for development (Silva-Soares et al. 2017; Deans & Hutchinson 2021). While taxonomy revealed broad evolutionary patterns in nutrient regulation, functional traits provided additional insights into the ecological strategies that shape intake targets. When examining feeding guilds, we found clear differences between herbivores and omnivores. Although both generalist and specialist herbivores regulated to a balanced P:C ratio, their intake distributions differed. Specialists clustered more tightly around their mean intake targets, while generalists exhibited broader, more variable distributions. Somewhat paradoxically, this pattern aligns with predictions about the scale of nutritional space used by generalists and specialist herbivores (Simpson & Raubenheimer 1999; Warbrick-Smith 2004). From an evolutionary perspective, broader intake targets in generalists likely reflect the need to cope with diverse and variable nutritional profiles of the plants they consume. Although generalists may share host plants, differential nutrient regulation can lead to niche partitioning allowing co-occupation and reduced interspecific competition (Behmer & Joern, 2008). In contrast, specialists typically feed on one or few host species and may fine-tune their nutrient regulation to match a narrower nutritional space. By specializing on distinct host plants or tissues, they may minimize resource overlap and reduce competitive pressure, thereby allowing greater convergence in nutritional strategies. In contrast to herbivores, omnivores regulated to significantly more carbohydrate-biased intake targets. Given that animal tissues, compared to plant tissues, are typically richer in protein and lipids (Goeriz Pearson et al. 2011; Chapman et al. 2012), this carbohydrate preference was initially surprising. However, previous studies suggest that insects often regulate protein relative to non-protein energy (i.e., carbohydrates + lipids) rather than carbohydrates alone (Noreika et al. 2016). Thus, in the absence of lipid-rich foods, carbohydrates may serve as a compensatory energy source. Additionally, omnivores may face periodic food shortages. Under such conditions, carbohydrate-derived lipids can be stored to improve survival (Rho & Lee 2014; Krabbe et al. 2019), potentially explaining the preference for carbohydrate-biased diets. Within omnivores, scavengers and predators exhibited markedly different intake targets and distributions. Predaceous omnivores can shift their intake by consuming more high-protein prey when plant quality is low (Agrawal & Klein 2001), whereas scavengers rely on decaying animal matter, which loses protein content over time (Shukla et al. 2018). These contrasting foraging constraints likely shape their distinct nutrient regulation strategies. While feeding guild explained much of the observed variation in intake regulation, developmental stage added another layer of complexity. Nutritional requirements are not static across the life and can vary with age, sex, physiology, and environmental context (Simpson & Simpson 1990; Boggs 2009; Fanson et al. 2013; Jensen et al. 2017). Across studies, we found consistent differences between immature and adult insects. Immatures generally chose protein-biased intake targets, reflecting the protein demands of somatic growth and tissue development (Wang et al. 2018). In contrast, adults favored carbohydrate-biased intake targets, likely a reflection of energetic demands tied to locomotion, mating, and lipid storage needed for survival and reproduction (South et al. 2011; Noreika et al. 2016; Reifer et al. 2018). We also compared nutrient regulation between insects with different developmental strategies – hemimetabolous versus holometabolous – but found no significant differences in their intake targets. This was unexpected, given prior evidence that holometabolous insects feed to a higher P:C, with higher growth rates, than hemimetabolous ones (Chambers et al. 1997; Lee et al. 2002). One possible explanation for similar intake targets across developmental types is that while nutrient selection may be similar, post-ingestive processes – such as nutrient assimilation, energy storage, or cuticle synthesis – could differ substantially (Bernays 1986; Arrese & Soulages 2010). These physiological differences may only become apparent under nutrient-restricted or imbalanced conditions, suggesting a promising direction for future research. While our synthesis reveals broad trends in insect protein-carbohydrate regulation, some limitations should be acknowledged. Of the 28 recognized insect Orders (Integrated Taxonomic Information System; https://itis.gov), only six were represented – these included five of the most speciose Orders, capturing a substantial portion of overall insect diversity. Some groups (e.g., Orthoptera, Lepidoptera) were well studied, while others – such as Coleoptera, despite being the most speciose Order – remain underrepresented. Even within well-sampled