Systemic Induction of Terpenoid Aldehydes in Cotton Pigment Glands by Feeding of Larval Spodoptera exigua

Pigment glands in cotton contain terpenoid aldehydes that are toxic and deterrent to feeding of several generalist lepidopteran insects. We hypothesized that previously observed systemically induced feeding deterrence may be associated with pigment glands. We conducted experiments to determine the dynamics and chemical nature of inducible feeding deterrents in leaves of cotton, Gossypium hirsutum L, to larvae of the beet armyworm, Spodoptera exigua. Production and/or filling of pigment glands was influenced by physiological age of Deltapine 90 cotton plants. In undamaged plants, successively formed leaves contained more pigment glands, up to the seventh or eighth true-leaf developmental stage. Feeding choice tests conducted one or seven days after initial feeding damage revealed that third instars of S. exigua consumed more of the two youngest leaves from control cotton plants than from plants whose two oldest leaves had been fed on previously for 24 hr by S. exigua. The preference for leaves from control plants was significant one day after initial damage and highly significant seven days after damage. Consumption of mature foliage (leaf immediately above initially damaged leaves) from control plants and damaged plants did not differ. More pigment glands were counted on the youngest leaf of damaged plants than on the youngest leaf of control plants one day after initial damage. HPLC analysis revealed greater amounts of hemigossypolone, heliocides 1 and 2 (H₁ and H₂), and total terpenoid aldehydes per gland in young foliage of damaged plants than control plants one day after initial injury. By seven days after initial injury, greater quantities of hemigossypolone and all heliocides except H₄ were detected in young foliage from damaged plants compared to control plants. Concentrations of H₁ per gland in young leaves from damaged plants increased the most of all terpenoid aldehydes measured (3.4× the amount found in leaves from control plants). Mature leaves from damaged plants did not contain more terpenoid aldehydes than mature leaves from control plants. We suggest that systemically induced feeding deterrence to S. exigua in young leaves of glanded cotton was due to increased amounts of terpenoid aldehydes in pigment glands.     

The Role of Indole and Other Shikimic Acid Derived Maize Volatiles in the Attraction of Two Parasitic Wasps

After herbivore attack, plants release a plethora of different volatile organic compounds (VOCs), which results in odor blends that are attractive to predators and parasitoids of these herbivores. VOCs in the odor blends emitted by maize plants (Zea mays) infested by lepidopteran larvae are well characterized. They are derived from at least three different biochemical pathways, but the relative importance of each pathway for the production of VOCs that attract parasitic wasps is unknown. Here, we studied the importance of shikimic acid derived VOCs for the attraction of females of the parasitoids Cotesia marginiventris and Microplitis rufiventris. By incubating caterpillar-infested maize plants in glyphosate, an inhibitor of the 5-enolpyruvylshikimate-3-phospate (EPSP) synthase, we obtained induced odor blends with only minute amounts of shikimic acid derived VOCs. In olfactometer bioassays, the inhibited plants were as attractive to naive C. marginiventris females as control plants that released normal amounts of shikimic acid derived VOCs, whereas naive M. rufiventris females preferred inhibited plants to control plants. By adding back synthetic indole, the quantitatively most important shikimic acid derived VOC in induced maize odors, to inhibited plants, we showed that indole had no effect on the attraction of C. marginiventris and that M. rufiventris preferred blends without synthetic indole. Exposing C. marginiventris females either to odor blends of inhibited or control plants during oviposition experiences shifted their preference in subsequent olfactometer tests in favor of the experienced odor. Further learning experiments with synthetic indole showed that C. marginiventris can learn to respond to this compound, but that this does not affect its choices between natural induced blends with or without indole. We hypothesize that for naïve wasps the attractiveness of an herbivore-induced odor blend is reduced due to masking by nonattractive compounds, and that during oviposition experiences in the presence of complex odor blends, parasitoids strongly associate some compounds, whereas others are largely ignored.     

