Sexta Caterpillars Research Articles

Caterpillars (Manduca sexta) 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 molt. These microstructures were compared to 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/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 subesophageal 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 was not due to damage to the central pattern generator (CPG) for chewing, motor nerves or chewing muscles, but was more likely due to a suppression of chewing CPG activity by ascending or descending inputs.

Culex tarsalis Is a Competent Host of the Insect-Specific Alphavirus Eilat Virus (EILV).

Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV's host competence and tissue tropism in five mosquito species: Aedes aegypti, Culex tarsalis, Anopheles gambiae, Anopheles stephensi, and Anopheles albimanus. Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host (Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis-a vector of harmful human pathogens, including West Nile virus-is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission-a crucial step in discerning how Eilat virus maintains itself in nature.

Adverse effects of inbreeding on the transgenerational expression of herbivore-induced defense traits in Solanum carolinense.

In addition to directly inducing physical and chemical defenses, herbivory experienced by plants in one generation can influence the expression of defensive traits in offspring. Plant defense phenotypes can be compromised by inbreeding, and there is some evidence that such adverse effects can extend to the transgenerational expression of induced resistance. We explored how the inbreeding status of maternal Solanum carolinense plants influenced the transgenerational effects of herbivory on the defensive traits and herbivore resistance of offspring. Manduca sexta caterpillars were used to damage inbred and outbred S. carolinense maternal plants and cross pollinations were performed to produced seeds from herbivore-damaged and undamaged, inbred and outbred maternal plants. Seeds were grown in the greenhouse to assess offspring defense-related traits (i.e., leaf trichomes, internode spines, volatile organic compounds) and resistance to herbivores. We found that feeding by M. sexta caterpillars on maternal plants had a positive influence on trichome and spine production in offspring and that caterpillar development on offspring of herbivore-damaged maternal plants was delayed relative to that on offspring of undamaged plants. Offspring of inbred maternal plants had reduced spine production, compared to those of outbred maternal plants, and caterpillars performed better on the offspring of inbred plants. Both herbivory and inbreeding in the maternal generation altered volatile emissions of offspring. In general, maternal plant inbreeding dampened transgenerational effects of herbivory on offspring defensive traits and herbivore resistance. Taken together, this study demonstrates that inducible defenses in S. carolinense can persist across generations and that inbreeding compromises transgenerational resistance in S. carolinense.

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.

Picking sides: feeding on the abaxial leaf surface is costly for caterpillars.

The study provides us with the evidence that caterpillars tend to feed on the abaxial leaf surface despite the damage caused to them because of higher trichome density. To defend against herbivory, plants have evolved physical and chemical defense mechanisms, including trichomes (hair like appendages on leaves and stem) being one of them. Caterpillars, a major group of insect herbivores are generally found to occupy the abaxial (underside) leaf surface, considered as an avoidance mechanism from biotic and abiotic stresses. Since trichomes are a first line of defense, we examined the correlation between abaxial vs adaxial (above side) trichomes and caterpillar feeding, behavior, and growth. A combination of field, lab and microscopy experiments were performed using tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae), a Solanaceae specialist caterpillar, and multiple host species. We found that M. sexta caterpillars overwhelmingly preferred to stay and feed on the abaxial leaf surface, but the abaxial leaf surface also had significantly more trichomes, and consequently, caterpillars took significantly longer to commence feeding. In addition, lab-based diet experiment containing shaved trichomes showed that feeding on the abaxial leaf surface with more trichomes also affected caterpillar growth. Taken together, our study shows that although caterpillars prefer to feed on the abaxial leaf surface, they accrue feeding delays and developmental constraints, indicating tradeoffs affecting performance, and exposure to predation and abiotic stressors.

Tobacco Hornworm (Manduca sexta) caterpillars as a novel host model for the study of fungal virulence and drug efficacy

ABSTRACT The two leading yeast pathogens of humans, Candida albicans and Cryptococcus neoformans, cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. albicans, C. neoformans, C. auris, and C. glabrata. Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar’s feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. M. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.

Transgenerational impacts of herbivory and inbreeding on reproductive output in Solanum carolinense.

