Mutualisms in a warming world.

Predicting the impacts of global warming on mutualisms poses a significant challenge given the functional and life history differences that usually exist among interacting species. However, this is a critical endeavour since virtually all species on Earth depend on other species for survival and/or reproduction. The field of thermal ecology can provide physiological and mechanistic insights, as well as quantitative tools, for addressing this challenge. Here, we develop a conceptual and quantitative framework that connects thermal physiology to species' traits, species' traits to interacting mutualists' traits and interacting traits to the mutualism. We first identify the functioning of reciprocal mutualism-relevant traits in diverse systems as the key temperature-dependent mechanisms driving the interaction. We then develop metrics that measure the thermal performance of interacting mutualists' traits and that approximate the thermal performance of the mutualism itself. This integrated approach allows us to additionally examine how warming might interact with resource/nutrient availability and affect mutualistic species' associations across space and time. We offer this framework as a synthesis of convergent and critical issues in mutualism science in a changing world, and as a baseline to which other ecological complexities and scales might be added.

The adaptive role of melanin plasticity in thermally variable environments.

Understanding the evolution of adaptive plasticity is fundamental to our knowledge of how organisms interact with their environments and cope with environmental change. Plasticity in melanin pigmentation is common in response to variable environments, especially thermal environments. Yet, the adaptive significance of melanin plasticity in thermally variable environments is often assumed, but rarely explicitly tested. Furthermore, understanding the role of plasticity when a trait is responsive to multiple environmental stimuli and plays many functional roles remains poorly understood. We test the hypothesis that melanin plasticity is an adaptation for thermally variable environments using Hyles lineata, the white-lined sphinx moth, which shows plasticity in melanin pigmentation during the larval stage. Melanin pigmentation influences thermal traits in H. lineata, as melanic individuals had higher heating rates and reached higher body temperatures than non-melanic individuals. Importantly, melanin pigmentation has temperature specific fitness consequences. While melanic individuals had an advantage in cold temperatures, neither phenotype had a clear fitness advantage at warm temperatures. Thus, the costs associated with melanin production may be unrelated to thermal context. Our results highlight the importance of explicitly testing the adaptive role of plasticity and considering all the factors that influence costs and benefits of plastic phenotypes across environments.

Context dependency of in-flight responses by Manduca sexta moths to ambient differences in relative humidity.

The use of sensory information to control behavior usually involves the integration of sensory input from different modalities. This integration is affected by behavioral states and experience, and it is also sensitive to the spatiotemporal patterns of stimulation and other general contextual cues. Following the finding that hawkmoths can use relative humidity (RH) as a proxy for nectar content during close-range foraging, we evaluate here whether RH could be used during locomotive flight under two simulated contexts in a wind tunnel: (1) dispersion and (2) search phase of the foraging behavior. Flying moths showed a bias towards air with a higher RH in a context devoid of foraging stimuli, but the addition of visual and olfactory floral stimuli elicited foraging responses that overrode the behavioral effects of RH. We discuss the results in relation to the putative adaptive value of the context-dependent use of sensory information.

More than just sugar: allocation of nectar amino acids and fatty acids in a Lepidopteran.

The ability to allocate resources, even when limited, is essential for survival and fitness. We examine how nutrients that occur in minute amounts are allocated among reproductive, somatic, and metabolic demands. In addition to sugar, flower nectars contain two macronutrients-amino acids and fatty acids. We created artificial nectars spiked with 13C-labelled amino acids and fatty acids and fed these to adult moths (Manduca sexta: Sphingidae) to understand how they allocate these nutrients among competing sinks (reproduction, somatic tissue, and metabolic fuel). We found that both essential and non-essential amino acids were allocated to eggs and flight muscles and were still detectable in early-instar larvae. Parental-derived essential amino acids were more conserved in the early-instars than non-essential amino acids. All amino acids were used as metabolic fuel, but the non-essential amino acids were oxidized at higher rates than essential amino acids. Surprisingly, the nectar fatty acids were not vertically transferred to offspring, but were readily used as a metabolic fuel by the moth, minimizing losses of endogenous nutrient stores. We conclude that the non-carbohydrate components of nectar may play important roles in both reproductive success and survival of these nectar-feeding animals.

