Chronic changes in developmental oxygen have little effect on mitochondria and tracheal density in the endothermic moth Manduca sexta.

Oxygen availability during development is known to impact the development of insect respiratory and metabolic systems. Drosophila adult tracheal density exhibits developmental plasticity in response to hypoxic or hyperoxic oxygen levels during larval development. Respiratory systems of insects with higher aerobic demands, such as those that are facultative endotherms, may be even more responsive to oxygen levels above or below normoxia during development. The moth Manduca sexta is a large endothermic flying insect that serves as a good study system to start answering questions about developmental plasticity. In this study, we examined the effect of developmental oxygen levels (hypoxia: 10% oxygen, and hyperoxia: 30% oxygen) on the respiratory and metabolic phenotype of adult moths, focusing on morphological and physiological cellular and intercellular changes in phenotype. Mitochondrial respiration rate in permeabilized and isolated flight muscle was measured in adults. We found that permeabilized flight muscle fibers from the hypoxic group had increased mitochondrial oxygen consumption, but this was not replicated in isolated flight muscle mitochondria. Morphological changes in the trachea were examined using confocal imaging. We used transmission electron microscopy to quantify muscle and mitochondrial density in the flight muscle. The respiratory morphology was not significantly different between developmental oxygen groups. These results suggest that the developing M. sexta trachea and mitochondrial respiration have limited developmental plasticity when faced with rearing at 10% or 30% oxygen.

Altering developmental oxygen exposure influences thermoregulation and flight performance of Manduca sexta.

Endothermic, flying insects are capable of some of the highest recorded metabolic rates. This high aerobic demand is made possible by the insect's tracheal system, which supplies the flight muscles with oxygen. Many studies focus on metabolic responses to acute changes in oxygen to test the limits of the insect flight metabolic system, with some flying insects exhibiting oxygen limitation in flight metabolism. These acute studies do not account for possible changes induced by developmental phenotypic plasticity in response to chronic changes in oxygen levels. The endothermic moth Manduca sexta is a model organism that is easy to raise and exhibits a high thorax temperature during flight (∼40°C). In this study, we examined the effects of developmental oxygen exposure during the larval, pupal and adult stages on the adult moth's aerobic performance. We measured flight critical oxygen partial pressure (Pcrit-), thorax temperature and thermoregulating metabolic rate to understand the extent of developmental plasticity as well as effects of developmental oxygen levels on endothermic capacity. We found that developing in hypoxia (10% oxygen) decreased thermoregulating thorax temperature when compared with moths raised in normoxia or hyperoxia (30% oxygen), when moths were warming up in atmospheres with 21-30% oxygen. In addition, moths raised in hypoxia had lower critical oxygen levels when flying. These results suggest that chronic developmental exposure to hypoxia affects the adult metabolic phenotype and potentially has implications for thermoregulatory and flight behavior.

Flexural rigidity of hawkmoth antennae depends on the bending direction.

To probe its environment, the flying insect controllably flexes, twists, and maneuvers its antennae by coupling mechanical deformations with the sensory output. We question how the materials properties of insect antennae could influence their performance. A comparative study was conducted on four hawkmoth species: Manduca sexta, Ceratomia catalpae, Manduca quinquemaculata, and Xylophanes tersa. The morphology of the antennae of three hawkmoths that hover while feeding and one putatively non-nectar-feeding hawkmoth (Ceratomia catalpa) do not fundamentally differ, and all the antennae are comb-like (i.e., pectinate), markedly in males but weakly in females. Applying different weights to the free end of extracted cantilevered antennae, we discovered anisotropy in flexural rigidity when the antenna is forced to bend dorsally versus ventrally. The flexural rigidity of male antennae was less than that of females. Compared with the hawkmoths that hover while feeding, Ceratomia catalpae has almost two orders of magnitude lower flexural rigidity. Tensile tests showed that the stiffness of male and female antennae is almost the same. Therefore, the differences in flexural rigidity are explained by the distinct shapes of the antennal pectination. Like bristles in a comb, the pectinations provide extra rigidity to the antenna. We discuss the biological implications of these discoveries in relation to the flight habits of hawkmoths. Flexural anisotropy of antennae is expected in other groups of insects, but the targeted outcome may differ. Our work offers promising new applications of shaped fibers as mechanical sensors. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. Our comparative analysis of mechanical properties of hawkmoth antennae revealed a new feature: antenna resistance to bending depends on the bending direction. Our discovery replaces the conventional textbook scenario considering hawkmoth antennae as rigid rods. We showed that the pectinate antennae of hawkmoths behave as a comb in which the bristles resist bending when they come together. This anisotropy of flexural resistance offers a new mode of environmental sensing that has never been explored. The principles we found apply to other insects with non-axisymmetric antennae. Our work offers new applications for shaped fibers that could be designed to sense the flows.