PremisePlant maternal effects on offspring phenotypes are well documented. However, little is known about how herbivory on maternal plants affects offspring fitness. Furthermore, while inbreeding is known to reduce plant reproductive output, previous studies have not explored whether and how such effects may extend across generations. Here, we addressed the transgenerational consequences of herbivory and maternal plant inbreeding on the reproduction of Solanum carolinense offspring.Methods Manduca sexta caterpillars were used to inflict weekly damage on inbred and outbred S. carolinense maternal plants. Cross‐pollinations were performed by hand to produce seed from herbivore‐damaged outbred plants, herbivore‐damaged inbred plants, undamaged outbred plants, and undamaged inbred plants. The resulting seeds were grown in the greenhouse to assess emergence rate and flower production in the absence of herbivores. We also grew offspring in the field to examine reproductive output under natural conditions.ResultsWe found transgenerational effects of herbivory and maternal plant inbreeding on seedling emergence and reproductive output. Offspring of herbivore‐damaged plants had greater emergence, flowered earlier, and produced more flowers and seeds than offspring of undamaged plants. Offspring of outbred maternal plants also had greater seedling emergence and reproductive output than offspring of inbred maternal plants, even though all offspring were outbred. Moreover, the effects of maternal plant inbreeding were more severe when plant offspring were grown in field conditions.ConclusionsThis study demonstrates that both herbivory and inbreeding have fitness consequences that extend across generations even in outbred progeny.

Cascading effects of polyphenol-rich purple corn pericarp extract on pupal, adult, and offspring of tobacco hornworm (Manduca sexta L.)

ABSTRACT A major bottleneck in the commercialization of plant-based pest management compounds is that the extraction methods are complex, time-consuming, and even highly expensive. Using a recently developed inexpensive extraction and quantification methodology to isolate polyphenols (including anthocyanins and condensed tannins) from purple corn pericarp, we examined their effects on Manduca sexta, a common insect herbivore. Following up on our previous work which demonstrated the negative impacts of polyphenol-rich extract on larval stages, we further examined whether there are any cascading effects on subsequent life stages (pupal and adult) including any possible transgenerational effects. Our results show that polyphenol-rich purple corn extract-fed caterpillars had significantly lower pupal mass and survival. Moreover, adult moths also had lower mass when eclosed from caterpillars reared on the extract diet. To test whether there were any transgenerational effects, we allowed male and female adults fed on purple corn extract diet and control diet to mate and lay eggs in a full factorial experiment. We found that purple corn extract-fed adult pair laid a lower number of eggs compared to other treatments. In addition, we also found that second instar M. sexta caterpillars hatched from eggs laid by any mating combination with at least one parent reared on purple corn extract gained significantly lower mass compared to caterpillars with both parents reared on the control diet. Taken together, our results show that there are cascading negative effects for feeding purple corn pericarp extract on pupal, adult, and second generation of M. sexta, reaffirming its potential application as a cost-effective and environmentally friendly pest deterrent.

Aboveground herbivory induced jasmonates disproportionately reduce plant reproductive potential by facilitating root nematode infestation.

Different plant feeders, including insects and parasitic nematodes, can influence each other by triggering systemic changes in their shared host plants. In most cases, however, the underlying mechanisms are unclear, and the consequences for plant fitness are not well understood. We studied the interaction between leaf feeding Manduca sexta caterpillars and root parasitic nematodes in Nicotiana attenuata. Simulated M.sexta attack increased the abundance of root parasitic nematodes in the field and facilitated Meloidogyne incognita reproduction in the glasshouse. Intact jasmonate biosynthesis was found to be required for both effects. Flower counts revealed that the jasmonate-dependent facilitation of nematode infestation following simulated leaf attack reduces the plant's reproductive potential to a greater degree than would be expected from the additive effects of the individual stresses. This work reveals that jasmonates mediate the interaction between a leaf herbivore and root parasitic nematodes and illustrates how plant-mediated interactions can alter plant's reproductive potential. The selection pressure resulting from the demonstrated fitness effects is likely to influence the evolution of plant defense traits in nature.

Protection via parasitism: Datura odors attract parasitoid flies, which inhibit Manduca larvae from feeding and growing but may not help plants.

Insect carnivores frequently use olfactory cues from plants to find prey or hosts. For plants, the benefits of attracting parasitoids have been controversial, partly because parasitoids often do not kill their host insect immediately. Furthermore, most research has focused on the effects of solitary parasitoids on growth and feeding of hosts, even though many parasitoids are gregarious (multiple siblings inhabit the same host). Here, we examine how a gregarious parasitoid, the tachinid fly Drino rhoeo, uses olfactory cues from the host plant Datura wrightii to find the sphingid herbivore Manduca sexta, and how parasitism affects growth and feeding of host larvae. In behavioral trials using a Y-olfactometer, female flies were attracted to olfactory cues emitted by attacked plants and by cues emitted from the frass produced by larval Manduca sexta. M. sexta caterpillars that were parasitized by D. rhoeo grew to lower maximum weights, grew more slowly, and ate less of their host plant. We also present an analytical model to predict how tri-trophic interactions change with varying herbivory levels, parasitization rates and plant sizes. This model predicted that smaller plants gain a relatively greater benefit compared to large plants in attracting D. rhoeo. By assessing the behavior, the effects of host performance, and the variation in ecological parameters of the system, we can better understand the complex interactions between herbivorous insects, the plants they live on and the third trophic level members that attack them.