Orders, data were often limited to a few families (e.g., ants in Hymenoptera). Specialist herbivores were also underrepresented relative to generalists, potentially influencing comparisons within feeding guilds. Collected intake targets came from terrestrial insects and focused on either immatures or adults, excluding aquatic taxa and full developmental coverage. These limitations reflect broader gaps in the literature and underscore the need for expanded sampling across taxa, life stages, and ecological contexts. Despite these constraints, our synthesis reveals that insect nutrient regulation is driven by both evolutionary lineage and functional traits, with consistent patterns across taxonomic Orders, feeding guilds, and developmental stages. An additional key finding is that performance isn’t constrained by protein intake alone but instead depends on species-specific balance of both protein and carbohydrate. Acknowlegements This research was supported by the National Science Foundation (2021795). References 1. Agrawal, A.A., Klein, C.N., 2000. What omnivores eat: direct effects of induced plant resistance on herbivores and indirect consequences for diet selection by omnivores. J. Anim. 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Differences among treatments were assessed using ANOVAs. Food-pairing treatments Results p7:c35 with p7:c35 with p14:c28 with Test Variable p28:c14 p35:c7 p35:c7 Statistic P -value Total Consumption (g) 1.05 (±0.08) 1.08 (±0.06) 1.14 (±0.09) 0.71 0.502 Stadium duration (days) 12.9 (±0.4) 12.9 (±0.4) 13.4 (±0.6) 20.4 0.313 Dry mass gain (mg) 117.8 (±9.0) 128.0 (±10.0) 115.9 (±12.6) 0.46 0.638 Figure 1 . Protein and carbohydrate consumption for S. americana nymphs. (A) Self-selected protein-carbohydrate intake target when individuals were given a choice between two suboptimal but nutritionally complementary foods. The dashed line originating from the origin indicates a balanced 1:1 P:C ratio. (B) Protein-carbohydrate intake targets for nymphs restricted to one of seven single diets varying in their protein-carbohydrate composition. Dashed lines represent the P:C rails of each diet; three diets share a 1:1 ratio. The solid line intersecting the five diets that have 42% macronutrient content reflects the ‘ equal distance rule ’ (nutrient maximization). Inset graphs in each panel show total food consumption. All values are presented as means (± SEM). Different letters above bars indicate significant differences among treatments ( P <0.05). Figure 2 . Efficiency of conversion of ingestion (E.C.I.) for S. americana nymphs fed diets with varying protein-carbohydrate content. (A) E.C.I. on five diets with equal total macronutrient content (42%) but differ P:C ratios. The target indicates the diet that matches the self-selected P:C ratio. (B) E.C.I on the three diets with identical P:C ratios (1:1) but differing absolute amounts of protein and carbohydrate. (C) E.C.I on the two diets with equal protein content (35%) but with differing carbohydrate levels. Values are presented as means (± SEM). Different letters above bars indicate significant differences among treatments ( P <0.05). Figure 3. Summary of protein-carbohydrate (P:C) intake targets across insect Orders. The main panel displays the log-transformed mean (± SEM) P:C intake target for six insect Orders, with individual species values shown as squares within a violin plots. The inset panel separates Orthoptera into its two sub-orders: Ensifera (katydids and crickets) and Caelifera (grasshoppers). The dotted line represents a balanced 1:1 P:C ratio. Intake target values for individual species are provided in Supplemental Table 1. Different letters indicate significant differences among treatments ( P <0.05). Figure 4. Protein-carbohydrate (P:C) intake targets as a function of consumer type. (A) Log-transformed mean (± SEM) P:C intake targets for insect herbivores and omnivores. (B) Frequency distribution of intake targets for specialist versus generalist herbivores. (C) Frequency distribution of intake targets for predaceous versus scavenging omnivores. Within the violin plots each square represents the intake target of an individual species; filled circles denote group means. The dotted line in each panel indicates a 1:1 P:C ratio. Different letters indicate significant differences between groups ( P <0.05). Figure 5. Protein-carbohydrate (P:C) intake targets as a function of developmental traits. (A) Log-transformed mean (± SEM) P:C intake target for life stage (immature vs. adult). (B) Log-transformed mean (± SEM) P:C intake target for development strategy (hemimetabolous vs. holometabolous). Within the violin plots each square represents the intake target of an individual species; filled circles indicate the group mean. The dotted line in each panel represents a balanced 1:1 P:C ratio. Different letters indicate significant differences between groups ( P <0.05). 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