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.

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.

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.

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.

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.

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.

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.

Within-host competition drives energy allocation trade-offs in an insect parasitoid.

Organismal body size is an important biological trait that has broad impacts across scales of biological organization, from cells to ecosystems. Size is also deeply embedded in life history theory, as the size of an individual is one factor that governs the amount of available resources an individual is able to allocate to different structures and systems. A large body of work examining resource allocation across body sizes (allometry) has demonstrated patterns of allocation to different organismal systems and morphologies, and extrapolated rules governing biological structure and organization. However, the full scope of evolutionary and ecological ramifications of these patterns have yet to be realized. Here, we show that density-dependent larval competition in a natural population of insect parasitoids (Drino rhoeo: Tachinidae) results in a wide range of body sizes (largest flies are more than six times larger (by mass) than the smallest flies). We describe strong patterns of trade-offs between different body structures linked to dispersal and reproduction that point to life history strategies that differ between both males and females and individuals of different sizes. By better understanding the mechanisms that generate natural variation in body size and subsequent effects on the evolution of life history strategies, we gain better insight into the evolutionary and ecological impacts of insect parasitoids in tri-trophic systems.

Size as a Proxy for Survival in Aedes aegypti (Diptera: Culicidae) Mosquitoes.

The Aedes aegypti mosquito is the primary vector of dengue, yellow fever, chikungunya, and Zika viruses. Infection with the dengue virus alone occurs in an estimated 400 million people each year. Likelihood of infection with a virus transmitted by Ae. aegypti is most commonly attributed to abundance of the mosquito. However, the Arizona-Sonora desert region has abundant Ae. aegypti in most urban areas, yet local transmission of these arboviruses has not been reported in many of these cities. Previous work examined the role of differential Ae. aegypti longevity as a potential explanation for these discrepancies in transmission. To determine factors that were associated with Ae. aegypti longevity in the region, we collected eggs from ovitraps in Tucson, AZ and reared them under multiple experimental conditions in the laboratory to examine the relative impact of temperature and crowding during development, body size, fecundity, and relative humidity during the adult stage. Of the variables studied, we found that the combination of temperature during development, relative humidity, and body size produced the best model to explain variation in age at death. El mosquito Aedes aegypti es el vector primario de los virus de dengue, fiebre amarilla, chikungunya y Zika. Solamente las infecciones con los virus de dengue ocurren en aproximadamente 400 millones de personas cada año. La probabilidad de infección con un virus transmitido por Ae. aegypti es frecuentemente atribuido a la abundancia del mosquito. No obstante, la región del desierto de Arizona-Sonora tiene una abundancia de Ae. aegypti en la mayoría de las áreas urbanas, pero la transmisión local de estos arbovirus no ha sido reportada en muchas de estas ciudades. Trabajos previos han examinado el rol de las diferencias de longevidad en Ae. aegypti como explicación potencial por estas discrepancias en la transmisión. Para determinar que factores fueron asociados con longevidad en Ae. aegypti en la región, colectamos huevos de ovitrampas en Tucson, Arizona y los criamos debajo de múltiples condiciones experimentales en el laboratorio para examinar el impacto relativo de temperatura y competencia para nutrición durante desarrollo, tamaño del cuerpo, capacidad reproductiva, y humedad relativa durante adultez. De las variables estudiados, encontramos que la combinación de temperatura durante desarrollo, humedad relativa, y tamaño del cuerpo produjo el mejor modelo para explicar variación en edad al tiempo de la muerte.

The nutritional landscape of host plants for a specialist insect herbivore.

Nutrition has far-reaching effects on both the ecology and evolution of species. A substantial body of work has examined the role of host plant quality on insect herbivores, with a particular focus on specialist-generalist dynamics, the interaction of growth and other physiological attributes on fitness and tritrophic effects. Measures of plant quality usually involve one or two axes of nutritional space: typically secondary metabolites or elemental proxies (N and C) of protein and carbohydrates, respectively.Here, we describe the nutrient space of seven host plants of the specialist insect herbivore, Manduca sexta, using an approach that measures physiologically relevant sources of nutrition, soluble protein and digestible carbohydrates. We show that plant species differ markedly in their nutrient content, offering developing insect herbivores a range of available nutrient spaces that also depend on the age of the leaves being consumed.The majority of host-plant species produce diets that are suboptimal to the herbivore, likely resulting in varying levels of compensatory feeding for M. sexta to reach target levels of protein to ensure successful growth and development. Low-quality diets can also impact immune function leading to complex patterns of optimization of plant resources that maximizes both growth and the ability to defend from parasitoids and pathogens. This study is the first to quantify the nutrient space of a suite of host plants used by an insect herbivore using physiologically relevant measures of nutrition.

Diversity and distribution of microbial communities in floral nectar of two night-blooming plants of the Sonoran Desert.

