Tobacco Hornworm Caterpillars Research Articles

Trophic transfer of PFAS from tomato (Solanum lycopersicum) to tobacco hornworm (Manduca sexta) caterpillars

PFASs are highly persistent in the environment and the potential exists for terrestrial biota to accumulate PFAS, which may result in exposure of higher trophic level organisms to these compounds through consumption. However, trophic transfer of proteinophilic compounds such as PFAS has not been extensively studied and the degree to which plant-accumulated PFAS will be transferred to herbivorous consumers is unclear. Here, we exposed Solanum lycopersicum (tomato) plants to a suite of 7 different PFAS, including 4 carboxylic acids (PFOA, PFHxA, PFHpA and PFDA) and 3 sulfonates (PFBS, PFHxS and PFOS). Exposed leaf tissues were subsequently fed to Manduca sexta (tobacco hornworm) caterpillars. Biomagnification factors (BMFs) were all below 1 and patterns of uptake and elimination were similar between the different PFAS. However, PFOS bioaccumulated in the hornworms to a much higher concentration, with approximately 5-fold higher BMFs and assimilation efficiencies (AEs) than other PFAS tested. AE and BMF, as well as PFAS uptake by the plants, were positively correlated with PFAS carbon chain length for both sulfonates and carboxylic acids, providing evidence that longer chain PFAS may be more efficiently accumulated (or less efficiently eliminated) than shorter-chain PFAS in some contexts.

Local adaptation to continuous mowing makes the noxious weed Solanum elaeagnifolium a superweed candidate by improving fitness and defense traits

The role of disturbance in accelerating weed growth is well understood. While most studies have focused on soil mediated disturbance, mowing can also impact weed traits. Using silverleaf nightshade (Solanum elaeagnifolium), a noxious and invasive weed, through a series of field, laboratory, and greenhouse experiments, we asked whether continuous mowing influences growth and plant defense traits, expressed via different avenues, and whether they cascade into offspring. We found that mowed plants produced significantly less number of fruits, and less number of total seeds per plant, but had higher seed mass, and germinated more and faster. When three herbivores were allowed to feed, tobacco hornworm (Manduca sexta) caterpillars, gained more mass on seedlings from unmowed plants, while cow pea aphid (Aphis craccivora), a generalist, established better on mowed seedlings; however, leaf trichome density was higher on unmowed seedlings, suggesting possible negative cross talk in defense traits. Texas potato beetle (Leptinotarsa texana), a co-evolved specialist on S. elaeagnifolium, did not show any differential feeding effects. We also found that specific root length, an indicator of nutrient acquisition, was significantly higher in first generation seedlings from mowed plants. Taken together, we show that mowing is a selective pressure that enhances some fitness and defense traits and can contribute to producing superweeds.

Tobacco Hornworm (Manduca sexta) Oral Secretion Elicits Reactive Oxygen Species in Isolated Tomato Protoplasts.

Plants are under constant attack by a suite of insect herbivores. Over millions of years of coexistence, plants have evolved the ability to sense insect feeding via herbivore-associated elicitors in oral secretions, which can mobilize defense responses. However, herbivore-associated elicitors and the intrinsic downstream modulator of such interactions remain less understood. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces reactive oxygen species (ROS) in tomato (Solanum lycopersicum) protoplasts. By using a dye-based ROS imaging approach, our study shows that application of plant-fed (PF) M. sexta OS generates significantly higher ROS while artificial diet-fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. Elevation in ROS generation was saturated after ~140 s of PF OS application. ROS production was also suppressed in the presence of an antioxidant NAC (N-acetyl-L-cysteine). Interestingly, PF OS-induced ROS increase was abolished in the presence of a Ca2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). These results indicate a potential signaling cascade involving herbivore-associated elicitors, Ca2+, and ROS in plants during insect feeding. In summary, our results demonstrate that plants incorporate a variety of independent signals connected with their herbivores to regulate and mount their defense responses.

Polyphenol-Rich Purple Corn Pericarp Extract Adversely Impacts Herbivore Growth and Development.