Sex differences in the utilization of essential and non-essential amino acids in Lepidoptera.

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva-derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

Synergism and Antagonism of Proximate Mechanisms Enable and Constrain the Response to Simultaneous Selection on Body Size and Development Time: An Empirical Test Using Experimental Evolution.

Natural selection acts on multiple traits simultaneously. How mechanisms underlying such traits enable or constrain their response to simultaneous selection is poorly understood. We show how antagonism and synergism among three traits at the developmental level enable or constrain evolutionary change in response to simultaneous selection on two focal traits at the phenotypic level. After 10 generations of 25% simultaneous directional selection on all four combinations of body size and development time in Manduca sexta (Sphingidae), the changes in the three developmental traits predict 93% of the response of development time and 100% of the response of body size. When the two focal traits were under synergistic selection, the response to simultaneous selection was enabled by juvenile hormone and ecdysteroids and constrained by growth rate. When the two focal traits were under antagonistic selection, the response to selection was due primarily to change in growth rate and constrained by the two hormonal traits. The approach used here reduces the complexity of the developmental and endocrine mechanisms to three proxy traits. This generates explicit predictions for the evolutionary response to selection that are based on biologically informed mechanisms. This approach has broad applicability to a diverse range of taxa, including algae, plants, amphibians, mammals, and insects.

Floral humidity as a reliable sensory cue for profitability assessment by nectar-foraging hawkmoths.

Most research on plant-pollinator communication has focused on sensory and behavioral responses to relatively static cues. Floral rewards such as nectar, however, are dynamic, and foraging animals will increase their energetic profit if they can make use of floral cues that more accurately indicate nectar availability. Here we document such a cue--transient humidity gradients--using the night blooming flowers of Oenothera cespitosa (Onagraceae). The headspace of newly opened flowers reaches levels of about 4% above ambient relative humidity due to additive evapotranspirational water loss through petals and water-saturated air from the nectar tube. Floral humidity plumes differ from ambient levels only during the first 30 min after anthesis (before nectar is depleted in wild populations), whereas other floral traits (scent, shape, and color) persist for 12-24 h. Manipulative experiments indicated that floral humidity gradients are mechanistically linked to nectar volume and therefore contain information about energy rewards to floral visitors. Behavioral assays with Hyles lineata (Sphingidae) and artificial flowers with appropriate humidity gradients suggest that these hawkmoth pollinators distinguish between subtle differences in relative humidity when other floral cues are held constant. Moths consistently approached and probed flowers with elevated humidity over those with ambient humidity levels. Because floral humidity gradients are largely produced by the evaporation of nectar itself, they represent condition-informative cues that facilitate remote sensing of floral profitability by discriminating foragers. In a xeric environment, this level of honest communication should be adaptive when plant reproductive success is pollinator limited, due to intense competition for the attention of a specialized pollinator.

Crop-emptying rate and nectar resource allocation in a nectivorous pollinator.

In nectivorous pollinators, timing and pattern of allocation of consumed nectar affects fitness traits and foraging behavior. Differences in male and female behaviors can influence these allocation strategies. These physiological patterns are not well studied in Lepidoptera, despite them being important pollinators. In this study we investigate crop-emptying rate and nectar allocation in Manduca sexta (Sphingidae), and how sex and flight influence these physiological patterns. After a single feeding event, moths were dissected at fixed time intervals to measure crop volume and analyze sugar allocation to flight muscle and fat body. Then we compared sedentary and flown moths to test how activity may alter these patterns. Sedentary males and females emptied their crops six hours after a feeding event. Both males and females preferentially allocated these consumed sugars to fat body over flight muscle. Moths began to allocate to the fat body during crop-emptying and retained these nutrients long-term (four and a half days after a feeding event). Males allocated consumed sugar to flight muscles sooner and retained these allocated nutrients in the flight muscle longer than did females. Flight initiated increased crop-emptying in females, but had no effect on males. Flight did not significantly affect allocation to flight muscle or fat body in either sex. This study showed that there are inherent differences in male and female nectar sugar allocation strategies, but that male and female differences in crop-emptying rate are context dependent on flight activity. These differences in physiology may be linked to distinct ways males and females maximize their own fitness.

