Effect of dietary macronutrients and immune challenge on gut microbiota, physiology and feeding behaviour in zebra finches.

Macronutrients play a vital role in host immunity and can influence host-pathogen dynamics, potentially through dietary effects on gut microbiota. To increase our understanding of how dietary macronutrients affect physiology and gut microbiota and investigate whether feeding behaviour is influenced by an immune threat, we conducted two experiments. First, we determined whether zebra finches (Taeniopygia guttata) exhibit shifts in physiology and gut microbiota when fed diets differing in macronutrient ratios. We found the type and amount of diet consumed affected gut microbiota alpha diversity, where microbial richness and Shannon diversity increased with caloric intake in birds fed a high-fat diet and decreased with caloric intake in birds fed a high protein diet. Diet macronutrient content did not affect physiological metrics, but lower caloric intake was associated with higher complement activity. In our second experiment, we simulated an infection in birds using the bacterial endotoxin lipopolysaccharide (LPS) and quantified feeding behaviour in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). We also examined whether social cues of infection alter physiological responses relevant to responding to an immune threat, an effect that could be mediated through shifts in feeding behaviour. LPS induced a reduction in caloric intake driven by a decrease in protein, but not fat consumption. No evidence was found for socially induced shifts in feeding behaviour, physiology or gut microbiota. Our findings carry implications for host health, as sickness-induced anorexia and diet-induced shifts in the microbiome could shape host-pathogen interactions.

Networking nutrients: How nutrition determines the structure of ecological networks.

Nutrients can shape ecological interactions but remain poorly integrated into ecological networks. Concepts such as nutrient-specific foraging nevertheless have the potential to expose the mechanisms structuring complex ecological systems. Nutrients also present an opportunity to predict dynamic processes, such as interaction rewiring and extinction cascades, and increase the accuracy of network analyses. Here, we propose the concept of nutritional networks. By integrating nutritional data into ecological networks, we envisage significant advances to our understanding of ecological processes from individual to ecosystem scales. We show that networks can be constructed with nutritional data to illuminate how nutrients structure ecological interactions in natural systems through an empirical example. Throughout, we identify fundamental ecological hypotheses that can be explored in a nutritional network context, alongside methods for resolving those networks. Nutrients influence the structure and complexity of ecological networks through mechanistic processes and concepts including nutritional niche differentiation, functional responses, landscape diversity, ecological invasions and ecosystem robustness. Future research on ecological networks should consider nutrients when investigating the drivers of network structure and function.

Multi-elemental consumer-driven nutrient cycling when predators feed on different prey.

Predators play a fundamental role in cycling nutrients through ecosystems, by altering the amount and compositions of waste products and uneaten prey parts available to decomposers. Different prey can vary in their elemental content and the deposition of elements in predator waste can vary depending on which elements are preferentially retained versus eliminated as waste products. We tested how feeding on different prey (caterpillars, cockroaches, crickets, and flies) affected the concentrations of 23 elements in excreta deposited by wolf spider across 2 seasons (spring versus fall). Spider excreta had lower concentrations of carbon and higher concentrations of many other elements (Al, B, Ba, K, Li, P, S, Si, and Sr) compared to prey remains and whole prey carcasses. In addition, elemental concentrations in unconsumed whole prey carcasses and prey remains varied between prey species, while spider excreta had the lowest variation among prey species. Finally, the concentrations of elements deposited differed between seasons, with wolf spiders excreting greater concentrations of Fe, Mg, Mn, Mo, S, and V in the fall. However, in the spring, spiders excreted higher concentrations of Al, B, Ba, Ca, Cd, Cu, K, P, Na, Si, Sr, and Zn. These results highlight that prey identity and environmental variation can determine the role that predators play in regulating the cycling of many elements. A better understanding of these convoluted nutritional interactions is critical to disentangle specific consumer-driven effects on ecosystem function.

Consequences of prey exoskeleton content for predator feeding and digestion: black widow predation on larval versus adult mealworm beetles.