Soil microbiota and herbivory drive the assembly of tomato plant-associated microbial communities through different mechanisms.

Plant-associated microbial communities are key to shaping many aspects of plant biology. In this study, we tested whether soil microbial communities and herbivory influence the bacterial community of tomato plants and whether their influence in different plant compartments is driven by microbial spillover between compartments or whether plants are involved in mediating this effect. We grew our plants in soils hosting three different microbial communities and covered (or not) the soil surface to prevent (or allow) passive microbial spillover between compartments, and we exposed them (or not) to herbivory by Manduca sexta. Here we show that the soil-driven effect on aboveground compartments is consistently detected regardless of soil coverage, whereas soil cover influences the herbivore-driven effect on belowground microbiota. Together, our results suggest that the soil microbiota influences aboveground plant and insect microbial communities via changes in plant metabolism and physiology or by sharing microorganisms via xylem sap. In contrast, herbivores influence the belowground plant microbiota via a combination of microbial spillover and changes in plant metabolism. These results demonstrate the important role of plants in linking aboveground and belowground microbiota, and can foster further research on soil microbiota manipulation for sustainable pest management.

Feeding rate in adult Manduca sexta is unaffected by proboscis submersion depth.

Adult moths from framily Spingidae (i.e. hawkmoths or sphinx moths) commonly feed on flower nectar through an extended proboscis, often several centimeters in length and longer than the body of the moth. Feeding on a viscous liquid (nectar) through a long and narrow tube is a challenging fluid dynamic problem and the subject of long-running scientific investigation. Here we characterized the relationship between proboscis submergence depth and nectar drinking rate in Manduca sexta hawkmoths. Video recordings of moth feeding bouts were collected and neural networks were used to extract data by object localization, tracking the location of the nectar meniscus and moths' proboscis tips. We found that although feeding rates vary among bouts, the variation was not associated with proboscis submergence depth. These results show that despite the theoretical possibility of fluid uptake through the walls of the proboscis, such effects do not have a substantial effect on nectar uptake rate, and suggest that nectar must traverse the full length of the proboscis.

Phenylalanine treatment induces tomato resistance to Tuta absoluta via increased accumulation of benzenoid/phenylpropanoid volatiles serving as defense signals.

Tuta absoluta ("leafminer"), is a major pest of tomato crops worldwide. Controlling this insect is difficult due to its efficient infestation, rapid proliferation, and resilience to changing weather conditions. Furthermore, chemical pesticides have only a short-term effect due to rapid development of T. absoluta strains. Here, we show that a variety of tomato cultivars, treated with external phenylalanine solutions exhibit high resistance to T. absoluta, under both greenhouse and open field conditions, at different locations. A large-scale metabolomic study revealed that tomato leaves absorb and metabolize externally given Phe efficiently, resulting in a change in their volatile profile, and repellence of T. absoluta moths. The change in the volatile profile is due to an increase in three phenylalanine-derived benzenoid phenylpropanoid volatiles (BPVs), benzaldehyde, phenylacetaldehyde, and 2-phenylethanol. This treatment had no effect on terpenes and green leaf volatiles, known to contribute to the fight against insects. Phe-treated plants also increased the resistance of neighboring non-treated plants. RNAseq analysis of the neighboring non-treated plants revealed an exclusive upregulation of genes, with enrichment of genes related to the plant immune response system. Exposure of tomato plants to either benzaldehyde, phenylacetaldehyde, or 2-phenylethanol, resulted in induction of genes related to the plant immune system that were also induced due to neighboring Phe-treated plants. We suggest a novel role of phenylalanine-derived BPVs as mediators of plant-insect interactions, acting as inducers of the plant defense mechanisms.