Jasmonate-dependent depletion of soluble sugars compromises plant resistance to Manduca sexta.

Jasmonates regulate plant secondary metabolism and herbivore resistance. How they influence primary metabolites and how this may affect herbivore growth and performance are not well understood. We profiled sugars and starch of jasmonate biosynthesis-deficient and jasmonate-insensitive Nicotiana attenuata plants and manipulated leaf carbohydrates through genetic engineering and invitro complementation to assess how jasmonate-dependent sugar accumulation affects the growth of Manduca sexta caterpillars. We found that jasmonates reduce the constitutive and herbivore-induced concentration of glucose and fructose in the leaves across different developmental stages. Diurnal, jasmonate-dependent inhibition of invertase activity was identified as a likely mechanism for this phenomenon. Contrary to our expectation, both inplanta and invitro approaches showed that the lower sugar concentrations led to increased M.sexta growth. As a consequence, jasmonate-dependent depletion of sugars rendered N.attenuata plants more susceptible to M.sexta attack. In conclusion, jasmonates are important regulators of leaf carbohydrate accumulation and this determines herbivore growth. Jasmonate-dependent resistance is reduced rather than enhanced through the suppression of glucose and fructose concentrations, which may contribute to the evolution of divergent resistance strategies of plants in nature.

Functional annotation of Cotesia congregata bracovirus: identification of viral genes expressed in parasitized host immune tissues.

Bracoviruses (BVs) from the Polydnaviridae family are symbiotic viruses used as biological weapons by parasitoid wasps to manipulate lepidopteran host physiology and induce parasitism success. BV particles are produced by wasp ovaries and injected along with the eggs into the caterpillar host body, where viral gene expression is necessary for wasp development. Recent sequencing of the proviral genome of Cotesia congregata BV (CcBV) identified 222 predicted virulence genes present on 35 proviral segments integrated into the wasp genome. To date, the expressions of only a few selected candidate virulence genes have been studied in the caterpillar host, and we lacked a global vision of viral gene expression. In this study, a large-scale transcriptomic analysis by 454 sequencing of two immune tissues (fat body and hemocytes) of parasitized Manduca sexta caterpillar hosts allowed the detection of expression of 88 CcBV genes expressed 24 h after the onset of parasitism. We linked the expression profiles of these genes to several factors, showing that different regulatory mechanisms control viral gene expression in the host. These factors include the presence of signal peptides in encoded proteins, diversification of promoter regions, and, more surprisingly, gene position on the proviral genome. Indeed, most genes for which expression could be detected are localized in particular proviral regions globally producing higher numbers of circles. Moreover, this polydnavirus (PDV) transcriptomic analysis also reveals that a majority of CcBV genes possess at least one intron and an arthropod transcription start site, consistent with an insect origin of these virulence genes. Bracoviruses (BVs) are symbiotic polydnaviruses used by parasitoid wasps to manipulate lepidopteran host physiology, ensuring wasp offspring survival. To date, the expressions of only a few selected candidate BV virulence genes have been studied in caterpillar hosts. We performed a large-scale analysis of BV gene expression in two immune tissues of Manduca sexta caterpillars parasitized by Cotesia congregata wasps. Genes for which expression could be detected corresponded to genes localized in particular regions of the viral genome globally producing higher numbers of circles. Our study thus brings an original global vision of viral gene expression and paves the way to the determination of the regulatory mechanisms enabling the expression of BV genes in targeted organisms, such as major insect pests. In addition, we identify sequence features suggesting that most BV virulence genes were acquired from insect genomes.

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.

Hypoxia‐induced compression of the tracheal system in the caterpillar, Manduca sexta

Abdominal pumping in caterpillars has only been documented during molting. Here, with synchrotron x‐ray imaging and high‐speed flow‐through respirometry, we show that Manduca sexta caterpillars contract their bodies in response to hypoxia, which results in significant compression of the tracheal system. Tracheal compression induced by abdominal contraction appears to be the driving force for external gas exchange, as evidenced by the high correlation between CO2 emission peaks and external body movements. Both frequency and diameter of tracheal compression increased with body mass as caterpillars increased in age. However, abdominal pumping was only observed in larger, older caterpillars (> 0.2 g body mass), suggesting that the hypoxia response varies with ontogeny. As insects increased in size, the fraction of tracheal system structures in the head increased, but not as much as would be predicted based on geometric scaling. The fraction of the body occupied by tracheae in the prothorax and last abdominal segment remain constant throughout ontogeny. Furthermore, diameter of major tracheae either do not vary with body mass or do not increase as much as expected, suggesting that trade‐offs between non‐respiratory structures result in smaller tracheae than would be expected based on geometric scaling. (Supported by NSF, NIH‐NCRR and US DOE)

Not all sugars are created equal: some mask aversive tastes better than others in an herbivorous insect