Plant secondary metabolites such as terpenes, phenolics, glycosides, and alkaloids play various functional roles including pigmentation, foliar and floral volatile synthesis, hormonal regulation, and direct and indirect defenses. Among these, phenolic compounds are commonly found in plants, but vary in the distribution of their specific compounds among plant families. Polyphenols, including anthocyanins and tannins, are widely distributed and have been well documented for their roles- primarily in plant pigmentation and also in plant defenses. However, commercialization of such compounds for use in insect pest management is severely hampered by expensive, inefficient, and time-consuming extraction protocols. Using a recently developed inexpensive and easy extraction method using the byproducts of pigmented (purple) corn processing, we examined whether the crude pericarp extract rich in polyphenols can affect the growth and development of tobacco hornworm (Manduca sexta L.) caterpillars. Our findings show that purple corn pericarp extract negatively affected M. sexta egg hatching and larval mass gain and prolonged developmental time compared to regular yellow corn extract or an artificial control diet. We also found that these effects were more severe during the early stages of caterpillar development. These results conclusively demonstrate that purple corn pericarp, an inexpensive by-product of the corn milling industry, is a valuable product with excellent potential as an insect antifeedant.

Caterpillar Oral Secretion Elicits Reactive Oxygen Species in Isolated Plant Protoplasts

Over millions of years, plants have evolved and developed several defense strategies such as spines, trichomes, toxins, and plant volatiles to either directly or indirectly defend against insect herbivores. Reactive Oxygen Species (ROS) is a signaling molecule that is suspected of signaling herbivory by activating defense cascade at the cellular level, leading to local and systemic defense gene expression. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces ROS in tomato (Solanum lycopersicum) protoplasts. We used a dye-based ROS imaging approach to dissect the interaction between a damaging herbivore and its host plant. Our study shows that application of Plant-Fed (PF) caterpillar OS increased ROS generation while artificial Diet-Fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. The elevation in ROS generation was observed after ˜100 s and was saturated after 165 s of PF-OS application. Interestingly, the PF-OS induced ROS increase was abolished in the presence of a Ca2+ chelator BAPTA-AM, suggesting possible crosstalk between Ca2+ and ROS signaling. Targeting the pathway that triggers ROS generation could be used to develop a herbivore origin potent elicitor of plant defense gene expression (ROS elicitor) pest management research.

Tobacco hornworm caterpillars held up by imaginal disc damage

![Figure][1] A tobacco hornworm ( Manduca sexta ) caterpillar. Photo credit: Fuse lab. Youngsters often bear more than a passing resemblance to their parents; but, the offspring of other creatures look nothing like their forebears. ‘Think maggot to fly, or caterpillar to butterfly or

Feeding on glandular and non-glandular leaf trichomes negatively affect growth and development in tobacco hornworm (Manduca sexta) caterpillars

Trichomes, the hair-like projections on plant leaves, have been well studied as an herbivore defense. However, whether variation in trichome type can have negative effects through the different stages of caterpillar life cycle is poorly understood. Using Solanum elaeagnifolium that produce non-glandular stellate trichomes, and Solanum lycopersicum that predominantly produce glandular, non-branched trichomes, we examined how trichomes affect choice and growth of Manduca sexta, a specialist herbivore of plant species belonging to the Solanaceae. To accomplish this, we removed leaf trichomes and added them into artificial diet for caterpillars, and allowed the caterpillars to grow and develop. Our results show that trichomes negatively affected caterpillar body mass and mass gain, although there was no preference or aversion for them in choice assays. Non-glandular trichomes were particularly damaging, as the consumption of non-glandular trichomes resulted in suppressed mass gain and increased time to pupation. While the consumption of glandular trichomes also affected growth, the effects were significantly lower compared to the consumption of non-glandular ones. Taken together, our results show that feeding on solanaceous trichomes can have negative effects on herbivore larval growth and development, and should be examined further.

Effects of High‐Fat Diets on Mortality and Feeding in the Tobacco Hornworm Caterpillar, Manduca sexta

