Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-β-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system. Received: 4 November 1999 / Accepted: 1 March 2000     

The Essential Role of Jasmonic Acid in Plant-Herbivore Interactions - Using the Wild Tobacco Nicotiana attenuata as a Model

The plant hormone jasmonic acid （JA） plays a central role in plant defense against herbivores. Herbivore damage elicits a rapid and transient JA burst in the wounded leaves and JA functions as a signal to mediate the accumulation of various secondary metabolites that confer resistance to herbivores. Nicotiana attenuata is a wild tobacco species that inhabits western North America. More than fifteen years of study and its unique interaction with the specialist herbivore insect Manduca sexta have made this plant one of the best models for studying plant-herbivore interactions. Here we review the recent progress in understanding the elicitation of JA accumulation by herbivore-specific elicitors, the regulation of JA biosynthesis, JA signaling, and the herbivore-defense traits in N. attenuata.     

Using nutritional indices to study LOX3-dependent insect resistance

Induced resistance to biotic attackers is thought to be mediated by responses elicited by jasmonic acid (JA), a subset of which are lipoxygenase 3 (LOX3) dependent. To understand the importance of LOX3-mediated insect resistance, we analysed the performance of Manduca sexta larvae on wild-type (WT) and on isogenic Nicotiana attenuata plants silenced in NaLOX3 expression and JA signalling, and we used Waldbauer nutritional indices to measure the pre- and post-ingestive effects. LOX3-mediated defenses reduced larval growth, consumption and frass production. These defenses reduced how efficiently late-instar larvae converted digested food to body mass (ECD). In contrast, LOX3-mediated defenses decreased approximate digestibility (AD) in early instar larvae without affecting the ECD and total food consumption. Larvae of all instars feeding on defended WT plants behaviourally compensate for their reduced body mass by consuming more food per unit of body mass gain. We suggest that larvae feeding on plants silenced in NaLOX3 expression (as-lox) initially increase their AD, which in turn enables them to consume more food in the later stages and consequently, to increase their ECD and efficiency of conversion of ingested food (ECI). We conclude that N. attenuata's oxylipin-mediated defenses are important for resisting attack from M. sexta larvae, and that Waldbauer nutritional assays reveal behavioural and physiological counter responses of insects to these plant defenses.     

Immunolocalization of a proton V-ATPase in ovarian follicles of the tobacco hornworm Manduca sexta

A monoclonal antibody, directed against an H⁺ translocating V-ATPase of the midgut of Manduca sexta, has been used for immunolocalization studies in ovarian follicles and testes of Manduca sexta. In testes, no distinct staining above background levels was observed. In vitellogenic follicles, V-ATPase immunoreactivity first appears in the cytoplasm of the trophocytes and then in the oocyte, but by far the strongest reaction is present in the region of the oolemma during endocytosis. All types of follicle cells surrounding both the oocyte and the trophocyte compartments show a distinct positive reaction. In the cylindrical follicle cells surrounding the oocyte, the immunoreactivity is clearly restricted to the basal part. Our results suggest an important role for V-ATPase in vitellogenin uptake in Manduca, similar to that suggested on electro-physiological grounds in Hyalophora cecropia. © 1995 Wiley-Liss, Inc.     

Variability in odor-modulated flight by moths

Based on previous studies of odor-modulated flight where track parameter data was lumped and averaged, the speed and orientation of the moths' movement along their flight tracks have been said to be controlled to maintain certain “preferred” values. The results from our fine-scaled analysis of this behavior show that none of the track parameters typically measured are held constant. The moths' speed along the flight track is modulated substantially and predictably: fastest along the straight legs and slowest around the turns. In addition, about half of the individuals studied progressively reduced the peak speed along the straight legs as they approached the pheromone source. While most of the track legs between the turns were directed upwind, their orientations were widely distributed, indicating no preferred direction. Small fluctuations of orientation along some straight legs suggest corrective maneuvers to stabilize flight direction about an internal set point. The visual inputs hypothesized to control steering and speed, transverse and longitudinal image flow, changed continuously during upwind flight in pheromone, but no regular relationship between them was observed. We found that the orientation of the longitudinal body axis and the direction of thrust (course angle) were only rarely coincident during upwind flight to the odor source, suggesting that moths receive sensory input which differs quantitatively from that calculated by conventional methods. Our results strongly suggest that the long-accepted hypothetical mechanisms of control for this behavior do not operate in the manner in which they have been proposed. Accepted: 11 July 1997     

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo . Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli . The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.     

Characterization of a 46 kda insect chitinase from transgenic tobacco

A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.     