The Role of Fresh <Emphasis Type="Italic">versus</Emphasis> Old Leaf Damage in the Attraction of Parasitic Wasps to Herbivore-Induced Maize Volatiles

The odor produced by a plant under herbivore attack is often used by parasitic wasps to locate hosts. Any type of surface damage commonly causes plant leaves to release so-called green leaf volatiles, whereas blends of inducible compounds are more specific for herbivore attack and can vary considerably among plant genotypes. We compared the responses of naïve and experienced parasitoids of the species Cotesia marginiventris and Microplitis rufiventris to volatiles from maize leaves with fresh damage (mainly green leaf volatiles) vs. old damage (mainly terpenoids) in a six-arm olfactometer. These braconid wasps are both solitary endoparasitoids of lepidopteran larvae, but differ in geographical origin and host range. In choice experiments with odor blends from maize plants with fresh damage vs. blends from plants with old damage, inexperienced C. marginiventris showed a preference for the volatiles from freshly damaged leaves. No such preference was observed for inexperienced M. rufiventris. After an oviposition experience in hosts feeding on maize plants, C. marginiventris females were more attracted by a mixture of volatiles from fresh and old damage. Apparently, C. marginiventris has an innate preference for the odor of freshly damaged leaves, and this preference shifts in favor of a blend containing a mixture of green leaf volatiles plus terpenoids, after experiencing the latter blend in association with hosts. M. rufiventris responded poorly after experience and preferred fresh damage odors. Possibly, after associative learning, this species uses cues that are more directly related with the host presence, such as volatiles from host feces, which were not present in the odor sources offered in the olfactometer. The results demonstrate the complexity of the use of plant volatiles by parasitoids and show that different parasitoid species have evolved different strategies to exploit these signals.     

An elicitor in caterpillar oral secretions that induces corn seedlings to emit chemical signals attractive to parasitic wasps

Regurgitate of corn-fed beet armyworm (BAW) caterpillars,Spodoptera exigua, when applied to damaged sites of corn (Zea mays) seedlings, causes the release of relatively large amounts of terpenes by the seedlings several hours later. This plant response could be induced by merely placing the cut stem of seedlings in a solution of BAW regurgitate for 12 hr, a response that could not be induced by placing seedlings in water only. Regurgitate of BAW fed various diets, including a minimal diet of filter paper, were all active. However, seedlings placed in corn leaf juice, BAW hemolymph, or BAW feces extract released significantly smaller amounts of terpenes than did seedlings placed in BAW regurgitate. These results indicate that the active components are present in relatively large concentrations in regurgitate and that they are not related to the food source. Furthermore, regurgitate from several other species of caterpillars (Spodoptera frugiperda, Helicoverpa zea,Trichoplusia ni, andAnticarsia gemmatalis) as well as from the grasshopperSchistocerca americana induced the release of significant amounts of terpenes in corn seedlings. The release of these volatiles, therefore, appears to be a general response to attack by phytophagous insects. The terpene-releasing corn seedlings were highly attractive to the generalist parasitoidCotesia marginiventris and to the specialized parasitoidMicroplitis croceipes. This study confirms a systemic herbivore-elicited release of terpenes in corn. It is proposed that such chemicals serve multifunctional purposes that directly and indirectly protect plants against herbivorous arthropods and pathogens.     

Systemic Induction of Allelochemicals in Glanded and Glandless Isogenic Cotton by Spodoptera exigua Feeding