No evidence for the melanin desiccation hypothesis in a larval Lepidopteran.

Water regulation is an important physiological challenge for insects due to their small body sizes and large surface area to volume ratios. Adaptations for decreasing cuticular water loss, the largest avenue of loss, are especially important. The melanin desiccation hypothesis states that melanin molecules in the cuticle may help prevent water loss, thus offering protection from desiccation. This hypothesis has much empirical support in Drosophila species, but remains mostly untested in other taxa, including Lepidoptera. Because melanin has many other important functions in insects, its potential role in desiccation prevention is not always clear. In this study we investigated the role of melanin in desiccation prevention in the white-lined Sphinx moth, Hyles lineata (Lepidoptera, Sphingidae), which shows high plasticity in the degree of melanin pigmentation during the late larval instars. We took advantage of this plasticity and used density treatments to induce a wide range of cuticular melanization; solitary conditions induced low melanin pigmentation while crowded conditions induced high melanin pigmentation. We tested whether more melanic larvae from the crowded treatment were better protected from desiccation in three relevant responses: i) total water loss over a desiccation period, ii) change in hemolymph osmolality over a desiccation period, and iii) evaporation rate of water through the cuticle. We did not find support for the melanin desiccation hypothesis in this species. Although treatment influenced total water loss, this effect did not occur via degree of melanization. Interestingly, this implies that crowding, which was used to induce high melanin phenotypes, may have other physiological effects that influence water regulation. There were no differences between treatments in cuticular evaporative water loss or change in hemolymph osmolality. However, we conclude that osmolality may not sufficiently reflect water loss in this case. This study emphasizes the context dependency of melanin's role in desiccation prevention and the importance of considering how it may vary across taxa. In lepidopteran larvae that are constantly feeding phytophagous insects with soft cuticles, melanin may not be necessary for preventing cuticular water loss.

The effect of ambient humidity on the foraging behavior of the hawkmoth Manduca sexta.

The foraging decisions of flower-visiting animals are contingent upon the need of an individual to meet both energetic and osmotic demands. Insects can alter their food preferences to prioritize one need over the other, depending on environmental conditions. In this study, preferences in nectar sugar concentrations (0, 12, 24 %) were tested in the hawkmoth Manduca sexta, in response to different levels of ambient humidity (20, 40, 60, and 80 % RH). Moths altered their foraging behavior when placed in low humidity environments by increasing the volume of nectar imbibed and by consuming more dilute nectar. When placed in high humidity environments the total volume imbibed decreased, because moths consumed less from dilute nectars (water and 12 % sucrose). Survivorship was higher with higher humidity. Daily foraging patterns changed with relative humidity (RH): moths maximized their nectar consumption earlier, at lower humidities. Although ambient humidity had an impact on foraging activity, activity levels and nectar preferences, total energy intake was not affected. These results show that foraging decisions made by M. sexta kept under different ambient RH levels allow individuals to meet their osmotic demands while maintaining a constant energy input.

Mass and volume growth of an insect tracheal system within a single instar.