Nectar-inhabiting microbes are increasingly appreciated as important components of plant-pollinator interactions. We quantified the incidence, abundance, diversity, and composition of bacterial and fungal communities in floral nectar of two night-blooming plants of the Sonoran Desert over the course of a flowering season: Datura wrightii (Solanaceae), which is pollinated by hawkmoths, and Agave palmeri (Agavaceae), which is pollinated by bats but visited by hawkmoths that forage for nectar. We examined the relevance of growing environment (greenhouse vs. field), time (before and after anthesis), season (from early to late in the flowering season), and flower visitors (excluded via mesh sleeves or allowed to visit flowers naturally) in shaping microbial assemblages in nectar. We isolated and identified bacteria and fungi from >300 nectar samples to estimate richness and taxonomic composition. Our results show that microbes were common in D. wrightii and A. palmeri nectar in the greenhouse but more so in field environments, both before and especially after anthesis. Bacteria were isolated more frequently than fungi. The abundance of microbes in nectar of D. wrightii peaked near the middle of the flowering season. Microbes generally were more abundant as time for floral visitation increased. The composition of bacterial and especially fungal communities differed significantly between nectars of D. wrightii and A. palmeri, opening the door to future studies examining their functional roles in shaping nectar chemistry, attractiveness, and pollinator specialization.

Molecular phylogeny, ecology and multispecies aggregation behaviour of bombardier beetles in Arizona.

Aggregations of conspecific animals are common and have been documented in most phyla. Multispecies aggregations are less common and less well studied. Eight species of Brachinus beetles -famous for their unique, highly effective, chemical defense-regularly settle together form large diurnal multispecies aggregations in dark, moist areas in riparian habitats in the Sonoran Desert Region. Here, we document these multispecies aggregations and investigate the incidence and dynamics of aggregation behavior. Analysis of species composition of 59 field-collected aggregations revealed that 71% contained more than one species, eight species regularly co-occurred in aggregations, and no two species showed a preference to aggregate with one another. We provide the first phylogenetic analyses of participants in multispecies aggregations, and find that Brachinus species found together in aggregations are not each other's closest relatives but rather are dispersed throughout the phylogeny of the genus. Further, we find no tendency for species to aggregate with close relatives more frequently than distant relatives. Laboratory experiments on B. elongatulus showed that it chose to settle in occupied shelters over empty shelters. Experiments with B. hirsutus and B. elongatulus showed that B. hirsutus prefers to settle under shelters housing heterospecifics over conspecifics. Our findings suggest that these multispecies aggregations do not form by chance, but rather are initiated by a genus-wide aggregation cue associated with the presence of individuals already in a shelter, which is likely to be chemical and potentially tactile in nature.

Flight duration and flight muscle ultrastructure of unfed hawk moths.

Flight muscle breakdown has been reported for many orders of insects, but the basis of this breakdown in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes across their lifespans, yet how this change affects the ability of these insects to complete their life cycles is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (Manduca sexta). Flight duration was examined in young, middle-aged, and advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorsolongitudinal muscle) was collected and histologically prepared for transmission electron microscopy to compare several measurements of muscle ultrastructure among moths of different ages. Muscle function assays revealed significant positive correlations between muscle ultrastructure and flight distance that were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more complete understanding of the ecophysiology and resource allocation strategies of this species.

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.

Life-extending dietary restriction and ovariectomy each increase leucine oxidation and alter leucine allocation in grasshoppers.

Reduced reproduction and dietary restriction each extend lifespan in many animal models, but possible contributions of nutrient oxidation and allocation are largely unknown. Ovariectomy and eating 70% of ad libitum-feeding each extend lifespan in lubber grasshoppers. When feeding levels between the two groups are matched, ovariectomy increases fat and protein storage while dietary restriction reduces fat storage. Because of these disparities in nutrient investment, metabolism may differ between these two life-extending treatments. Therefore, we examined the allocation and organismal oxidation of one representative of each macronutrient class: leucine, oleic acid, and glucose. Ovariectomy and dietary restriction each increased oxidation of dietary leucine. Dietary leucine may play a special role in aging because amino acids stimulate cellular growth. Speeding oxidation of leucine may attenuate cellular growth. Allocation of leucine to muscle was the clearest difference between ovariectomy and dietary restriction in this study. Ovariectomy reduced allocation of leucine to femur muscle, whereas dietary restriction increased allocation of leucine to femur muscle. This allocation likely corresponds to muscle maintenance for locomotion, suggesting dietary restriction increases support for locomotion, perhaps to search for food. Last, ovariectomy decreased oxidation of dietary oleic acid and glucose, perhaps to save them for storage, but the site of storage is unclear. This study suggests that the oxidation of branched-chain amino acids may be an underappreciated mechanism underlying lifespan extension.

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.

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.