Woolly mutation with the Get02 locus overcomes the polygenic nature of trichome-based pest resistance in tomato.

Type-IV glandular trichomes, which only occur in the juvenile developmental phase of the cultivated tomato (Solanum lycopersicum), produce acylsugars that broadly protect against arthropod herbivory. Previously, we introgressed the capacity to retain type-IV trichomes in the adult phase from the wild tomato, Solanum galapagense, into the cultivated species cv. Micro-Tom (MT). The resulting MT-Galapagos enhanced trichome (MT-Get) introgression line contained 5 loci associated with enhancing the density of type-IV trichomes in adult plants. We genetically dissected MT-Get and obtained a subline containing only the locus on Chromosome 2 (MT-Get02). This genotype displayed about half the density of type-IV trichomes compared to the wild progenitor. However, when we stacked the gain-of-function allele of WOOLLY, which encodes a homeodomain leucine zipper IV transcription factor, Get02/Wo exhibited double the number of type-IV trichomes compared to S. galapagense. This discovery corroborates previous reports positioning WOOLLY as a master regulator of trichome development. Acylsugar levels in Get02/Wo were comparable to the wild progenitor, although the composition of acylsugar types differed, especially regarding fewer types with medium-length acyl chains. Agronomical parameters of Get02/Wo, including yield, were comparable to MT. Pest resistance assays showed enhanced protection against silverleaf whitefly (Bemisia tabaci), tobacco hornworm (Manduca sexta), and the fungus Septoria lycopersici. However, resistance levels did not reach those of the wild progenitor, suggesting the specificity of acylsugar types in the pest resistance mechanism. Our findings in trichome-mediated resistance advance the development of robust, naturally resistant tomato varieties, harnessing the potential of natural genetic variation. Moreover, by manipulating only 2 loci, we achieved exceptional results for a highly complex, polygenic trait, such as herbivory resistance in tomato.

Olfaction in the Anthropocene: NO3 negatively affects floral scent and nocturnal pollination.

There is growing concern about sensory pollutants affecting ecological communities. Anthropogenically enhanced oxidants [ozone (O3) and nitrate radicals (NO3)] rapidly degrade floral scents, potentially reducing pollinator attraction to flowers. However, the physiological and behavioral impacts on pollinators and plant fitness are unknown. Using a nocturnal flower-moth system, we found that atmospherically relevant concentrations of NO3 eliminate flower visitation by moths, and the reaction of NO3 with a subset of monoterpenes is what reduces the scent's attractiveness. Global atmospheric models of floral scent oxidation reveal that pollinators in certain urban areas may have a reduced ability to perceive and navigate to flowers. These results illustrate the impact of anthropogenic pollutants on an animal's olfactory ability and indicate that such pollutants may be critical regulators of global pollination.

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.

Manduca sexta caterpillars parasitized by the wasp Cotesia congregata stop chewing despite an intact motor system.

The parasitic wasp Cotesia congregata suppresses feeding in its host, the caterpillar Manduca sexta, during specific periods of wasp development. We examined both feeding behaviour and the neurophysiology of the mandibular closer muscle in parasitized and unparasitized control M. sexta to determine how the wasp may accomplish this. To test whether the wasps activated a pre-existing host mechanism for feeding cessation, we examined the microstructure of feeding behaviour in caterpillars that stopped feeding due to illness-induced anorexia or an impending moult. These microstructures were compared with that shown by parasitized caterpillars. While there were overall differences between parasitized and unparasitized caterpillars, the groups showed similar progression in feeding microstructure as feeding ended, suggesting a common pattern for terminating a meal. Parasitized caterpillars also consumed less leaf area in 100 bites than control caterpillars at around the same time their feeding microstructure changed. The decline in food consumption was accompanied by fewer spikes per burst and shorter burst durations in chewing muscle electromyograms. Similar extracellular results were obtained from the motorneuron of the mandibular closer muscle. However, chewing was dramatically re-activated in non-feeding parasitized caterpillars if the connectives posterior to the suboesophageal ganglion were severed. The same result was observed in unparasitized caterpillars given the same treatment. Our results suggest that the reduced feeding in parasitized caterpillars is not due to damage to the central pattern generator (CPG) for chewing, motor nerves or chewing muscles, but is more likely to be due to a suppression of chewing CPG activity by ascending or descending inputs.

An information theoretic method to resolve millisecond-scale spike timing precision in a comprehensive motor program.

Sensory inputs in nervous systems are often encoded at the millisecond scale in a precise spike timing code. There is now growing evidence in behaviors ranging from slow breathing to rapid flight for the prevalence of precise timing encoding in motor systems. Despite this, we largely do not know at what scale timing matters in these circuits due to the difficulty of recording a complete set of spike-resolved motor signals and assessing spike timing precision for encoding continuous motor signals. We also do not know if the precision scale varies depending on the functional role of different motor units. We introduce a method to estimate spike timing precision in motor circuits using continuous MI estimation at increasing levels of added uniform noise. This method can assess spike timing precision at fine scales for encoding rich motor output variation. We demonstrate the advantages of this approach compared to a previously established discrete information theoretic method of assessing spike timing precision. We use this method to analyze the precision in a nearly complete, spike resolved recording of the 10 primary wing muscles control flight in an agile hawk moth, Manduca sexta. Tethered moths visually tracked a robotic flower producing a range of turning (yaw) torques. We know that all 10 muscles in this motor program encode the majority of information about yaw torque in spike timings, but we do not know whether individual muscles encode motor information at different levels of precision. We demonstrate that the scale of temporal precision in all motor units in this insect flight circuit is at the sub-millisecond or millisecond-scale, with variation in precision scale present between muscle types. This method can be applied broadly to estimate spike timing precision in sensory and motor circuits in both invertebrates and vertebrates.

Muscle in the caterpillar Manduca sexta responds to an immune challenge, but at a cost, suggesting a physiological trade-off.

Although skeletal muscle is a specialized tissue that provides the motor for movement, it also participates in other functions, including the immune response. However, little is known about the effects of this multitasking on muscle. We show that muscle loses some of its capacity while it is participating in the immune response. Caterpillars (Manduca sexta) were exposed to an immune challenge, predator stress or a combination of immune challenge and predator stress. The expression of immune genes (toll-1, domeless, cactus, tube and attacin) increased in body wall muscle after exposure to an immune challenge. Muscle also showed a reduction in the amount of the energy storage molecule glycogen. During an immune challenge, the force of the defensive strike, an important anti-predator behaviour in M. sexta, was reduced. Caterpillars were also less able to defend themselves against a common enemy, the wasp Cotesia congregata, suggesting that the effect on muscle is biologically significant. Our results support the concept of an integrated defence system in which life-threatening events activate organism-wide responses. We suggest that increased mortality from predation is a non-immunological cost of infection in M. sexta. Our study also suggests that one reason non-immunological costs of infection exist is because of the participation of diverse organs, such as muscle, in immunity.

Structural damping renders the hawkmoth exoskeleton mechanically insensitive to non-sinusoidal deformations.

Muscles act through elastic and dissipative elements to mediate movement, which can introduce dissipation and filtering which are important for energetics and control. The high power requirements of flapping flight can be reduced by an insect's exoskeleton, which acts as a spring with frequency-independent material properties under purely sinusoidal deformation. However, this purely sinusoidal dynamic regime does not encompass the asymmetric wing strokes of many insects or non-periodic deformations induced by external perturbations. As such, it remains unknown whether a frequency-independent model applies broadly and what implications it has for control. We used a vibration testing system to measure the mechanical properties of isolated Manduca sexta thoraces under symmetric, asymmetric and band-limited white noise deformations. The asymmetric and white noise conditions represent two types of generalized, multi-frequency deformations that may be encountered during steady-state and perturbed flight. Power savings and dissipation were indistinguishable between symmetric and asymmetric conditions, demonstrating that no additional energy is required to deform the thorax non-sinusoidally. Under white noise conditions, stiffness and damping were invariant with frequency, suggesting that the thorax has no frequency-dependent filtering properties. A simple flat frequency response function fits our measured frequency response. This work demonstrates the potential of materials with frequency-independent damping to simplify motor control by eliminating any velocity-dependent filtering that viscoelastic elements usually impose between muscle and wing.