Predators often feed on a wide range of prey that can vary in behavior, morphology, and physiology. The net benefits that predators gain from prey are likely related to both prey nutrient content and prey morphology or defenses. For invertebrates, the exoskeleton is a morphological trait that varies widely among species and during ontogeny and could affect nutrient extraction by predators. The goal of this study was to determine how prey exoskeleton content affected predator nutrient intake, assimilation, and excretion by comparing spiders feeding on either larval or adult mealworms of similar size. We found that the proportion of prey energy invested in digestion was greatest in spiders consuming adult mealworm beetles which had higher amounts of exoskeleton than larvae. Further, spiders extracted a greater proportion of elements, macronutrients, and energy from the larval mealworms, which had lower amounts of exoskeleton. Interestingly, total nitrogen content of prey was not a predictor of nitrogen assimilation as spiders assimilated more nitrogen from the larval mealworms, which had lower total nitrogen content. While adult beetles had higher total nitrogen content, their discarded remains of prey had large amounts of nitrogen that was nutritionally unavailable for spiders (i.e., exoskeleton). These results suggest that prey exoskeleton can affect assimilation efficiency by predators, and that a combination of macronutrient and elemental analyses may be needed to examine the quality of prey for predators and the potential consequences of predation for nutrient flows (e.g., consumer assimilation, egestion, and excretion) in ecosystems.

Merging elemental and macronutrient approaches for a comprehensive study of energy and nutrient flows.

Global warming and predation risk can have important impacts on animal physiology and life histories that can have consequences for ecosystem function. Zhang et al. () recently tested the separate and interactive effects of warming and predation risk on the body composition of Daphnia magna. By measuring both the elemental and biochemical composition of individuals, they showed that D. magna body elemental composition responded opposite to theoretical predictions and previous studies but that these changes were explained by adaptive life-history shifts in allocation to protein in eggs versus body lipid reserves. Photograph by Joachim Mergeay. Zhang, C., Jansen, M., De Meester, L. & Stoks, R. (2016) Energy storage and fecundity explain deviations from ecological stoichiometry predictions under global warming and size-selective predation. Journal of Animal Ecology 85, 1431-1441. Understanding the mechanisms through which energy and nutrients flow through ecosystems is critical to predicting and mitigating the consequences of climate change and other ecological disturbances. Ecological stoichiometry and nutritional geometry, using data on elements and macromolecules, respectively, have independently made major contributions towards this goal. Zhang et al. () provide data demonstrating that these two major frameworks can provide complementary insight into the consequences of global warming and predation risk for the physiology and life-history traits of a key aquatic herbivore, Daphnia magna. This study should catalyse further work to unite these two parallel and complementary frameworks.

Recent advances in the integrative nutrition of arthropods.

In this review we highlight recent advances in four areas in which nutrition shapes the relationships between organisms: between plants and herbivores, between hosts and their microbiota, between individuals within groups and societies, and between species within food webs. We demonstrate that taking an explicitly multidimensional view of nutrition and employing the logic of the geometric framework for nutrition provide novel insights and offer a means of integration across different levels of organization, from individuals to ecosystems.

Intercontinental differences in resource use reveal the importance of mutualisms in fire ant invasions.

Mutualisms play key roles in the functioning of ecosystems. However, reciprocally beneficial interactions that involve introduced species also can enhance invasion success and in doing so compromise ecosystem integrity. For example, the growth and competitive ability of introduced plant species can increase when fungal or microbial associates provide limiting nutrients. Mutualisms also may aid animal invasions, but how such systems may promote invasion success has received relatively little attention. Here we examine how access to food-for-protection mutualisms involving the red imported fire ant (Solenopsis invicta) aids the success of this prominent invader. Intense interspecific competition in its native Argentina constrained the ability of S. invicta to benefit from honeydew-producing Hemiptera (and other accessible sources of carbohydrates), whereas S. invicta dominated these resources in its introduced range in the United States. Consistent with this strong pattern, nitrogen isotopic data revealed that fire ants from populations in the United States occupy a lower trophic position than fire ants from Argentina. Laboratory and field experiments demonstrated that honeydew elevated colony growth, a crucial determinant of competitive performance, even when insect prey were not limiting. Carbohydrates, obtained largely through mutualistic partnerships with other organisms, thus represent critical resources that may aid the success of this widespread invasive species. These results illustrate the potential for mutualistic interactions to play a fundamental role in the establishment and spread of animal invasions.

Arthropod food webs become increasingly lipid-limited at higher trophic levels.