Manduca sexta caterpillars are unusual because they exhibit strong peripheral gustatory responses to sugars, but nevertheless fail to show immediate appetitive responses to them. We hypothesized that the primary function of the peripheral gustatory response to sugars is to mask the taste of noxious compounds, which abound in host plants of M. sexta. We compared 10 s biting responses to water with those to mixtures of a noxious compound [caffeine (Caf) or aristolochic acid (AA)] and various combinations of sugars [i.e. sucrose (Suc), glucose (Glu), inositol (Ino), Suc+Glu, Suc+Ino or Glu+Ino]. The biting assays indicated that the aversive taste of AA was completely masked by Suc+Ino, and partially masked by Suc+Glu, Glu+Ino and Suc, whereas that of Caf was completely masked by Suc+Ino and Suc+Glu, and partially masked by Glu+Ino, Suc and Ino. To examine the contribution of the peripheral taste system to the masking phenomenon, we recorded responses of the maxillary gustatory sensilla to each stimulus mixture. The sugars differed greatly in their capacity to suppress peripheral gustatory responses to AA and Caf: Suc+Ino and Suc+Glu produced the greatest suppression, and Glu and Ino the least. Further, the extent to which each sugar stimulus suppressed the peripheral gustatory responses to AA reliably predicted the extent to which it masked the taste of AA in biting assays; no such predictive relationship was observed for the sugar/Caf mixtures. We conclude that some, but not all, sugars act on both peripheral and central elements of the gustatory system to mask the taste of noxious compounds.

The role of jasmonic acid and ethylene crosstalk in direct defense of Nicotiana attenuata plants against chewing herbivores

We examined performance of herbivores on plants lacking either jasmonate (JA, asLOX3) or ethylene (ET, mETR1) signaling, or both (mETR1asLOX3). Plant defenses against Manduca sexta caterpillars were strongly impaired in JA-deficient asLOX3 plants; however, making asLOX3 plants ethylene insensitive did not further increase the performance of the larvae on a mETR1asLOX3 genetic cross. This result demonstrates the dominant role of JA over ET in the regulation of plant defenses against herbivores. However, ET-insensitivity combined with otherwise normal levels of JA in mETR1 plants promoted faster caterpillar growth, which correlated with reduced accumulation of the alkaloidal direct defense nicotine in mETR1 compared to WT plants. Our data points to an important accessory function of ET in the activation of JA-regulated plant defenses against herbivores at the level of alkaloid biosynthesis in the roots and/or accumulation in the leaves.

Plants’ Modified SOS Call

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

Plants' Modified SOS Call

Plants have several ways of defending themselves against insect attack. The release of distinctive volatile chemicals betrays the location of herbivores to their predators, but volatile production can be slow and the pests may escape. Compounds known as green leaf volatiles are released immediately after damage, and Allmann and Baldwin (p. [1075][1]) have found that, when attacked by tobacco hornworm ( Manduca sexta ) caterpillars, Nicotiana attenuata plants emit a compound that, when combined with an oral secretion from the caterpillar, is transformed into an attractant for the generalist hemipteran predator Geocoris pallidens , which preys on the hornworm eggs and young larvae. Thus, insect feeding activity can begin the process of plant defense before other protective volatiles are synthesized and emitted. [1]: /lookup/doi/10.1126/science.1191634

Visceral-Locomotory Pistoning in Crawling Caterpillars

Animals with an open coelom do not fully constrain internal tissues, and changes in tissue or organ position during body movements cannot be readily discerned from outside of the body. This complicates modeling of soft-bodied locomotion, because it obscures potentially important changes in the center of mass as a result of internal tissue movements. We used phase-contrast synchrotron X-ray imaging and transmission light microscopy to directly visualize internal soft-tissue movements in freely crawling caterpillars. Here we report a novel visceral-locomotory piston in crawling Manduca sexta larvae, in which the gut slides forward in advance of surrounding tissues. The initiation of gut sliding is synchronous with the start of the terminal prolegs' swing phase, suggesting that the animal's center of mass advances forward during the midabdominal prolegs' stance phase and is therefore decoupled from visible translations of the body. Based on synchrotron X-ray data and transmission light microscopy results, we present evidence for a two-body mechanical system with a nonlinear elastic gut that changes size and translates between the anterior and posterior of the animal. The proposed two-body system--the container and the contained--is unlike any form of legged locomotion previously reported and represents a new feature in our emerging understanding of crawling.

R2R3-NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores in Nicotiana attenuata.

Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine, increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY-2 cells regulates PPC biosynthesis, and silenced its expression by RNA interference in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPC biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and dicaffeoylspermidine showed no changes in reproductive parameters of the plants. In contrast, a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically preinduced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared with wild-type plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatiotemporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the optimal defense theory: plants preferentially protect their most fitness-enhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness.