The Nutritional Geometry Framework (NGF) has been instrumental in revealing how animals regulate nutrient intake. In honeybees (Apis mellifera), most research has emphasized protein-carbohydrate regulation, even though pollen is rich in both protein and lipid. We used NGF-based no-choice and choice experiments to examine how nurse bees, responsible for brood care, regulate protein and lipid intake. Bees consumed the most and developed the largest hypopharyngeal glands on diets with 30% protein and 20% lipid. When given dietary options, they regulated intake toward this 1.5:1 protein-lipid ratio. Feeding typically stopped once either a protein or lipid threshold was reached, consistent with a “strict restraint” rule that may reflect physiological costs of even slight overconsumption. These findings underscore the importance of protein-lipid regulation in nurse bees. This work broadens understanding of nutrient regulation in animals that consume protein- and lipid-rich foods and highlights the importance of diet quality for bee health.

Sterols are essential for eukaryotic cell structure and metabolism, yet insects cannot synthesize them de novo and must acquire them through their diet. For insect herbivores, plant-derived sterols are typically converted into cholesterol to support development and reproduction. We previously engineered Arabidopsis thaliana lines with silenced HYD1, resulting in altered sterol composition. In this study, we evaluated the performance of the phloem-feeding aphid Myzus persicae on these sterol-modified plants. Aphids reared on the modified lines (HYD1RNAi10, 12, 25) exhibited significantly reduced growth, reproduction, and survival compared to those on wild-type Col-0. However, choice assays and electropenetrography (EPG) revealed no differences in host preference or probing and feeding behaviors. Unlike the chewing insect Plutella xylostella, M. persicae did not accumulate atypical sterols but instead showed a significant reduction of total sterol content. Phloem-sap analysis mirrored aphid sterol profiles, lacking the atypical sterols found in leaf tissue of the modified lines. RNA-seq of HYD1RNAi lines revealed no induction of known plant defense pathways; instead, genes involved in translation and nitrate metabolism were upregulated. These findings show that HYD1 silencing reduces host suitability for aphids by limiting sterol availability for insect development and reproduction. Our results highlight the potential of sterol-modified plants as a promising strategy for managing phloem sap-feeding insect pests.

Virus-induced gene silencing (VIGS) has been a crucial tool for elucidating gene function in Upland cotton (Gossypium hirsutum) due to its complex allotetraploid genome. Reverse-transcription quantitative PCR is routine for measuring gene expression in VIGS studies, yet reference gene stability has not been adequately evaluated when using VIGS especially under biotic stress in cotton. Here, we employed several statistical methods (∆Ct, geNorm, BestKeeper, NormFinder, and weighted rank aggregation) to evaluate the stability of six candidate reference genes (GhACT7, GhPP2A1, GhUBQ7, GhUBQ14, GhTMN5, and GhTBL6) in wild-type and VIGS-infiltrated plants under cotton aphid herbivory stress over time in a fully factorial experiment. Ranked stability analyses overwhelmingly indicated that the frequently used reference genes GhUBQ7 and GhUBQ14 were the least stable whereas GhACT7 and GhPP2A1 were the most stable under VIGS and cotton aphid herbivory stress. Results were validated by comparing normalization methods of the phytosterol biosynthesis gene GhHYDRA1 in response to aphid herbivory. Normalization using GhACT7/GhPP2A1 revealed significant upregulation of GhHYDRA1 in aphid-infested plants. Conversely, normalization using GhUBQ7 reduced sensitivity to detect expression changes, highlighting the importance of stable reference gene selection for accurate expression normalization and interpretation. This study will facilitate future investigations of the genetics underpinning cotton-herbivore interactions necessary for novel pest management biotechnology discovery.

The Nutritional Geometry Framework (NGF) has been instrumental in revealing how animals regulate nutrient intake. In honeybees (Apis mellifera), most research has emphasized protein-carbohydrate regulation, even though pollen is rich in both protein and lipid. We used NGF-based no-choice and choice experiments to examine how nurse bees, responsible for brood care, regulate protein and lipid intake. Bees consumed the most and developed the largest hypopharyngeal glands on diets with 30% protein and 20% lipid. When given dietary options, they regulated intake toward this 1.5:1 protein-lipid ratio. Feeding typically stopped once either a protein or lipid threshold was reached, consistent with a "strict restraint" rule that may reflect physiological costs of even slight overconsumption. These findings underscore the importance of protein-lipid regulation in nurse bees. This work broadens understanding of nutrient regulation in animals that consume protein- and lipid-rich foods and highlights the importance of diet quality for bee health.

In response to the growing worldwide demand for enhanced agricultural output and sustainable farming practices, nanopesticides have become a significant area of investigation in agricultural research. Importantly, the fate, distribution, and efficacy of any nanopesticide is linked to the interfacial attributes and dynamic interactions between the outer surfaces – cuticle – of plants and insects. This review starts with an outline of the diverse pathways facilitating the accumulation of nanopesticides on plant cuticles, including their eventual transfer to the cuticles of insect pests. Subsequently, a comprehensive overview is provided of the micro- and nano-scale morphological features characteristic of plant and insect cuticles, along with the implications these features hold for their interactions with various nanopesticides. The review then focuses on interactions between nanopesticides and insect cuticles mediated through the plant cuticle. Finally, nanoscale mechanistic processes are discussed, with an emphasis on aspects such as wetting dynamics, critical length scales (e.g., inter-crystal spacing of waxes and surface wavelengths), and interdigitation and molecular adhesion processes of long-chain and macromolecular nanocarriers. Collectively, the review elucidates the essential interfacial processes governing the transfer and adhesion of nanopesticides between entities. The concluding section provides an overview of the prevailing challenges and potential avenues for understanding the transport and deposition mechanisms of nanopesticides to plants and insects.

The ability to keep stable, healthy colonies in laboratory settings is fundamental for conducting research on ants and particularly important for developing management tools for pest species. An important component for successful ant rearing is a suitable diet and a number of diets for feeding ants have been developed. Calcium caseinate as a protein supplement has been a widely used ingredient in ant diets, but it has become difficult to obtain. Therefore, there is a need to find an alternative protein supplement for rearing ants in captivity. Using the ant Nylanderia fulva as a model, the suitability of four alternative diets was tested against a calcium caseinate containing diet: (1) whey protein isolate fortified with calcium, (2) whey protein isolate, (3) sodium caseinate, and (4) cricket powder. We tested the performance of colony fragments fed on diets by comparing worker and queen mortality, as well as brood production for 6 weeks. Diet containing cricket powder performed better than all other diets, reflected by lower worker mortality and the addition of new workers and brood to the colony fragment, resulting in colony growth. Considering recent developments in the production of cricket powder making it a low cost and readily accessible ingredient, it should prove an effective protein supplement for rearing ants in captivity for other species.

Abstract Herbivorous insects and their host plants comprise most known species on Earth. Illuminating how herbivory repeatedly evolved in insects from nonherbivorous lineages is critical to understanding how this biodiversity is created and maintained. We characterized the trophic niche of Scaptomyza flava, a representative of a lineage nested within the Drosophila that transitioned to herbivory ~10–15 million years ago. We used natural history studies to determine whether S. flava is a true herbivore or a cryptic microbe‐feeder, given that the ancestral character state for the family Drosophilidae is likely microbe‐feeding. Specifically, we quantified oviposition substrate choice and larval viability across food types, trophic‐related morphological traits, and nitrogen isotope and sterol profiles across putatively herbivorous and nonherbivorous drosophilids. The results of these studies show that S. flava is an obligate herbivore of living plants. Paired with its genetic model host, Arabidopsis thaliana, S. flava is a novel and powerful system for exploring mechanisms underlying the evolution of herbivory, a complex trait that enabled the exceptional diversification of insects.

All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they cannot synthesize sterols de novo and must acquire sterols through diet. Cholesterol is the dominant sterol in most insects; however, because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. An additional challenge for plant-feeding insects is that only limited types of phytosterols can be converted to cholesterol. In this study, we used RNA interference to modify the plant sterol profile to generate phytosterols that would negatively impact insect herbivores. Specifically, we knocked down the expression of Arabidopsis thaliana Δ8–Δ7-sterol isomerase gene (HYD1), responsible for the conversion of Δ8-sterols into Δ7-sterols. Silenced lines with > 95

Dietary protein and digestible carbohydrates are two key macronutrients for insect herbivores, but the amounts and ratios of these two macronutrients in plant vegetative tissues can be highly variable. Typically, insect herbivores regulate their protein-carbohydrate intake by feeding selectively on nutritionally complementary plant tissues, but this may not always be possible. Interestingly, lab experiments consistently demonstrate that performance - especially growth and survival - does not vary greatly when caterpillars and nymphal grasshoppers are reared on diets that differ in their protein-carbohydrate content. This suggests insect herbivores employ postingestive physiological mechanisms to compensate for variation in diet protein-carbohydrate profile. However, the molecular mechanisms that underlie this compensation are not well understood. Here we explore, for the first time in an insect herbivore, the transcriptional effects of two dietary factors: protein-to-carbohydrate ratio (p:c) and total macronutrient (p + c) content. Specifically, we reared Helicoverpa zea caterpillars on three diets that varied in diet p:c ratio and one diet that varied in total p + c concentration, all within an ecologically-relevant range. We observed two key findings. Caterpillars reared on diets with elevated total p + c content showed large differences in gene expression. In contrast, only small differences in gene expression were observed when caterpillars were reared on diets with different p:c ratios (spanning from protein-biased to carbohydrate-biased). The invariable expression of many metabolic genes across these variable diets suggests that H. zea caterpillars employ a strategy of constitutive expression to deal with protein-carbohydrate imbalances rather than dietspecific changes. This is further supported by two findings. First, few genes were uniquely associated with feeding on a protein- and carbohydrate-biased diet. Second, many differentially-expressed genes were shared across protein-biased, carbohydrate-biased, and concentrated diet treatments. Our study provides insights into the post-ingestive physiological mechanisms insect herbivores employ to regulate protein-carbohydrate intake. Most notably, it suggests that H. zea, and perhaps other generalist species, use similar post-ingestive mechanisms to deal with protein-carbohydrate imbalances - regardless of the direction of the imbalance.

Abstract All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they are unable to synthesize sterols de novo and must acquire sterols through their diet. Cholesterol is the dominant sterol in most insects, but because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. However, metabolic constraints limit which phytosterols insect herbivores can convert to cholesterol. In this study we used RNA interference to modify the sterol profile of Arabidopsis thaliana to generate phytosterols that would be problematic for insect herbivores. We did this by silencing the Δ8-Δ7-sterol isomerase gene (HYD1) that is responsible for the conversion of Δ8-sterols into Δ7-sterols. Sterol production decreased in Arabidopsis lines with > 95% transcript reduction and half of the total sterol profile in these plants showed a Δ8 configuration. These sterol-modified lines exhibited normal growth, but caterpillars (Plutella xylostella) reared on these plants showed reduced growth and survival, while adults showed decreased reproductive output. Sterol analysis of the pupae reared on sterol-modified plants suggests these negative effects are a function of reduced cholesterol levels and the accumulation of Δ8-sterols. Finally, we used our collective performance and reproduction data to model the effects of sterol-modified plants at the population level. We show that population numbers are significantly reduced, and generation time extended, after only two generations. Our results suggest that modifying plant sterols has real potential for management of pest caterpillars, especially multivoltine species.

Plants attacked by insects may induce defenses locally in attacked plant tissues and/or systemically in non-attacked tissues, such as aboveground herbivory affecting belowground roots or belowground herbivory modifying aboveground tissues (i.e., cross-compartment systemic defense). Through induced systemic plant defenses, above-and belowground insect herbivores indirectly interact when feeding on a shared host plant. However, determining the systemic effects of herbivory on cross-compartment plant tissues and cascading consequences for herbivore communities remains underexplored. The goal of this study was to determine how belowground striped cucumber beetle ( Acalymma vittatum ) larval herbivory alters aboveground zucchini squash ( Cucurbita pepo subsp. pepo ) defenses and interactions with herbivores, including adult cucumber beetles and squash bugs ( Anasa tristis ). To explore this question, field and laboratory experiments were conducted to compare responses of aboveground herbivores to belowground larvae-damaged plants and non-damaged control plants. We also characterized changes in defensive chemicals and nutritional content of aboveground plant structures following belowground herbivory. We discovered belowground herbivory enhanced aboveground plant resistance and deterred aboveground foraging herbivores. We also found that larvae-damaged plants emitted higher amounts of a key volatile compound, ( E )-β-ocimene, compared to non-damaged controls. Further investigation suggests that other mechanisms, such as plant nutrient content, may additionally contribute to aboveground herbivore foraging decisions. Collectively, our findings underscore connections between above-and belowground herbivore communities as mediated through induced systemic defenses of a shared host plant. Specifically, these findings indicate that belowground larval herbivory systemically enhances plant defenses and deters a suite of aboveground herbivores, suggesting larvae may manipulate aboveground plant defenses for their own benefit, while plants may benefit from enhanced systemic defenses against multi-herbivore attack.