Substrate use and temperature effects in flight muscle mitochondria from an endothermic insect, the hawkmoth Manduca sexta.

Manduca sexta are endothermic insects, requiring adult thorax temperatures to be elevated above 35 °C for flight muscles to produce the wing beat frequencies necessary for flight. During flight, these animals rely on aerobic production of ATP by flight muscle mitochondria with several potential metabolic pathways providing the fuel. Along with typical carbohydrate substrates, mitochondria of other endothermic insects including bumblebees and wasps can use the amino acid proline or glycerol 3-phosphate (G3P) as metabolic fuel for prewarm up and flight. Here we examine flight muscle mitochondria physiology and the role of temperature and substrates in oxidative phosphorylation from 3-day old adult Manduca sexta. Mitochondria oxygen flux from flight muscle fibers were temperature sensitive with Q10 values ranging from 1.99 to 2.90, with a large increase in LEAK respiration with increased temperature. Mitochondria oxygen flux was stimulated by carbohydrate-based substrates, with flux through Complex I substrates providing the greatest oxygen flux. Neither proline nor G3P produced an increase in oxygen flux of the flight muscle mitochondria. Unlike other endothermic insects, Manduca are unable to supplement carbohydrate oxidation with either proline or G3P entering through Coenzyme Q and rely on substrates entering at complex I and II.

Hawkmoths regulate flight torques with their abdomen for yaw control.

Many animals use body parts such as tails to stabilize posture while moving at high speed. In flying insects, leg or abdominal inertia can influence flight posture. In the hawkmoth Manduca sexta, the abdomen contributes ∼50% of the total body weight and it can therefore serve to inertially redirect flight forces. How do torques generated by the wings and abdomen interact for flight control? We studied the yaw optomotor response of M. sexta by using a torque sensor attached to their thorax. In response to yaw visual motion, the abdomen moved antiphase with the stimulus, head and total torque. By studying moths with ablated wings and a fixed abdomen, we resolved abdomen and wing torques and revealed their individual contribution to total yaw torque production. Frequency-domain analysis revealed that the abdomen torque is overall smaller than wing torque, although the abdomen torque is ∼80% of the wing torque at higher visual stimulus temporal frequency. Experimental data and modeling revealed that the wing and abdomen torque are transmitted linearly to the thorax. By modeling the thorax and abdomen as a two-link system, we show that abdomen flexion can inertially redirect the thorax to add constructively to wing steering efforts. Our work argues for considering the role of the abdomen in tethered insect flight experiments that use force/torque sensors. Taken together, the hawkmoth abdomen can regulate wing torques in free flight, which could modulate flight trajectories and increase maneuverability.

Exploring the metabolic basis of growth/defense trade-offs in complex environments with Nicotiana attenuata plants cosilenced in NaMYC2a/b expression.

In response to challenges from herbivores and competitors, plants use fitness-limiting resources to produce (auto)toxic defenses. Jasmonate signaling, mediated by MYC2 transcription factors (TF), is thought to reconfigure metabolism to minimize these formal costs of defense and optimize fitness in complex environments. To study the context-dependence of this metabolic reconfiguration, we cosilenced NaMYC2a/b by RNAi in Nicotiana attenuata and phenotyped plants in the field and increasingly realistic glasshouse setups with competitors and mobile herbivores. NaMYC2a/b had normal phytohormonal responses, and higher growth and fitness in herbivore-reduced environments, but were devastated in high herbivore-load environments in the field due to diminished accumulations of specialized metabolites. In setups with competitors and mobile herbivores, irMYC2a/b plants had lower fitness than empty vector (EV) in single-genotype setups but increased fitness in mixed-genotype setups. Correlational analyses of metabolic, resistance, and growth traits revealed the expected defense/growth associations for most sectors of primary and specialized metabolism. Notable exceptions were some HGL-DTGs and phenolamides that differed between single-genotype and mixed-genotype setups, consistent with expectations of a blurred functional trichotomy of metabolites. MYC2 TFs mediate the reconfiguration of primary and specialized metabolic sectors to allow plants to optimize their fitness in complex environments.