Absorption and storage of phosphorus by larval Manduca sexta

The role of phosphorus (P) in numerous important biological structures, coupled with the observation that P-content of many insect foods is disproportionately low, suggests that P may be a critical nutrient for growing insects — however, the few studies examining the effects of dietary P on insect performance have generally found only weak relationships. This mismatch may be reconciled by understanding the physiological mechanisms by which insects handle P. Here we describe P processing by larvae of Manduca sexta. When given un-manipulated leaves of a common host plant, Datura wrightii, fifth-instar larvae retained about 85% of P consumed; when given P-enriched leaves larvae retained only 25% of P consumed. Analysis of gut concentrations of P at four sites along the digestive tract, and in leaves and feces, indicates that the rectum is the primary site of P transport between the gut and body and that differences in P retention may be accounted for by differential rates of rectal P transport. Larvae given P-enriched leaves also showed an eightfold increase in the concentration of P in the hemolymph, primarily as α-glycerophosphate — but only a 12% increase in the concentration of P in body tissues, suggesting that hemolymph plays a central role in storage and buffering of P.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Glomerulus development in the absence of a set of mitral-like neurons in the insect olfactory lobe

Mitral cells are the first neurons in the mammalian olfactory bulb to synapse with olfactory receptor axons during glomerulus development, and in an invertebrate, the moth Manduca sexta, mitral-like neurons overlap very early with olfactory receptor axons as they begin to form protoglomeruli. The possibility for early interaction between receptor neurons and mitral-like neurons led us to ask whether such an interaction plays an essential role in glomerulus development. In the current study in the moth, we surgically removed a major class of these mitral-like neurons before glomeruli began to form and asked: (a) Is the formation of the array of olfactory glomeruli triggered by an interaction of the first-arriving receptor axons with the dendrites of mitral-like neurons? (b) At the level of individual glomeruli, must the mitral-like dendrites be in place either to maintain receptor axons in a glomerular arrangement, or to guide later-growing dendrites of other types into the developing glomeruli? Our results indicate that even without the participation of this group of mitral-like neurons, the array of sexually isomorphic ordinary glomeruli forms and the basic substructure of individual glomeruli develops apparently normally. We conclude that the mitral-like neurons in Manduca are not essential for the formation of ordinary olfactory glomeruli during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 41–52, 1998     

Behavioral strategies underlying pheromone-modulated flight in moths: lessons from simulation studies

Several mechanisms have been proposed to underlie the characteristic `zigzag' tracks produced by moths flying up an odor plume. To test which, if any, of these are capable of reproducing the animals' behavior, we constructed behavioral-level simulations. The simulations are as tightly constrained as possible by the known biology, and incorporate realistic physical effects such as air turbulence, and delays due to sensory transduction and axonal condition, to mimic the dynamics of sensory information encountered by real moths. Formulated as schemas, the models all share a common set of sensory and motor systems, but differ in the interposed control systems. We analyzed the behavior of the models with the same methods we use for real moths. Even the simplest of the models was capable of successful orientation some of the time, and of producing flight tracks similar to those of moths. Individuals which succeeded in tracking the odor plume produced average behavior not significantly different from that of real moths. As a population, however, none of the models was as successful as the moths. The best of the models had a success rate in tracking the plume of about 30%, compared to the average of 70% seen in the insects. Accepted: 25 March 1998     

Steroid control of muscle remodeling during metamorphosis in Manduca sexta

During metamorphosis in the tobacco hornworm, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. The degeneration of muscle DEO1 involves the dismantling of its contractile apparatus followed by the degeneration of muscle nuclei. As some nuclei are degenerating, others begin to incorporate 5-bromodeoxyuridine (BrdU), indicating the onset of nuclear proliferation. This proliferation is initially most evident at the site where the motoneuron contacts the muscle remnant. The developmental events involved in muscle remodeling are under the control of the steroid hormones, the ecdysteroids. The loss of the contractile elements of the larval muscle requires the rise and fall of the prepupal peak of ecdysteroids, whereas the subsequent loss of muscle nuclei is influenced by the slight rise in ecdysteroids seen after pupal ecdysis. Incorporation of BrdU by muscle nuclei depends on both the adult peak of the ecdysteroids and contact with the motoneuron. Unilateral axotomy blocks proliferation within the rudiment, but it does not block its subsequent differentiation into a very thin muscle in the adult. © 1996 John Wiley & Sons, Inc.     

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths,Manduca sexta (L.)

Analysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effects of prey quantity and quality on predatory wasps

1. The simultaneous effects of prey quantity and prey quality on fitness correlates of the predatory wasp Polistes fuscatus were examined in a glasshouse study. Prey quantity was manipulated by providing prey in excess (high quantity) or one‐third of that (low quantity). Prey quality was manipulated by providing either palatable (Manduca sexta) caterpillars or unpalatable (Junonia coenia) caterpillars. 2. The effect of prey quality on wasp production depended on prey quantity. Nests given unpalatable prey produced few wasps whereas nests given palatable prey increased wasp production with increased prey. 3. The low production of nests given unpalatable prey reflected the low acceptability of those prey. The wasps preferred the palatable prey and learned to reject the unpalatable prey. With no choice of prey, they took only enough unpalatable prey to develop a small nest or colony. 4. A diet of unpalatable prey also resulted in smaller wasps and reduced the proportion of males produced, from about 40% to just 8–14%, depending on the year.