Poor nutrition and landscape changes are regularly cited as key factors causing the decline of wild and managed bee populations. However, what constitutes 'poor nutrition' for bees currently is inadequately defined. Bees collect and eat pollen: it is their only solid food source and it provides a broad suite of required macro- and micronutrients. Bees are also generalist foragers and thus the different pollen types they collect and eat can be highly nutritionally variable. Therefore, characterizing the multidimensional nutrient content of different pollen types is needed to fully understand pollen as a nutritional resource. Unfortunately, the use of different analytical approaches to assess pollen nutrient content has complicated between-studies comparisons and blurred our understanding of pollen nutrient content. In the current study, we start by reviewing the common methods used to estimate protein and lipids found in pollen. Next, using monofloral Brassica and Rosa pollen, we experimentally reveal biases in results using these methods. Finally, we use our collective data to propose a unifying approach for analysing pollen nutrient content. This will help researchers better study and understand the nutritional ecology-including foraging behaviour, nutrient regulation and health-of bees and other pollen feeders.This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

BACKGROUND:The widespread adoption of genetically modified crops, including Bacillius thuringensis (Bt) crops that target chewing insects, has transformed agricultural pest management. This increased use of Bt has raised concerns about the onset of resistance amongst target pests. Recent studies have shown that for some caterpillars, nutritional foraging (e.g. the ratio of proteins and carbohydrates consumed) can affect the insect susceptibility to the Bt toxin Cry1Ac. However, studies on both nutritional foraging and Bt susceptibility tend to rely on laboratory colonies without specifically addressing physiological differences that may occur between populations of the same species. Here, we used choice assays, no choice assays and dose response assays to address two overarching questions: Do populations of Spodoptera frugiperda (J.E. Smith) vary in their protein-carbohydrate foraging behavior? and Does protein-carbohydrate intake impact S. frugiperda's susceptibility to the Bt toxin Cry1F?RESULTS:All three of our S. frugiperda populations actively regulated their protein-carbohydrate intake, but we observed significant differences between populations with respect to their self-selected protein-carbohydrate intake. We also found that feeding at the protein-carbohydrate intake target slightly increased Cry1F susceptibility for one S. frugiperda population, but had no effect on the other two populations.CONCLUSIONS:Our findings indicate that inherent differences exist in the nutritional physiology of three S. frugiperda populations, possibly related to the time spent in culture. This suggests that population-level differences are an important consideration when drawing parallels between field-collected and laboratory-reared insects.

Supplementary Methods: Animal material; Genomic DNA extraction; Library construction, sequencing for RNA-Seq and de novo transcriptome assembly Supplementary Table S1. Available Polyneopteran genomes (incl. Schistocerca gregaria for comparison) Supplementary Table S2. Software parameter settings Supplementary Table S3. Transfer RNA (tRNA), microRNA (miRNA), small nuclear RNA (snRNA) and ribosomal RNA (rRNA) content of the desert locust genome Supplementary Table S4. Desert locust genome annotation details Supplementary Table S5. BUSCO assessments for the genomes of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria (Wang et al., 2014) Supplementary Table S6. Functional annotation of the proteome of the desert locust

