Cascading Effects of Root Microbial Symbiosis on the Development and Metabolome of the Insect Herbivore Manduca Sexta L.

Root mutualistic microbes can modulate the production of plant secondary metabolites affecting plant–herbivore interactions. Still, the main mechanisms underlying the impact of root mutualists on herbivore performance remain ambiguous. In particular, little is known about how changes in the plant metabolome induced by root mutualists affect the insect metabolome and post-larval development. By using bioassays with tomato plants (Solanum lycopersicum), we analyzed the impact of the arbuscular mycorrhizal fungus Rhizophagus irregularis and the growth-promoting fungus Trichoderma harzianum on the plant interaction with the specialist insect herbivore Manduca sexta. We found that root colonization by the mutualistic microbes impaired insect development, including metamorphosis. By using untargeted metabolomics, we found that root colonization by the mutualistic microbes altered the secondary metabolism of tomato shoots, leading to enhanced levels of steroidal glycoalkaloids. Untargeted metabolomics further revealed that root colonization by the mutualists affected the metabolome of the herbivore, leading to an enhanced accumulation of steroidal glycoalkaloids and altered patterns of fatty acid amides and carnitine-derived metabolites. Our results indicate that the changes in the shoot metabolome triggered by root mutualistic microbes can cascade up altering the metabolome of the insects feeding on the colonized plants, thus affecting the insect development.

Host Plant Constancy in Ovipositing Manduca sexta

Many pollinating insects exhibit flower constancy, i.e. they target flower species they have already experienced and fed from. While the insects might profit from reduced handling costs when revisiting similar flowers, flower constancy, in addition, is of benefit for the plants as it guarantees pollen transfer to conspecifics. Here we investigate whether the previous experience of an insect can also result in oviposition constancy, i.e. whether ovipositing on a given plant species will drive future oviposition preference in a female insect. We show that female hawkmoths (Manduca sexta), after having oviposited on a given plant species only once, indeed will prefer this plant in future oviposition choices. As oviposition preference is even affected 24 h after the moth has oviposited on a given plant, long term memory seems to be involved in this oviposition constancy. Our data furthermore suggest that, as shown for flower constancy, ovipositing moths increase their handling efficiency by targeting those host plants they have already experienced.

Learning to feed in the dark: how light level influences feeding in the hawkmoth Manduca sexta

Nocturnal insects like moths are essential for pollination, providing resilience to the diurnal pollination networks. Moths use both vision and mechanosensation to locate the nectary opening in the flowers with their proboscis. However, increased light levels due to artificial light at night (ALAN) pose a serious threat to nocturnal insects. Here, we examined how light levels influence the efficacy by which the crepuscular hawkmoth Manduca sexta locates the nectary. We used three-dimensional-printed artificial flowers fitted with motion sensors in the nectary and machine vision to track the motion of hovering moths under two light levels: 0.1 lux (moonlight) and 50 lux (dawn/dusk). We found that moths in higher light conditions took significantly longer to find the nectary, even with repeated visits to the same flower. In addition to taking longer, moths in higher light conditions hovered further from the flower during feeding. Increased light levels adversely affect learning and motor control in these animals.

Moths sense, but do not learn flower odors with their proboscis during flower investigation

Insect pollinators, like the tobacco hawkmoth Manduca sexta, are known for locating flowers and learning floral odors by using their antennae. A recent study revealed, however, that the tobacco hawkmoth additionally possesses olfactory sensilla at the tip of its proboscis. Here, we ask whether this second “nose” of the hawkmoth is similarly involved in odor learning as are the antennae. We first show that Manduca foraging efficiency at Nicotiana attenuata flowers increases with experience. This raises the question whether olfactory learning with the proboscis is playing a role during flower handling. By rewarding the moths at an artificial flower, we show that – while moths learn an odor easily when they perceive it with their antennae – experiencing the odor just with the proboscis is not sufficient for odor learning. Furthermore, experiencing the odor with the antennae during training does not affect the behavior of the moths when they later can detect the learned odor with the proboscis only. Therefore, there seems to be no cross-talk between antennae and proboscis and information learnt by the antennae cannot be retrieved by the proboscis.