Nutritionally‐balanced diets are important for fitness, but for insects the high energy value of fat storage is crucial for reproduction and development. Although studies have shown that insects fed high carbohydrate diets have increased fat storage, and excess fat storage in insects may have a fitness cost, little is known about how insects regulate fat consumption when presented with diets containing varying lipid content. To determine how insects respond to high fat diets, we fed caterpillars of different ages high (5.6%), medium (3.4%), or low (0.4%) fat diets. Young Manduca sexta larvae reared on high fat diet had 80% mortality and 43% lower body mass compared to those reared on medium or low fat diets. Older larvae showed no difference in mortality, but those fed a high fat diet had a lower average body mass by the third day of the fifth instar. Growth rates and development time also differed. High fat‐fed caterpillars wandered later than those fed low and medium fat diets. To test the hypothesis that the increased survival of older larvae was due to increased lipid transport, we measured mRNA expression of Apolipoprotein I and II (APO1 and 2), proteins responsible for transporting lipids to the fat body and to various tissues. APO1 and 2 expression did not differ with dietary fat content. In addition, we tested the hypothesis that caterpillars preferentially metabolize fat during the fifth instar. We measured oxygen consumption and carbon dioxide emission of caterpillars fed a high or low fat diet and calculated the respiratory quotient. To determine the mechanism underlying the decrease in body size, we tested the hypothesis that the high fat diet altered feeding. Caterpillars fed a high fat diet indeed ate less, as indicated by a decrease in food consumption and the number and mass of fecal pellets produced. These results suggest that increased dietary fat results in cessation of feeding in these insects. Due to conserved fat metabolism pathways in vertebrates and invertebrates, better understanding of the role of lipids in insects could contribute to knowledge of human metabolic diseases and provide support for using insect models for these studies.Support or Funding InformationThis research was funded in part by NSF EPS‐0447679 and IOS‐0953297 to K.J.G. and NIH National Center for Research Resources 2P20RR0l5566. The contents of this study are solely the responsibility of the authors and do not necessarily reflect the views of the NIH.

Hypoxia-induced compression in the tracheal system of the tobacco hornworm caterpillar,Manduca sextaL.

Abdominal pumping in caterpillars has only been documented during molting. Using synchrotron X-ray imaging in conjunction with high-speed flow-through respirometry, we show that Manduca sexta caterpillars cyclically contract their bodies in response to hypoxia, resulting in significant compressions of the tracheal system. Compression of tracheae induced by abdominal pumping drives external gas exchange, as evidenced by the high correlation between CO2 emission peaks and body movements. During abdominal pumping, both the compression frequency and fractional change in diameter of tracheae increased with body mass. However, abdominal pumping and tracheal compression were only observed in larger, older caterpillars (>0.2 g body mass), suggesting that this hypoxic response increases during ontogeny. The diameters of major tracheae in the thorax increased isometrically with body mass. However, tracheae in the head did not scale with mass, suggesting that there is a large safety margin for oxygen delivery in the head in the youngest animals. Together, these results highlight the need for more studies of tracheal system scaling and suggest that patterns of tracheal investment vary regionally in the body.

Bioaccumulation of Gold Nanomaterials by Manduca sextathrough Dietary Uptake of Surface Contaminated Plant Tissue

We investigated the potential for bioaccumulation of engineered nanomaterials (ENMs) by tobacco hornworm (Manduca sexta) caterpillars resulting from the ingestion of plant tissue surface contaminated with ENMs. Caterpillars were fed tomato leaf tissue that had been surface contaminated with 12 nm tannate coated Au ENMs. After dosing was complete, bulk Au concentrations in individual caterpillars were measured after 0, 1, 4, and 7 days of elimination. Growth, mortality, and ingestion rate were monitored. This experiment revealed (1) no evidence that caterpillars were affected by ingestion of ENM contaminated plant tissue; (2) low bioaccumulation factors (BAF = 0.16) compared to a previous study where hornworm caterpillars were fed plants that had previously bioaccumulated Au ENMs (BAF = 6.2-11.6); (3) inefficient elimination of accumulated Au ENMs not associated with hornworm gut contents; and (4) regional differences in translocation of Au ENMs into tissues surrounding the hornworm gut, possibly the result of the interaction between ENM surface chemistry and regional differences in hornworm gut chemistry. These data, along with previous findings, indicate that although ENMs resuspended from soil onto plant surfaces by wind, water, biota, and/or mechanical disturbances are bioavailable to terrestrial consumers, bioaccumulation efficiency may be much lower via this pathway than through direct trophic exposure.

Compensatory mechanisms for ameliorating the fundamental trade-off between predator avoidance and foraging

Most organisms face the problem of foraging and maintaining growth while avoiding predators. Typical animal responses to predator exposure include reduced feeding, elevated metabolism, and altered development rate, all of which can be beneficial in the presence of predators but detrimental in their absence. How then do animals balance growth and predator avoidance? In a series of field and greenhouse experiments, we document that the tobacco hornworm caterpillar, Manduca sexta, reduced feeding by 30-40% owing to the risk of predation by stink bugs, but developed more rapidly and gained the same mass as unthreatened caterpillars. Assimilation efficiency, extraction of nitrogen from food, and percent body lipid content all increased during the initial phase (1-3 d) of predation risk, indicating that enhanced nutritional physiology allows caterpillars to compensate when threatened. However, we report physiological costs of predation risk, including altered body composition (decreased glycogen) and reductions in assimilation efficiency later in development. Our findings indicate that hornworm caterpillars use temporally dynamic compensatory mechanisms that ameliorate the trade-off between predator avoidance and growth in the short term, deferring costs to a period when they are less vulnerable to predation.