Understanding why food chains are relatively short in length has been an area of research and debate for decades. We tested if progressive changes in the nutritional content of arthropods with trophic position limit the availability of a key nutrient, lipid, for carnivores. Arthropods at higher trophic levels had progressively less lipid and more protein in their bodies compared with arthropods at lower trophic levels. The nutrients present in arthropod bodies were directly related to the nutrients that predators extracted when feeding on those arthropods. As a consequence, nutrient assimilation shifted from lipid-biased to protein-biased as arthropods ascended trophic levels from herbivores to secondary carnivores. Successive changes in the nutritional consequences of predation may ultimately set an upper limit on the number of trophic levels in arthropod communities. Further work is needed to examine the influence of lipid and other nutrients on food web traits in a range of ecosystems.

Introduced fire ants can exclude native ants from critical mutualist-provided resources.

Animals frequently experience resource imbalances in nature. For ants, one resource that may be particularly valuable for both introduced and native species is high-carbohydrate honeydew from hemipteran mutualists. We conducted field and laboratory experiments: (1) to test if red imported fire ants (Solenopsis invicta) competed with native ants for access to mutualisms with aphids, and (2) to quantify the effects of aphid honeydew presence or absence on colony growth of native ants. We focused on native dolichoderine ants (Formicidae, Dolichoderinae) because they are abundant ants that have omnivorous diets that frequently include mutualist-provided carbohydrates. At two sites in the southeastern US, native dolichoderine ants were far less frequent, and fire ants more frequent, at carbohydrate baits than would be expected based on their frequency in pitfall traps. A field experiment confirmed that a native ant species, Dorymyrmex bureni, was only found tending aphids when populations of S. invicta were suppressed. In the laboratory, colonies of native dolichoderine ants with access to both honeydew and insect prey had twice as many workers and over twice as much brood compared to colonies fed only ad libitum insect prey. Our results provide the first experimental evidence that introduced ants compete for access to mutualist-provided carbohydrates with native ants and that these carbohydrates represent critical resources for both introduced and native ants. These results challenge traditional paradigms of arthropod and ant nutrition and contribute to growing evidence of the importance of nutrition in mediating ecological interactions.

Comparing the accuracy of protein measures for arthropods.

Data on the protein content of arthropods can be useful for addressing a variety of ecological, behavioral, and physiological hypotheses. Yet, the most accurate method for measuring protein content (i.e., amino acid analysis) is expensive and the accuracy of less expensive measures of protein is unclear. We analyzed a diversity of arthropods to test for relationships between digestible protein content as measured by amino acid analysis and several common protein measures: crude protein, Bradford assay, BCA assay, and Lowry assay. In the full dataset, the closest relationship to the amino acid data was found for the Lowry assay and the average of the Bradford and Lowry assays. However, one species, Blattella germanica, appeared to be an outlier in some analyses. When the data were analyzed without B. germanica, the closest relationships to the amino acid data were found for the Lowry assay. Our results suggest that not all protein measures are equal in their ability to estimate amino acid content. Some arthropod species can also contain chemicals that interfere with the accuracy of protein assays. Given that it is unclear how often interfering compounds are found in invertebrates, it may be best to conduct multiple assays when analyzing the protein content of arthropods, especially the Bradford and Lowry assays.

Variation among arthropod taxa in the amino acid content of exoskeleton and digestible tissue.

Arthropod consumption provides amino acids to invertebrates and vertebrates alike, but not all amino acids in arthropods may be digestible as some are bound in the exoskeleton. Consumers may not be able to digest exoskeleton in significant amounts or avoid it entirely (e.g., extraoral digestion). Hence, measures that do not separate digestible amino acids from those in exoskeleton may not accurately represent the amino acids available to consumers. Additionally, arthropods are taxonomically diverse, and it remains unclear if taxonomic differences also reflect differences in amino acid availability. Thus, we tested: (1) if there were consistent differences in the content and balance of amino acids between the digestible tissue and exoskeleton of arthropods and (2) if arthropod Orders differ in amino acid content and balance. We measured the amino acid content (mg/100 mg dry mass) and balance (mg/100 mg protein) of whole bodies and exoskeleton of a variety of arthropods using acid hydrolysis. Overall, there was higher amino acid content in digestible tissue. There were also significant differences in the amino acid balance of proteins in digestible tissue and exoskeleton. Amino acid content and balance also varied among Orders; digestible tissues of Hemiptera contained more of some essential amino acids than other Orders. These results demonstrate that arthropod taxa vary in amino acid content, which could have implications for prey choice by insectivores. In addition, exoskeleton and digestible tissue content differ in arthropods, which means that whole body amino acid content of an arthropod is not necessarily a predictor of amino acid intake of a predator that feeds on that arthropod.