Organisms must accommodate oxygen delivery to developing tissues as body mass increases during growth. In insects, the growth of the respiratory system has been assumed to occur only during molts, whereas body mass and volume increase during the larval stages between molts. This decouples whole-body growth from the growth of the oxygen supply system. This assumption is derived from the observation that the insect respiratory system is an invagination of the exoskeleton, which must be shed during molts for continued growth to occur. Here, we provide evidence that this assumption is incorrect. We found that the respiratory system increases substantially in both mass and volume within the last larval instar of Manduca sexta larvae, and that the growth of the respiratory system changes with diet quality, potentially as a consequence of shifting metabolic demands.

Herbivory of wild Manduca sexta causes fast down-regulation of photosynthetic efficiency in Datura wrightii: an early signaling cascade visualized by chlorophyll fluorescence.

Plants experiencing herbivory suffer indirect costs beyond direct loss of leaf area, but differentially so based on the herbivore involved. We used a combination of chlorophyll fluorescence imaging and gas exchange techniques to quantify photosynthetic performance, the efficiency of photochemistry, and heat dissipation to examine immediate and longer-term physiological responses in the desert perennial Datura wrightii to herbivory by tobacco hornworm, Manduca sexta. Herbivory by colony-reared larvae yielded no significant reduction in carbon assimilation, whereas herbivory by wild larvae induced a fast and spreading down-regulation of photosynthetic efficiency, resulting in significant losses in carbon assimilation in eaten and uneaten leaves. We found both an 89 % reduction in net photosynthetic rates in herbivore-damaged leaves and a whole-plant response (79 % decrease in undamaged leaves from adjacent branches). Consequently, herbivory costs are higher than previously estimated in this well-studied plant-insect interaction. We used chlorophyll fluorescence imaging to elucidate the mechanisms of this down-regulation. Quantum yield decreased up to 70 % in a small concentric band surrounding the feeding area within minutes of the onset of herbivory. Non-photochemical energy dissipation by the plant to avoid permanent damage was elevated near the wound, and increased systematically in distant areas of the leaf away from the wound over subsequent hours. Together, the results underscore not only potential differences between colony-reared and wild-caught herbivores in experimental studies of herbivory but also the benefits of quantifying physiological responses of plants in unattacked leaves.

Flight-Fecundity Trade-offs: A Possible Mechanistic Link in Plant-Herbivore-Pollinator Systems.

Plant-herbivore and plant-pollinator interactions are both well-studied, but largely independent of each other. It has become increasingly recognized, however, that pollination and herbivory interact extensively in nature, with consequences for plant fitness. Here, we explore the idea that trade-offs in investment in insect flight and reproduction may be a mechanistic link between pollination and herbivory. We first provide a general background on trade-offs between flight and fecundity in insects. We then focus on Lepidoptera; larvae are generally herbivores while most adults are pollinators, making them ideal to study these links. Increased allocation of resources to flight, we argue, potentially increases a Lepidopteran insect pollinator's efficiency, resulting in higher plant fitness. In contrast, allocation of resources to reproduction in the same insect species reduces plant fitness, because it leads to an increase in herbivore population size. We examine the sequence of resource pools available to herbivorous Lepidopteran larvae (maternally provided nutrients to the eggs, as well as leaf tissue), and to adults (nectar and nuptial gifts provided by the males to the females), which potentially are pollinators. Last, we discuss how subsequent acquisition and allocation of resources from these pools may alter flight-fecundity trade-offs, with concomitant effects both on pollinator performance and the performance of larval herbivores in the next generation. Allocation decisions at different times during ontogeny translate into costs of herbivory and/or benefits of pollination for plants, mechanistically linking herbivory and pollination.

Sex differences in the foraging behavior of a generalist hawkmoth.