Modeling locomotion of a soft-bodied arthropod using inverse dynamics

Most bio-inspired robots have been based on animals with jointed, stiff skeletons. There is now an increasing interest in mimicking the robust performance of animals in natural environments by incorporating compliant materials into the locomotory system. However, the mechanics of moving, highly conformable structures are particularly difficult to predict. This paper proposes a planar, extensible-link model for the soft-bodied tobacco hornworm caterpillar, Manduca sexta, to provide insight for biologists and engineers studying locomotion by highly deformable animals and caterpillar-like robots. Using inverse dynamics to process experimentally acquired point-tracking data, ground reaction forces and internal forces were determined for a crawling caterpillar. Computed ground reaction forces were compared to experimental data to validate the model. The results show that a system of linked extendable joints can faithfully describe the general form and magnitude of the contact forces produced by a crawling caterpillar. Furthermore, the model can be used to compute internal forces that cannot be measured experimentally. It is predicted that between different body segments in stance phase the body is mostly kept in tension and that compression only occurs during the swing phase when the prolegs release their grip. This finding supports a recently proposed mechanism for locomotion by soft animals in which the substrate transfers compressive forces from one part of the body to another (the environmental skeleton) thereby minimizing the need for hydrostatic stiffening. The model also provides a new means to characterize and test control strategies used in caterpillar crawling and soft robot locomotion.

Plants’ Modified SOS Call

Damage caused by a caterpillar releases compounds that are transformed by the pest’s saliva into a predator attractant.

Insects Betray Themselves in Nature to Predators by Rapid Isomerization of Green Leaf Volatiles

Plants emit green leaf volatiles (GLVs) in response to herbivore damage, thereby attracting predators of the herbivores as part of an indirect defense. The GLV component of this indirect defense was thought to be a general wound signal lacking herbivore-specific information. We found that Manduca sexta-infested Nicotiana attenuata attract the generalist hemipteran predator Geocoris spp. as the result of an herbivore-induced decrease in the (Z)/(E) ratio of released GLVs, and that these changes in the volatile bouquet triple the foraging efficiency of predators in nature. These (E)-isomers are produced from plant-derived (Z)-isomers but are converted by a heat-labile constituent of herbivore oral secretions. Hence, attacking herbivores initiate the release of an indirect defense a full day before the attacked plants manufacture their own defensive compounds.

Matrix Metalloproteinases Control Hemocyte Trafficking in Innate Immunity of Manduca sexta

Matrix metalloproteinases (MMPs) are a family of conserved, proteolytic enzymes. MMPs can degrade most extracellular matrix proteins. As such; they are key enzymes in remodeling processes, such as molting and metamorphosis. They are also known to be upregulated in many disease states, where they may cause tissue damage. Yet their presence may be beneficial, as their inhibition may result in worsening of symptoms or increased mortality. We tested the hypothesis that MMPs play a role in innate immunity in the tobacco hornworm caterpillar, Manduca sexta. We infected 5th instar caterpillars with E. coli DH5α in the presence or absence of the MMP inhibitor, GM6001. Larvae infected with E. coli DH5α had significantly more circulating hemocytes when compared to the larvae which received an MMP inhibitor, suggesting that MMPs may be important for hemocyte recruitment from the fat body. The majority of the circulating hemocytes were granulocytes, a cell type that phagocytoses forein bodies, including pathogens. In addition, we used flow cytometry to assess phagocytic ability. Hemocytes in the presence of the MMP inhibitor were less able to phagocytose sephadex beads, supporting the hypothesis that this MMP may contribute to cellular innate immune function.This work was funded in part by NDSU, NSFEPSCoR #EPS‐00447679, and NIH 2P20RR015566.

Proton Sponge: A Novel and Versatile MALDI Matrix for the Analysis of Metabolites Using Mass Spectrometry

Here, we show the usefulness of a strong base, 1,8-bis(dimethyl-amino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-TOF/MS analysis of anions. Several strong and weakly acidic low-molecular-weight analytes (fatty acids, amino acids, fatty acid-amino acid conjugates, plant and animal hormones, vitamins, and short peptides) were measured at physiologically relevant concentrations. Clear negative-mode MALDI-TOF/MS spectra of all analytes using DMAN as the matrix show only deprotonated analyte signals at a low picomole/femtomole limit-of-detection. Moreover, the spectra were totally devoid of any matrix-related signals. Standard calibration curves gave good linearity over the entire picomole range: over two concentration orders in most cases and over three orders for peptides. Using this method, the crude regurgitate of the tobacco hornworm caterpillars (Manduca sexta, Lepidoptera, Sphingidae) was analyzed. As many as 11 different components were identified from a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids and 5:0-Glu, 6:0-Glu, 18:2-Glu, 18:3-Glu, 16:0-Glu, and 16:3-Glu fatty acid-amino acid conjugates (FACs) in complete qualitative agreement with previously reported anion exchange-HPLC analyses. The identity of these components was confirmed by negative ion collision-induced dissociation (CID) MS2 spectra.