Sterols are essential membrane components and are critical for many physiological processes in all eukaryotes. Insects and other arthropods are sterol auxotrophs that typically rely on a dietary source of sterols. Herbivorous insects generally obtain sterols from plants and then metabolize them into cholesterol, the dominant sterol in most insects. However, there is significant variation in phytosterol structure, and not all phytosterols are equally suitable for insects. In the current study, we used seven Arabidopsis thaliana lines that display altered sterol profiles due to mutations in the sterol biosynthetic pathway or to overexpression of key enzymes of the pathway, and investigated how plant sterol profiles affected green peach aphid (Myzus persicae) growth and reproduction. We also characterized the sterol profile of aphids reared on these Arabidopsis genotypes. Aphids on two mutant lines (14R/fk and ste1-1) that accumulated biosynthetic sterol intermediates (Δ8,14-sterols, and Δ7-sterols, respectively) all showed significantly reduced growth and reproduction. Aphids on SMT2COSUP plants (which have decreased β-sitosterol but increased campesterol) also displayed significantly reduced growth and reproduction. However, aphids on SMT2OE plants (which have increased β-sitosterol but decreased campesterol) performed similarly to aphids on wild-type plants. Finally, Arabidopsis plants that had an overproduction of sterols (CD-HMGROE) or decreased sterol esters (psat1-2) had no impact on aphid performance. Two noteworthy results come from the aphid sterol profile study. First, β-sitosterol, cholesterol and stigmasterol were recovered in all aphids. Second, we did not detect Δ8,14-sterols in aphids reared on 14R/fk plants. We discuss the implications of our findings, including how aphid sterol content does not appear to reflect plant leaf sterol profiles. We also discuss the potential of modifying plant sterol profiles to control insect herbivore pests, including aphids.

This work deduces principles of bioinspired product architecture to effectively leverage biological function-sharing in engineering design. Function-sharing enables a single structure to perform multiple functions and can improve the performance characteristics of a system. The process of evolution via natural selection has led to the emergence of function-sharing adaptations in biological systems. However, the current practice of bioinspired function-sharing is largely limited to the solution-driven imitation of biological structures. This work aims to overcome such limitations by performing a function-based analysis of biological product architectures. First, a phylogenetic approach is used to select generalized case studies from the animal kingdom. Next, the product architectures of the selected case studies are then modeled using function modeling and analyzed by clustering the identified functions into modules. A function-based categorization of the sampled biological modules reveals the presence of four types of modules in the biological case studies. Analyzing the function-sharing scenarios associated with each type of biological module enables us to deduce four guidelines for bioinspired development and arrangement of function-sharing modules. Finally, a demonstration study applies the guidelines to the design of an inlet-outlet port for a washer-dryer system. The deduced guidelines can enable engineers to identify function-sharing scenarios in the early stages of product design and reduce the need to imitate biological structures for function-sharing.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust’s use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.

Insects, like all eukaryotes, require sterols for structural and metabolic purposes. However, insects, like all arthropods, cannot make sterols. Cholesterol is the dominant tissue sterol for most insects; insect herbivores produce cholesterol by metabolizing phytosterols, but not always with high efficiency. Many insects grow on a mixed-sterol diet, but this ability varies depending on the types and ratio of dietary sterols. Dietary sterol uptake, transport, and metabolism are regulated by several proteins and processes that are relatively conserved across eukaryotes. Sterol requirements also impact insect ecology and behavior. There is potential to exploit insect sterol requirements to ( a) control insect pests in agricultural systems and ( b) better understand sterol biology, including in humans. We suggest that future studies focus on the genetic mechanism of sterol metabolism and reverse transportation, characterizing sterol distribution and function at the cellular level, the role of bacterial symbionts in sterol metabolism, and interrupting sterol trafficking for pest control.

The proximate forces that create omnivores out of herbivores and predators have long fascinated ecologists, but the causal reasons for a shift to omnivory are poorly understood. Determining what factors influence changes in trophic position are essential as omnivory plays a central role in theoretical and applied ecology. We used sevenspotted lady beetles (Coccinella septempunctata) to test how prey nutrient content affects beetles' propensity to engage in herbivory. We show that beetles consuming an all-prey diet demonstrate normal growth and development, but suffer a complete loss of fitness (spermatogenic failure) that is restored via herbivory and supplementation with phytosterols and cholesterol. Furthermore, we show that lady beetles possess a state-dependent sterol-specific appetite and redressed their sterol deficit by feeding on foliage. These results demonstrate that predators balance their nutrient intake via herbivory when prey quality is low, and reveal a selective force (sterol nutrition) that drives predatory taxa to omnivory.