Within-species variation in pollinator behavior is widely observed, but its causes have been minimally investigated. Pollinator sex is associated with large differences in behavior that may lead to predictable differences in flower foraging, but this expectation has not been explicitly tested. We investigate sex-associated differences in nectar-foraging behavior of the hawkmoth Hyles lineata, using pollen on the proboscis as a proxy for flower visitation. We tested two predictions emerging from the literature: (1) the sexes differ in the flower species they visit, (2) females are more specialized in flower choice. We also examined potential drivers underlying these predictions by performing field and laboratory experiments to test whether males (3) switch among flower species more frequently, or (4) fly farther and therefore encounter more species than females. Consistent with prediction (1), pollen load composition differed between the sexes, indicative of visitation differences. Contrary to prediction (2), females consistently carried more species-rich pollen loads than males. (3) Both sexes switched between flower species at similar rates, suggesting that differences in floral fidelity are unlikely to explain why females are less specialized than males. (4) Males flew longer distances than females; coupled with larger between-site differences in pollen composition for females, this result suggests that sex differences in mobility influence foraging, and that females may forage more frequently and in smaller areas than males. Together, our results demonstrate that sex-associated foraging differences can be large and consistent over time, and highlight the importance of sex as a driver of variation in pollinator behavior.

Eat, Drink, Live: Foraging behavior of a nectarivore when relative humidity varies but nectar resources do not.

To meet energetic and osmotic demands, animals make dynamic foraging decisions about food quality and quantity. In the wild, foraging animals may be forced to consume a less preferred or sub-optimal food source for long periods of time. Few choice feeding assays in laboratory settings approximate such contingencies. In this study the foraging behaviors of the hawkmoth Manduca sexta were measured when adult moths were placed within different relative humidity (RH) environments (20%, 40%, 60% and 80% RH) and provided with only one of the following experimental nectars: 0% (water), 12% or 24 % w/V sucrose solutions. Overall, ambient humidity influenced survivorship and foraging behaviors. Moth survivorship increased at higher ambient humidity regardless of experimental nectar. Moths that had access to experimental nectar imbibed large volumes of fluid regardless of what nectar was offered when placed at the lowest humidity (20% RH). However, when placed at the highest humidity (80% RH), moths imbibed higher volumes of fluid when given access to experimental nectar with sucrose in comparison with water. RH also influenced daily foraging behaviors: peak nectar consumption occurred earlier at lower RH levels. Consistent with previous studies in which moths could choose among nectar solutions, total energy intake was not affected by ambient RH under no-choice conditions. However, the proportion of time spent foraging and total energy consumption were significantly reduced across all RH levels in no-choice assays, when compared with previous studies of choice assays under the same conditions. Our results show that even when M. sexta moths are presented with limited options, they can alter their foraging behavior in response to environmental changes, enabling them to meet osmotic and/or energetic demands.

Behavioral consequences of innate preferences and olfactory learning in hawkmoth-flower interactions.

Spatiotemporal variability in floral resources can have ecological and evolutionary consequences for both plants and the pollinators on which they depend. Seldom, however, can patterns of flower abundance and visitation in the field be linked with the behavioral mechanisms that allow floral visitors to persist when a preferred resource is scarce. To explore these mechanisms better, we examined factors controlling floral preference in the hawkmoth Manduca sexta in the semiarid grassland of Arizona. Here, hawkmoths forage primarily on flowers of the bat-adapted agave, Agave palmeri, but shift to the moth-adapted flowers of their larval host plant, Datura wrightii, when these become abundant. Both plants emit similar concentrations of floral odor, but scent composition, nectar, and flower reflectance are distinct between the two species, and A. palmeri flowers provide six times as much chemical energy as flowers of D. wrightii. Behavioral experiments with both naïve and experienced moths revealed that hawkmoths learn to feed from agave flowers through olfactory conditioning but readily switch to D. wrightii flowers, for which they are the primary pollinator, based on an innate odor preference. Behavioral flexibility and the olfactory contrast between flowers permit the hawkmoths to persist within a dynamic environment, while at the same time to function as the major pollinator of one plant species.

Sex differences in phenotypic plasticity affect variation in sexual size dimorphism in insects: from physiology to evolution.