A scorpion neurotoxin increases the potency of a fungal insecticide

The low virulence of the insecticidal fungus Metarhizium anisopliae has stymied its widespread use in controlling insect pests. We show that high-level expression of an insect-specific neurotoxin from the scorpion Androctonus australis in hemolymph by M. anisopliae increases fungal toxicity 22-fold against tobacco hornworm (Manduca sexta) caterpillars and ninefold against adult yellow fever mosquitoes (Aedes aegypti) without compromising host specificity. Prelethal effects include reduced mobility and feeding of the insects targeted.

Muscle performance in a soft-bodied terrestrial crawler: constitutive modelling of strain-rate dependency

Experimental data on the passive mechanical properties of the ventral interior lateral muscle of the tobacco hornworm caterpillar, Manduca sexta, are reported. The stress-deformation response of the Manduca muscle is shown to be nonlinear pseudo-elastic, capable of large deformations and subject to stress softening during initial loading cycles. The muscle passive mechanical properties also depend on multiple time-dependent processes. In particular, we show new experimental data from cyclic loading tests of an unstimulated muscle with constant maximum stretch and different, constant engineering strain rates. Then, on the basis of these data a constitutive model is derived to reproduce the main characteristics of this behaviour. In formulating the constitutive model, we consider the muscle as a complex macromolecular structure with fibrous components at numerous size scales. The model uses a phenomenological approach to account for different mechanisms by which passive force changes during applied deformation and how the muscle properties recover after unloading.

A constitutive model for muscle properties in a soft-bodied arthropod

In this paper, we examine the mechanical properties of muscles in a soft-bodied arthropod under both passive and stimulated conditions. In particular, we examine the ventral interior lateral muscle of the tobacco hornworm caterpillar, Manduca sexta, and show that its response is qualitatively similar to the behaviour of particle-reinforced rubber. Both materials are capable of large nonlinear elastic deformations, show a hysteretic behaviour and display stress softening during the first few cycles of repeated loading. The Manduca muscle can therefore be considered as different elastic materials during loading and unloading and is best described using the theory of pseudo-elasticity. We summarize the basic equations for transversely isotropic pseudo-elastic materials, first for general deformations and then for the appropriate uniaxial specialization. The constitutive relation proposed is in good agreement with the experimental data for both the passive and the stimulated conditions.

Resource availability and the trichome defenses of tomato plants

We conducted two experiments to determine how resource availability influenced allocation by tomato (Lycopersicon esculentum) to trichomes, and how different patterns of trichome allocation by plants grown in different resource environments might then influence the behavior of tobacco hornworm (Manduca sexta) caterpillars. In the first experiment we used high and low levels of light and water, and then, using scanning electron microscopy, determined trichome densities on the leaves and stems. We sampled leaves and stems at several places throughout the plant to determine whether there were within-plant differences in allocation to trichomes. The results of the first experiment showed that resource availability influenced allocation to trichome growth. Patterns in high and low-light supported both the growth-differentiation balance hypothesis (GDBH) and the carbon-nutrient balance hypothesis (CNBH). However, the GDBH was not supported by differences among water treatments. Contrary, to predictions of the GDBH, plants with intermediate growth did not have the highest trichome densities, and plants with similar growth differed in trichome density. Possible biological and artifactual explanations are discussed. The first experiment also showed that there was within-plant variation in allocation to trichomes, and that plant resource availability may influence within-plant variation in allocation to trichomes. In the second experiment, we grew plants in high and low-light, and then monitored the behavior of tobacco hornworms on the stems of these plants in the laboratory. This experiment demonstrated that the light environment that tomato plants were grown in influenced the resting behavior of caterpillars. Furthermore, it demonstrated that both glandular and non-glandular trichomes impeded caterpillars from searching for food. Overall, this study indicated that plant resource availability can influence allocation to trichome defenses, and that these differences may affect insect herbivores.