Assessing pollen nutrient content: a unifying approach for the study of bee nutritional ecology.

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'.

Quantity versus quality: Effects of diet protein-carbohydrate ratios and amounts on insect herbivore gene expression.

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 post-ingestive 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 diet-specific 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.

Effect of queen number on colony-level nutrient regulation, food collection and performance in two polygynous ant species.

There is growing appreciation for how social interactions influence animal foraging behavior, especially with respect to key nutrients. Ants, given their eusocial nature and ability to be reared and manipulated in the laboratory, offer unique opportunities to explore how social interactions influence nutrient regulation and related processes. At the colony-level, ants simultaneously regulate their protein and carbohydrate intake; a regulation tied to the presence of larvae. However, even though 45% of the approximately 10,000 ant species are polygynous, we know little about the influence of queen number on colony-level foraging behavior and performance. Here we explored the direct effects of queen number on colony-level protein-carbohydrate regulation, food collection, survival, and brood production in two polygynous ant species (Nylanderia fulva and Solenopsis invicta). For both species we conducted choice and no-choice experiments using small experimental colonoids (20 workers) with 0, 1, or 2 queens. Both species regulated their relative intake of protein and carbohydrate around a P1:C2 mark. However, only N. fulva responded to the addition of queens, increasing overall food collection, biasing intake towards carbohydrates, and over-collecting imbalanced foods. N. fulva also exhibited reduced survival and reproduction on protein-biased foods. In contrast, S. invicta showed no response to queen number and reduced food collection on the protein-biased diet while maintaining high survival and reproduction. Our results demonstrate the potential for queens of some ant species to impact colony-level foraging and performance, with interspecific variation likely being shaped by differences in life history traits.

Predator Performance and Fitness Is Dictated by Herbivore Prey Type Plus Indirect Effects of their Host Plant.

Animals, including herbivores and predators, use diet-mixing to balance their macro- and micronutrient intake. Recent work demonstrated that lady beetles fed only pea aphids from fava beans had reduced fitness caused by a deficiency of dietary sterols. However, beetles redressed this deficit by eating fava bean leaves. In the current study we used Coccinella septempunctata as a model to test the hypotheses that pea aphids are a poor sterol resource independent of their host plant, and that fava beans produce low quality prey regardless of aphid species. Additionally, we tested the reproductive rescue capacity of alfalfa and barley foliage compared to fava, and profiled the sterols of phloem exudates, foliage, and aphids reared on these different hosts. Beetle fecundity and egg viability was significantly better when provided pea aphids reared on alfalfa (compared to fava beans) and green peach aphids reared on fava plants. Alfalfa and barley leaves were not consumed by beetles and did not support beetle reproduction. The sterol profile of aphids largely reflected their host plant phloem. However, green peach aphids from fava acquired 125-times more sterol than pea aphids from fava. Our findings show how the sterol content of different host-plants can affect the third trophic level. Our results suggest that 1) prey quality varies depending on prey species, even when they occur on the same plant, 2) plant species can mediate prey quality, 3) host plant-mediated effects on prey quality partially drive omnivory, and 4) diet-mixing benefits growth and reproduction by redressing micronutrient deficits.

Protein-carbohydrate regulation and nutritionally mediated responses to Bt are affected by caterpillar population history.

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.

Herbivory improves the fitness of predatory beetles.

While many predatory arthropods consume non-prey foods from lower trophic levels, little is known about what drives the shift from predator to omnivore. Predatory lady beetles often consume non-prey foods like plant foliage and pollen. One species, Coccinella septempunctata, eats foliage to redress sterol deficits caused by eating sterol-deficient prey. Here we explore how omnivory benefits lady beetle fitness. We reared seven species of lady beetles-from five genera distributed across the tribe Coccinellini-on pea aphids in the presence or absence of fava bean foliage; pea aphids have very low sterol content. Foliage supplements lengthened the development times of four species and decreased survival to adulthood of two species; it had no effect on adult mass. We mated beetles in a 2 × 2 factorial design (males with or without foliage paired with females with or without foliage). For each species, we observed a profound paternal effect of foliage supplements on fitness. Females mated to foliage-supplemented males laid more eggs and more viable eggs compared to females mated to non-supplemented males. Foliage-supplemented males produced 2.9-4.6 times more sperm compared to non-supplemented males for the three species we examined. We analysed the sterol profile of four beetle species reared on pea aphids-with or without foliage-and compared their sterol profile to field-collected adults. For two laboratory-reared species, sterols were not detected in adult male beetles, and overall levels were generally low (total ng of sterol/beetle range: 3-33 ng); the exception being Propylea quatuordecimpunctata females (total ng of sterol/beetle range: 50-157 ng). Laboratory-reared lady beetle sterol content was not significantly affected by the presence of foliage. Field-collected beetles had higher levels of sterols compared to laboratory-reared beetles (2,452-145,348 ng per beetle); cholesterol and sitosterol were the dominant sterols in both field-collected and laboratory-reared beetles. Our findings indicate that herbivory benefits lady beetle fitness across the Coccinellini, and that this was entirely a paternal effect. Our data provide a rare example of a nutritional constraint impacting fitness in a sex-specific manner. It also shows, more broadly, how a nutritional constraint can drive predators towards omnivory.

Aphid growth and reproduction on plants with altered sterol profiles: Novel insights using Arabidopsis mutant and overexpression lines.

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 ß-sitosterol but increased campesterol) also displayed significantly reduced growth and reproduction. However, aphids on SMT2OE plants (which have increased ß-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, ß-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.

First draft genome assembly of the desert locust, Schistocerca gregaria.

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.

Insect Sterol Nutrition: Physiological Mechanisms, Ecology, and Applications.

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.

First evidence of protein-carbohydrate regulation in a plant bug (Lygus hesperus).

Lygus bugs are highly polyphagous piercing/sucking insects found throughout North America. Collectively, they have been reported to feed on over 330 plant species (one of the broadest host range ever documented for a group of insects); they also feed on many economically important crops. Despite its prevalence across North America and status as a common pest in many agroecosystems, very little is known about how Lygus bugs regulate their intake of nutrients. In reality, little is known about nutrient regulation for most hemipterans, specifically non-phloem feeding species in the suborder Heteroptera. This likely reflects difficulties in developing adequate artificial diets for insects with piercing/sucking mouthparts. There is, however, an artificial diet for L. hersperus, and in this study we modified it and performed choice and no-choice experiments to determine how L. hesperus regulates its intake of two macronutrients - protein (p) and carbohydrates (c) - that are tightly linked to survival and performance in other insect herbivores. In choice experiments L. hesperus was allowed to select between two foods with different protein:carbohydrate ratios. We documented strong regulation for protein and carbohydrates, with late instar nymphs selecting a slightly protein-biased intake target (protein-carbohydrate ratio = 1.5:1). We also performed no-choice experiments, where nymphs were restricted to a single food. Here, the protein-carbohydrate ratio of their food had a strong impact on survival, which was highest for nymphs reared on the treatment with a protein-carbohydrate ratio closest to the self-selected intake target (determined by the choice experiments), but no significant impact on developmental time or mass gain. Our data are the first of their kind for a non-phloem feeding hemipteran and provide a starting point for more broadly understanding and further investigating the nutritional ecology/physiology of Lygus bugs. Our study also provides a framework for exploring nutrient regulation in other hemipterans and for optimizing artificial diets for piercing/sucking insects, especially heteropterans.

Prey nutrient content creates omnivores out of predators.

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.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Protein-carbohydrate regulation in Helicoverpa amigera and H. punctigera and how diet protein-carbohydrate content affects insect susceptibility to Bt toxins.

Many animals, including insects, demonstrate a remarkable ability to regulate their intake of key macronutrients (e.g., soluble protein and digestible carbohydrates), which allows them to optimize fitness and performance. Additionally, regulating the intake of these two macronutrients enhances an animal's ability to defend itself against pathogens, mitigate the effects of secondary plant metabolites, and decrease susceptibility to toxins. In this study, we first compared how Bt-resistant and -susceptible lines of Helicoverpa armigera and Helicoverpa punctigera regulate their intake of protein (p) and digestible carbohydrates (c). We found that there was no difference in the self-selected protein-carbohydrate intake target between resistant and susceptible genotypes of either species. We then explored the extent to which food protein-carbohydrate content altered the susceptibility of these species to three Bt toxins: Cry1Ac, Cry2Ab, and Vip3Aa. We found that H. armigera on diets that had protein-carbohydrate profiles that matched their self-selected protein-carbohydrate intake target were significantly less susceptible to Cry1Ac. In contrast, diet protein-carbohydrate content did not affect H. punctigera susceptibility to Cry1Ac. For both H. armigera and H. punctigera, susceptibility to Cry2Ab and Vip3Aa toxins did not change as a function of diet protein-carbohydrate profile. These results, when combined with earlier work on H. zea, suggest food protein-carbohydrate content can modify susceptibility to some Bt toxins, but not others. An increased understanding of how the nutritional environment can modify susceptibility to different Bt toxins could help improve pest management and resistance management practices.

Overturning dogma: tolerance of insects to mixed-sterol diets is not universal.

Insects cannot synthesize sterols de novo, but like all eukaryotes they use them as cell membrane inserts where they influence membrane fluidity and rigidity. They also use a small amount for metabolic purposes, most notably as essential precursors for steroid hormones. It has been a long-held view that most insects require a small amount of specific sterol (often cholesterol) for metabolic purposes, but for membrane purposes (where the bulk of sterols are used) specificity in sterol structure was less important. Under this model, it was assumed that insects could tolerate mixed-sterol diets as long as a small amount of cholesterol was available. In the current paper this dogma is overturned, using data from plant-feeding insects that were fed mixed-sterol diets with different amounts and ratios of dietary sterols.

Structural, tribological, and mechanical properties of the hind leg joint of a jumping insect: Using katydids to inform bioinspired lubrication systems.

This study investigates the structural properties of the hind leg femur-tibia joint in adult katydids (Orthoptera: Tettigoniidae), including its tribological and mechanical properties. It is of particular interest because the orthopteran (e.g., grasshoppers, crickets, and katydids) hind leg is highly specialized for jumping. We show that the katydid hind leg femur-tibia joint had unique surfaces and textures, with a friction coefficient (µ) at its coupling surface of 0.053±0.001. Importantly, the sheared surfaces at this joint showed no sign of wear or damage, even though it had undergone thousands of external shearing cycles. We attribute its resiliency to a synergistic interaction between the hierarchical surface texture/pattern on the femoral surfaces, a nanograded internal nanostructure of articulating joints, and the presence of lubricating lipids on the surface at the joint interface. The micro/nanopatterned surface of the katydid hind leg femur-tibia joint enables a reduction in the total contact area, and this significantly reduces the adhesive forces between the coupling surfaces. In our katydids, the femur and tibia joint surfaces had a maximum effective elastic modulus (Eeff) value of 2.6GPa and 3.9GPa, respectively. Presumably, the decreased adhesion through the reduction of van der Waals forces prevented adhesive wear, while the contact between the softer textured surface and harder smooth surface avoided abrasive wear. The results from our bioinspired study offer valuable insights that can inform the development of innovative coatings and lubrication systems that are both energy efficient and durable. STATEMENT OF SIGNIFICANCE: Relative to body length, insects can outjump most animals. They also accelerate their bodies at a much faster rate. Orthopterans (e.g., grasshoppers, crickets, and katydids) have hind legs that are specialized for jumping. Over an individual's lifetime, the hind leg joint endures repeated cycles of flexing and extending, including jumping, and its efficiency and durability easily surpass that of most mechanical devices. Although the efficient functioning of insect joints has long been recognized, the mechanism by which insect joints experience friction/adhesion/wear, and operate efficiently/reliably is still largely unknown. Our study on the structural, tribological, and mechanical properties of the orthopteran hind leg joints reveals the potential of katydid bioinspired research leading to more effective coatings and lubrication systems.

Nutrition affects insect susceptibility to Bt toxins.

Pesticide resistance represents a major challenge to global food production. The spread of resistance alleles is the primary explanation for observations of reduced pesticide efficacy over time, but the potential for gene-by-environment interactions (plasticity) to mediate susceptibility has largely been overlooked. Here we show that nutrition is an environmental factor that affects susceptibility to Bt toxins. Protein and carbohydrates are two key macronutrients for insect herbivores, and the polyphagous pest Helicoverpa zea self-selects and performs best on diets that are protein-biased relative to carbohydrates. Despite this, most Bt bioassays employ carbohydrate-biased rearing diets. This study explored the effect of diet protein-carbohydrate content on H. zea susceptibility to Cry1Ac, a common Bt endotoxin. We detected a 100-fold increase in LC50 for larvae on optimal versus carbohydrate-biased diets, and significant diet-mediated variation in survival and performance when challenged with Cry1Ac. Our results suggest that Bt resistance bioassays that use ecologically- and physiologically-mismatched diets over-estimate susceptibility and under-estimate resistance.

Spatio-Temporal, Genotypic, and Environmental Effects on Plant Soluble Protein and Digestible Carbohydrate Content: Implications for Insect Herbivores with Cotton as an Exemplar.

Plant soluble protein and digestible carbohydrate content significantly affect insect herbivore fitness, but studies reporting plant protein and carbohydrate content are rare. Instead, the elements nitrogen and carbon often are used as surrogates for plant protein and digestible carbohydrate content, respectively. However, this is problematic for two reasons. First, carbon is found in all organic molecules, which precludes strong correlations with ecologically important dietary macronutrients (e.g., digestible carbohydrates, the primary energy source for most insect herbivores). Second, some elements (e.g., nitrogen) are present in both macronutrients (e.g., protein) and non-nutritive secondary compounds (e.g., alkaloids, protease inhibitors); in these cases N values would greatly overestimate protein available for an insect herbivore. Thus, the objective of this study was to explicitly document plant protein-carbohydrate content and assess its variation in cotton (Gossypium hirsutum and G. barbadense), which is a nutritional resource for a number of insect herbivores. We did this by measuring plant soluble protein (P) and digestible carbohydrate (C) content across seven plant tissues, five varieties, and two growing environments. Significant differences in P and C concentration, total macronutrient content (P + C), and P:C ratio were observed across plant tissues, plant age and environment; smaller differences were seen across plant genotype. Foliar tissues had higher total P + C content compared to reproductive tissues, except for developing seeds and developing flowers, which contained twice the total P + C content; these two tissues also had the highest P content. Our data show that even agricultural monocultures offer a highly heterogeneous protein-carbohydrate landscape for insect herbivores. Characterizing plant resources using nutritional currencies (e.g., protein and carbohydrates) that are ecologically and physiologically-relevant to insect herbivores can be used to enhance our understanding of plant-insect interactions.

Summer and fall ants have different physiological responses to food macronutrient content.

Seasonally, long-lived animals exhibit changes in behavior and physiology in response to shifts in environmental conditions, including food abundance and nutritional quality. Ants are long-lived arthropods that, at the colony level, experience such seasonal shifts in their food resources. Previously we reported summer- and fall-collected ants practiced distinct food collection behavior and nutrient intake regulation strategies in response to variable food protein and carbohydrate content, despite being reared in the lab under identical environmental conditions and dietary regimes. Seasonally distinct responses were observed for both no-choice and choice dietary experiments. Using data from these same experiments, our objective here is to examine colony and individual-level physiological traits, colony mortality and growth, food processing, and worker lipid mass, and how these traits change in response to variable food protein-carbohydrate content. For both experiments we found that seasonality per se exerted strong effects on colony and individual level traits. Colonies collected in the summer maintained total worker mass despite high mortality. In contrast, colonies collected in the fall lived longer, and accumulated lipids, including when reared on protein-biased diets. Food macronutrient content had mainly transient effects on physiological responses. Extremes in food carbohydrate content however, elicited a compensatory response in summer worker ants, which processed more protein-biased foods and contained elevated lipid levels. Our study, combined with our previously published work, strongly suggests that underlying physiological phenotypes driving behaviors of summer and fall ants are likely fixed seasonally, and change circannually.

Physiological Status Drives Metabolic Rate in Mediterranean Geckos Infected with Pentastomes.

Negative effects of parasites on their hosts are well documented, but the proximate mechanisms by which parasites reduce their host's fitness are poorly understood. For example, it has been suggested that parasites might be energetically demanding. However, a recent meta-analysis suggests that they have statistically insignificant effects on host resting metabolic rate (RMR). It is possible, though, that energetic costs associated with parasites are only manifested during and/or following periods of activity. Here, we measured CO2 production (a surrogate for metabolism) in Mediterranean geckos (Hemidactylus turcicus) infected with a lung parasite, the pentastome Raillietiella indica, under two physiological conditions: rested and recently active. In rested geckos, there was a negative, but non-significant association between the number of pentastomes (i.e., infection intensity) and CO2 production. In recently active geckos (chased for 3 minutes), we recorded CO2 production from its maximum value until it declined to a stationary phase. We analyzed this decline as a 3 phase function (initial decline, secondary decline, stationary). Geckos that were recently active showed, in the secondary phase, a significant decrease in CO2 production as pentastome intensity increased. Moreover, duration of the secondary phase showed a significant positive association with the number of pentastomes. These results suggest that the intensity of pentastome load exerts a weak effect on the metabolism of resting geckos, but a strong physiological effect on geckos that have recently been active; we speculate this occurs via mechanical constraints on breathing. Our results provide a potential mechanism by which pentastomes can reduce gecko fitness.