Males and females of nearly all animals differ in their body size, a phenomenon called sexual size dimorphism (SSD). The degree and direction of SSD vary considerably among taxa, including among populations within species. A considerable amount of this variation is due to sex differences in body size plasticity. We examine how variation in these sex differences is generated by exploring sex differences in plasticity in growth rate and development time and the physiological regulation of these differences (e.g., sex differences in regulation by the endocrine system). We explore adaptive hypotheses proposed to explain sex differences in plasticity, including those that predict that plasticity will be lowest for traits under strong selection (adaptive canalization) or greatest for traits under strong directional selection (condition dependence), but few studies have tested these hypotheses. Studies that combine proximate and ultimate mechanisms offer great promise for understanding variation in SSD and sex differences in body size plasticity in insects.

Habitat-centric versus species-centric approaches to edible insects for food and feed.

The current paradigm of the edible insects for food and feed industry uses a species-centric approach in which an insect species is chosen first and development of rearing practices follows. The goal is to optimize production to maximize the yield of that species in that facility. In contrast, the habitat-centric approach first chooses a habitat, either natural or artificial, then develops harvesting or rearing protocols within that habitat. The goal of this approach is to maximize the yield derived from that habitat. The habitat-centric approach eliminates potential threats from invasive species, and can repurpose local food and agricultural waste into protein derived from local insect species. This approach can increase food security by increasing the diversity of insects that are mass-produced. The species-centric and habitat-centric approaches address different issues and offer advantages in different situations. Further development of the edible insect industry will likely use a combination of both approaches.

Coevolutionary transitions from antagonism to mutualism explained by the Co-Opted Antagonist Hypothesis.

There is now good evidence that many mutualisms evolved from antagonism; why or how, however, remains unclear. We advance the Co-Opted Antagonist (COA) Hypothesis as a general mechanism explaining evolutionary transitions from antagonism to mutualism. COA involves an eco-coevolutionary process whereby natural selection favors co-option of an antagonist to perform a beneficial function and the interacting species coevolve a suite of phenotypic traits that drive the interaction from antagonism to mutualism. To evaluate the COA hypothesis, we present a generalized eco-coevolutionary framework of evolutionary transitions from antagonism to mutualism and develop a data-based, fully ecologically-parameterized model of a small community in which a lepidopteran insect pollinates some of its larval host plant species. More generally, our theory helps to reconcile several major challenges concerning the mechanisms of mutualism evolution, such as how mutualisms evolve without extremely tight host fidelity (vertical transmission) and how ecological context influences evolutionary outcomes, and vice-versa.

Sex differences in phenotypic plasticity of a mechanism that controls body size: implications for sexual size dimorphism.

The degree and/or direction of sexual size dimorphism (SSD) varies considerably among species and among populations within species. Although this variation is in part genetically based, much of it is probably due to the sexes exhibiting differences in body size plasticity. Here, we use the hawkmoth, Manduca sexta, to test the hypothesis that moths reared on different diet qualities and at different temperatures will exhibit sex-specific body size plasticity. In addition, we explore the proximate mechanisms that potentially create sex-specific plasticity by examining three physiological variables known to regulate body size in this insect: the growth rate, the critical weight (which measures the cessation of juvenile hormone secretion from the corpora allata) and the interval to cessation of growth (ICG; which measures the time interval between the critical weight and the secretion of the ecdysteroids that regulate pupation and metamorphosis). We found that peak larval mass of males and females did not exhibit sex-specific plasticity in response to diet or temperature. However, the sexes did exhibit sex-specific plasticity in the mechanism that controls size; males and females exhibited sex-specific plasticity in the growth rate and the critical weight in response to both diet and temperature, whereas the ICG only exhibited sex-specific plasticity in response to diet. Our results suggest it is important for the sexes to maintain the same degree of SSD across environments and that this is accomplished by the sexes exhibiting differential sensitivity of the physiological factors that determine body size to environmental variation.