A signal-like role for floral humidity in a nocturnal pollination system.

Previous studies have considered floral humidity to be an inadvertent consequence of nectar evaporation, which could be exploited as a cue by nectar-seeking pollinators. By contrast, our interdisciplinary study of a night-blooming flower, Datura wrightii, and its hawkmoth pollinator, Manduca sexta, reveals that floral relative humidity acts as a mutually beneficial signal in this system. The distinction between cue- and signal-based functions is illustrated by three experimental findings. First, floral humidity gradients in Datura are nearly ten-fold greater than those reported for other species, and result from active (stomatal conductance) rather than passive (nectar evaporation) processes. These humidity gradients are sustained in the face of wind and are reconstituted within seconds of moth visitation, implying substantial physiological costs to these desert plants. Second, the water balance costs in Datura are compensated through increased visitation by Manduca moths, with concomitant increases in pollen export. We show that moths are innately attracted to humid flowers, even when floral humidity and nectar rewards are experimentally decoupled. Moreover, moths can track minute changes in humidity via antennal hygrosensory sensilla but fail to do so when these sensilla are experimentally occluded. Third, their preference for humid flowers benefits hawkmoths by reducing the energetic costs of flower handling during nectar foraging. Taken together, these findings suggest that floral humidity may function as a signal mediating the final stages of floral choice by hawkmoths, complementing the attractive functions of visual and olfactory signals beyond the floral threshold in this nocturnal plant-pollinator system.

Single gene mutation in a plant MYB transcription factor causes a major shift in pollinator preference.

Understanding the molecular basis of reproductive isolation and speciation is a key goal of evolutionary genetics. In the South American genus Petunia, the R2R3-MYB transcription factor MYB-FL regulates the biosynthesis of UV-absorbing flavonol pigments, a major determinant of pollinator preference. MYB-FL is highly expressed in the hawkmoth-pollinated P. axillaris, but independent losses of its activity in sister taxa P. secreta and P. exserta led to UV-reflective flowers and associated pollinator shifts in each lineage (bees and hummingbirds, respectively). We created a myb-fl CRISPR mutant in P. axillaris and studied the effect of this single gene on innate pollinator preference. The mutation strongly reduced the expression of the two key flavonol-related biosynthetic genes but only affected the expression of few other genes. The mutant flowers were UV reflective as expected but additionally contained low levels of visible anthocyanin pigments. Hawkmoths strongly preferred the wild-type P. axillaris over the myb-fl mutant, whereas both social and solitary bee preference depended on the level of visible color of the mutants. MYB-FL, with its specific expression pattern, small number of target genes, and key position at the nexus of flavonol and anthocyanin biosynthetic pathways, provides a striking example of evolution by single mutations of large phenotypic effect.

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.

The larval scaffold controls fascicle number but is not required for formation of the dorsolongitudinal flight muscles in Manduca sexta.

During metamorphosis, the dorsolongitudinal flight muscles (DLMs) of both the moth Manduca sexta and the fly Drosophila melanogaster develop from the remnants of larval muscles called larval scaffolds. Although this developmental program has been conserved across highly disparate taxa, the role of the larval scaffold remains unclear. Ablation experiments have demonstrated that the Drosophila DLM does not require the scaffold, but the resulting de novo muscles vary highly in fiber number, and their functional characteristics were not examined. To address this question in Manduca, we have surgically ablated the DLM precursors in Manduca sexta larvae and assayed the resulting DLMs in pharate adults using X-ray micro-CT and phalloidin histology. Following ablation, animals were able to form de novo DLMs with normal myofibril alignment, but these muscles had an altered shape and highly variable number of fascicles. Our results suggest that the larval scaffold is not required for DLM development in Manduca sexta, but appears to define the number of fascicles in the adult muscle, as previously found in Drosophila. Additionally, our ablated animals were able to generate flight, further suggesting that the use of a larval scaffold is a modification on the more ancestral myogenesis program.