Carbohydrates complement high-protein diets to maximize the growth of an actively hunting predator.

In nature, food is often variable in composition and availability. As a consequence, predators may need to seek non-prey food sources. Some predators are known to feed on nectar when food is limited. Nectar and other carbohydrate resources could also be beneficial when prey are more abundant if it helps predators balance protein-biased diets. We tested if an actively hunting predator, the jumping spider, Phidippus audax, benefited from liquid carbohydrates when prey were not limited. We also tested if the benefit of carbohydrates varied with the nutrient content of prey (i.e., from protein to lipid biased). Spiders were reared on one of six live prey, Drosophila melanogaster, treatments that ranged from high protein to high lipid. Half of the spiders were given access to a 20% sucrose solution. After 2 months, we measured spider mass, cephalothorax width, instar duration, percent body fat, survival, and estimated number of prey eaten. Spiders reared on high-protein diets with carbohydrates were larger and heavier than spiders on other treatments. Access to carbohydrates also increased percent body fat and survival across prey treatments. Our results suggest that carbohydrates may be a valuable component of spider diets, especially when prey have high protein and low lipid content as is commonly observed in prey in the field. Our results highlight the importance of diet balancing for predators, and that liquid carbohydrates can be an important nutrient to supplement a diet of prey rather than just being an energy supplement during periods of starvation.

Climate alters the movement ecology of a non-migratory bird.

Global climate change is causing increased climate extremes threatening biodiversity and altering ecosystems. Climate is comprised of many variables including air temperature, barometric pressure, solar radiation, wind, relative humidity, and precipitation that interact with each other. As movement connects various aspects of an animal's life, understanding how climate influences movement at a fine-temporal scale will be critical to the long-term conservation of species impacted by climate change. The sedentary nature of non-migratory species could increase some species risk of extirpation caused by climate change. We used Northern Bobwhite (Colinus virginianus; hereafter bobwhite) as a model to better understand the relationship between climate and the movement ecology of a non-migratory species at a fine-temporal scale. We collected movement data on bobwhite from across western Oklahoma during 2019-2020 and paired these data with meteorological data. We analyzed movement in three different ways (probability of movement, hourly distance moved, and sinuosity) using two calculated movement metrics: hourly movement (displacement between two consecutive fixes an hour apart) and sinuosity (a form of tortuosity that determines the amount of curvature of a random search path). We used generalized linear-mixed models to analyze probability of movement and hourly distance moved, and used linear-mixed models to analyze sinuosity. The interaction between air temperature and solar radiation affected probability of movement and hourly distance moved. Bobwhite movement increased as air temperature increased beyond 10°C during low solar radiation. During medium and high solar radiation, bobwhite moved farther as air temperature increased until 25-30°C when hourly distance moved plateaued. Bobwhite sinuosity increased as solar radiation increased. Our results show that specific climate variables alter the fine-scale movement of a non-migratory species. Understanding the link between climate and movement is important to determining how climate change may impact a species' space use and fitness now and in the future.

Macronutrient content of plant-based food affects growth of a carnivorous arthropod.

Many arthropods engage in mutualisms in which they consume plant-based foods including nectar, extrafloral nectar, and honeydew. However, relatively little is known about the manner in which the specific macronutrients in these plant-based resources affect growth, especially for carnivorous arthropods. Using a combination of laboratory and field experiments, we tested (1) how plant-based foods, together with ad libitum insect prey, affect the growth of a carnivorous ant, Solenopsis invicta, and (2) which macronutrients in these resources (i.e., carbohydrates, amino acids, or both) contribute to higher colony growth. Access to honeydew increased the production of workers and brood in experimental colonies. This growth effect appeared to be due to carbohydrates alone as colonies provided with the carbohydrate component of artificial extrafloral nectar had greater worker and brood production compared to colonies deprived of carbohydrates. Surprisingly, amino acids only had a slight interactive effect on the proportion of a colony composed of brood and negatively affected worker survival. Diet choice in the laboratory and field matched performance in the laboratory with high recruitment to carbohydrate baits and only slight recruitment to amino acids. The strong, positive effects of carbohydrates on colony growth and the low cost of producing this macronutrient for plants and hemipterans may have aided the evolution of food-for-protection mutualisms and help explain why these interactions are so common in ants. In addition, greater access to plant-based resources in the introduced range of S. invicta may help to explain the high densities achieved by this species throughout the southeastern United States.

The sublethal effects of neonicotinoids on spiders are independent of their nutritional status.

Spiders were recently shown to be adversely affected by field-realistic concentrations of a broad scale of neonicotinoid insecticides. Among the reported effects of neonicotinoids on invertebrates were declines in lipid biosynthesis and upregulation of ß-oxidation, while vertebrate models suggest increased adipogenesis following treatment with neonicotinoids. Therefore, we hypothesized that there exists synergy between the effects of diet and concurrent exposure to field-realistic concentrations of neonicotinoid insecticides. To address this hypothesis, we fed first instars of the large wolf spider Hogna antelucana with two types of diets and exposed them to field-realistic concentrations of three formulations of neonicotinoids (thiamethoxam, thiacloprid and acetamiprid). We then measured the growth of the tested spiders; the lipid and protein content of their bodies; and their behavior, including ballooning, rappelling, and locomotor parameters. The two tested diets consisted of casein-treated and sucrose-treated Drosophila melanogaster. The dietary treatments affected the lipid and protein content of the spiders, their body weight and carapace length but did not affect any of the measured behavioral parameters. Surprisingly, we did not find any effects of acute exposure to neonicotinoid insecticides on the lipid or protein reserves of spiders. Exposure to neonicotinoids altered the behavior of the spiders as reported previously in other spider species; however, these effects were not affected by dietary treatments. Overall, the dietary treatments did not have any major synergy with acute exposure to field-realistic concentrations of neonicotinoid insecticides.

Effects of fire on ground-dwelling arthropods in a shrub-dominated grassland.

Arthropods are abundant and diverse animals in many terrestrial food webs. In western Oklahoma, some shrublands are interspersed with discrete, dense thickets of tall, woody vegetation, known as mottes. Some of these shrublands are managed with prescribed burning. The goal of this study was to examine whether prescribed burning interacted with habitat type (i.e., shrubland versus mottes) to affect ground-dwelling arthropod communities. Arthropods were collected in pitfall traps at four sampling locations in relation to mottes; in the center of mottes, and three plot location in shrublands; 1 m, 15 m, and 50 m away from the edge of the motte. There were three treatment levels for burning: one year postburn (burned in dormant months of 2017), two years postburn (burned in dormant months of 2016), and unburned (burned in dormant season of 2014 and prior). There were no significant interactions between prescribed burning and habitat type. Mottes had a different community of arthropods compared with the surrounding shrubland. Mottes also had lower overall abundance, but a higher diversity of arthropods. In terms of fires, arthropod communities one year after burning were different from those two or more years after burning. There was no effect of burning on overall arthropod abundance, but plots that were one year since burning had significantly lower diversity compared with plots that were two or more years postburn. The results of this study suggest that both fire and mottes can independently facilitate heterogeneity in arthropod communities, but they do not appear to interact with one another.

Arthropod prey vary among orders in their nutrient and exoskeleton content.