Variation in Manduca sexta Pollination-Related Floral Traits and Reproduction in a Wild Tobacco Plant

Most flowering plants depend on animal pollination for successful sexual reproduction. Floral signals such as color, shape, and odor are crucial in establishing this (often mutualistic) interaction. Plant and pollinator phenotypes can vary temporally but also spatially, thus creating mosaic-like patterns of local adaptations. Here, we investigated natural variation in floral morphology, flower volatile emission, and phenology in four accessions of a self-compatible wild tobacco, Nicotiana attenuata, to assess how these traits match the sensory perception of a known pollinator, the hawkmoth Manduca sexta. These accessions differ in floral traits and also in their habitat altitudes. Based on habitat temperatures, the accession occurring at the highest altitude (California) is less likely to be visited by M. sexta, while the others (Arizona, Utah 1, and Utah 2) are known to receive M. sexta pollinations. The accessions varied significantly in flower morphologies, volatile emissions, flower opening, and phenology, traits likely important for M. sexta perception and floral handling. In wind tunnel assays, we assessed the seed set of emasculated flowers after M. sexta visitation and of natural selfed and hand-pollinated selfed flowers. After moth visitations, plants of two accessions (Arizona and Utah 2) produced more capsules than the other two, consistent with predictions that accessions co-occurring with M. sexta would benefit more from the pollination services of this moth. We quantified flower and capsule production in four accessions in a glasshouse assay without pollinators to assess the potential for self-pollination. The two Utah accessions set significantly more seeds after pollen supplementation compared with those of autonomous selfing flowers, suggesting a greater opportunistic benefit from efficient pollinators than the other two. Moreover, emasculated flowers of the accession with the most exposed stigma (Utah 2) produced the greatest seed set after M. sexta visitation. This study reveals intraspecific variation in pollination syndromes that illuminate the potential of a plant species to adapt to local pollinator communities, changing environments, and altered pollination networks.

Proleg retractor muscles in Manduca sexta larvae are segmentally different suggesting anteroposterior specialization

Manduca sexta larvae are an important model system for studying the neuromechanics of soft body locomotion. They climb on plants using the abdominal prolegs to grip and maneuver in any orientation and on different surfaces. The prolegs grip passively with an array of cuticular hooks and grip release is actively controlled by retractor muscles inserted into the soft planta membrane at the proleg tip. Until now, the principal planta retractor muscles (PPRMs) in each body segment were thought to be a single fiber bundle originating on the lateral body wall. Here, using high resolution X-ray microtomography of intact animals, we show that PPRM is a more complex muscle consisting of multiple contractile fibers originating at several distinct sites on the proleg. Furthermore, we show that there are segmental differences in the number and size of some of these fiber groups which suggests that the prolegs may operate differently along the anterior-posterior axis.

Rapid frequency modulation in a resonant system: aerial perturbation recovery in hawkmoths

Centimetre-scale fliers must contend with the high power requirements of flapping flight. Insects have elastic elements in their thoraxes which may reduce the inertial costs of their flapping wings. Matching wingbeat frequency to a mechanical resonance can be energetically favourable, but also poses control challenges. Many insects use frequency modulation on long timescales, but wingstroke-to-wingstroke modulation of wingbeat frequencies in a resonant spring-wing system is potentially costly because muscles must work against the elastic flight system. Nonetheless, rapid frequency and amplitude modulation may be a useful control modality. The hawkmoth Manduca sexta has an elastic thorax capable of storing and returning significant energy. However, its nervous system also has the potential to modulate the driving frequency of flapping because its flight muscles are synchronous. We tested whether hovering hawkmoths rapidly alter frequency during perturbations with vortex rings. We observed both frequency modulation (32% around mean) and amplitude modulation (37%) occurring over several wingstrokes. Instantaneous phase analysis of wing kinematics revealed that more than 85% of perturbation responses required active changes in neurogenic driving frequency. Unlike their robotic counterparts that abdicate frequency modulation for energy efficiency, synchronous insects use wingstroke-to-wingstroke frequency modulation despite the power demands required for deviating from resonance.