Insectivores gain macronutrients and elements from consuming arthropod prey, but must also deal with indigestible components (i.e., exoskeleton) of prey. For example, avian chicks (e.g., northern bobwhites; Colinus virginianus) have limited gut space, and ingesting prey with relatively higher proportions of indigestible components may impact assimilation efficiency, growth, and survival. The ability of insectivores to choose higher quality prey would depend on prey taxa varying consistently in nutritional content. We tested whether there were consistent differences among taxonomic orders of arthropod prey in their macronutrient (protein and lipid), elemental (C and N), and exoskeleton content. We used northern bobwhite chicks as our focal insectivore and focused on their potential prey as a case study. We also tested the influence of indigestible exoskeleton on the measurement of macronutrient content and the ability of elemental content to predict macronutrients. We found large and consistent variation in macronutrient and elemental content between arthropod orders. Some orders had consistently high protein content and low exoskeleton content (i.e., Araneae) and are likely higher quality prey for insectivores. Abundant orders common in the diets of insectivores, like Hymenoptera and Coleoptera, had high exoskeleton content and low protein content. We also found support for the ability of elements to predict macronutrients and found that metabolizable (i.e., exoskeleton removed) elemental content better predicted macronutrient content. A better understanding of arthropod nutrient content is critical for elucidating the role of spatial and temporal variation in prey communities in shaping the growth and survival of insectivores.

MEDI: Macronutrient Extraction and Determination from invertebrates, a rapid, cheap and streamlined protocol.

Macronutrients, comprising carbohydrates, proteins and lipids, underpin many ecological processes, but their quantification in ecological studies is often inaccurate and laborious, requiring large investments of time and bulk samples, which make individual-level studies impossible. This study presents Macronutrient Extraction and Determination from Invertebrates (MEDI), a protocol for the direct, rapid and relatively low-cost determination of macronutrient content from single small macroinvertebrates.Macronutrients were extracted by a sequential process of soaking in 1:12 chloroform:methanol solution to remove lipid and then solubilising tissue in 0.1 M NaOH. Proteins, carbohydrates and lipids were determined by colorimetric assays from the same individual specimens.The limits of detection of MEDI with the equipment and conditions used were 0.067, 0.065 and 0.006 mg/ml for proteins, carbohydrates and lipids respectively. Adjusting the volume of reagents used for extraction and determination can broaden the range of concentrations that can be detected. MEDI successfully identified taxonomic differences in macronutrient content between five insect species.Macronutrient Extraction and Determination from Invertebrates can directly and rapidly determine macronutrient content in tiny (dry mass ~3 mg) and much larger individual invertebrates. Using MEDI, the total macronutrient content of over 50 macroinvertebrates can be determined within around 3 days of collection at a cost of ~$1.35 per sample.

Extrafloral nectar content alters foraging preferences of a predatory ant.

We tested whether the carbohydrate and amino acid content of extrafloral nectar affected prey choice by a predatory ant. Fire ants, Solenopsis invicta, were provided with artificial nectar that varied in the presence of carbohydrates and amino acids and were then provided with two prey items that differed in nutritional content, female and male crickets. Colonies of fire ants provided with carbohydrate supplements consumed less of the female crickets and frequently did not consume the high-lipid ovaries of female crickets. Colonies of fire ants provided with amino acid supplements consumed less of the male crickets. While a number of studies have shown that the presence of extrafloral nectar or honeydew can affect ant foraging activity, these results suggest that the nutritional composition of extrafloral nectar is also important and can affect subsequent prey choice by predatory ants. Our results suggest that, by altering the composition of extrafloral nectar, plants could manipulate the prey preferences of ants foraging on them.

Males make poor meals: a comparison of nutrient extraction during sexual cannibalism and predation.

Cannibalism is hypothesized to have evolved as a way to obtain a high-quality meal. We examined the extraction of lipid and protein by female wolf spiders, Hogna helluo, during sexual cannibalism of males and predation of crickets. Most food-limited females did not cannibalize males but immediately consumed a size-matched cricket. When consuming male H. helluo and crickets, female H. helluo only consumed 51% of the male body while they consumed 72% of the cricket body. While males had higher protein content in their bodies than crickets and other insects, female H. helluo ingested similar amounts of protein from male H. helluo and crickets. Female H. helluo extracted 47% of the protein present in male H. helluo and 67% of the protein present in crickets. Females were able to extract nearly all of the lipid present in male H. helluo and crickets. However, crickets and other insects had almost 4 times higher lipid content than male H. helluo. The ratio of lipid to protein consumed from crickets appeared more similar to the nutritional requirements of egg production than that of males. Taken together, female hesitancy to engage in cannibalism, low extraction of nutrients from males and a low ratio of lipid to protein in the food extracted from males suggest that males may be poor-quality prey items compared to common insects such as crickets.