Induction of systemic resistance to feeding of beet armyworm, Spodoptera exigua, was investigated in two isogenic lines of Stoneville 213 cotton, Gossypium hirsutum, that differed in the presence of pigment glands. In laboratory bioassays, larvae strongly preferred to feed on glandless cotton plants when presented a choice between undamaged terminal leaves of undamaged glanded and glandless plants. Feeding damage inflicted by S. exigua larvae on the two oldest leaves of glanded plants seven days prior to feeding bioassays caused larvae to prefer by 33-fold the undamaged terminal foliage from undamaged plants compared to that from damaged plants. Feeding damage on glandless plants caused only a 2.6-fold greater preference for terminal foliage from undamaged plants over foliage from previously damaged plants. Extracts of terminal foliage from glanded cotton damaged seven days earlier had significantly greater quantities of terpenoid aldehydes (hemigossypolone, gossypol, and heliocides) than did foliage from undamaged glanded plants. Terpenoid aldehydes were undetectable in extracts of both undamaged and previously damaged glandless plants. The profile of volatile compounds collected from the headspace of mechanically damaged terminal leaves of undamaged glanded and glandless plants differed. Both cotton isolines released large quantities of lipoxygenase products (hexenyl alcohols, acetates, and butyrates), but glandless plants released only small amounts of mono- and sesquiterpenes compared to glanded plants. Glandless plants damaged seven days prior to volatile collection released significantly greater quantities of lipoxygenase products, -ocimene, and - and -farnesene than did undamaged glandless plants. Previously damaged glanded plants released significantly greater quantities of all mono- and sesquiterpenes and hexenyl acetates and butyrates, but not alcohols. The relative importance of volatile compounds versus terpenoid aldehydes in induced feeding deterrence in cotton to S. exigua larvae is still unclear.     

Systemic induction of feeding deterrents in cotton plants by feeding ofSpodoptera SPP. Larvae

Cotton,Gossypium hirsutum L., has been shown to exhibit systemic induced resistance to arthropods under certain conditions. We conducted experiments to determine the effects of previous feeding ofSpodoptera exigua Hübner andSpodoptera littoralis (Boisd.) larvae on feeding behavior, growth, and survival of larvae subsequently feeding on cotton. In one feeding choice test,S. exigua larvae preferred young leaves from undamaged control plants to undamaged young leaves from a previously damaged plant. Feeding deterrence was noticeable after only 6 hr of initial feeding damage by larvae, and there was almost complete deterrence after 30 and 54 hr of continuous feeding. In a second feeding choice test,S. littoralis larvae fed more on mature leaves from undamaged control plants than on undamaged mature leaves from previously damaged plants. In no-choice tests, third instars ofS. littoralis fed undamaged young leaves from damaged plants did not gain weight and died by the seventh day, whereas larvae fed young leaves from undamaged control plants gained weight and pupated within 11 days. Sixth instars ofS. littoralis fed either old damaged leaves, old undamaged leaves, or young undamaged leaves all from previously damaged plants gained weight slowly and took more than 12 days to pupate, whereas larvae fed young leaves from undamaged plants gained weight rapidly and pupated within five days of the beginning of the experiment.     

Differences in Effects of Pyrrolizidine Alkaloids on Five Generalist Insect Herbivore Species

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.     

Effects of Water Deficit Stress, Shade, Weed Competition, and Kaolin Particle Film on Selected Foliar Free Amino Acid Accumulations in Cotton, Gossypium hirsutum (L.)

Leaves of cotton plants, Gossypium hirsutum L., stressed by water deficit, reduced daylight, and weed competition, or treated with a kaolin wettable powder formulation were analyzed for levels of 17 free amino acids (FAAs) using reversed-phase high-performance liquid chromatography. Water deficit stress resulted in heightened free proline levels (49.9-fold, P < 0.001) that were correlated with diffusive resistance (seconds per centimeter). Five other FAAs increased, and the amounts of total free essential (for insect growth and development) amino acids and total FAAs also increased (P 0.05). Cotton grown in 50% shade accumulated significantly more free arginine than control plants. In a small-plot weed competition assay, four FAAs increased and three FAAs decreased in association with weed competition, but because free proline levels were not altered and free arginine levels increased, other stresses aside from water deficit, possibly including shading by tall weeds, appear to have caused the changes. In a small-plot kaolin particle film assay, five FAAs were lower in cotton foliage sprayed weekly with kaolin. Because free proline was unaffected and free arginine was lower, it is possible that kaolin's reflectivity heightened light reception. The responses of free proline and arginine to the treatments used in these assays demonstrate that types and degrees of some stresses to cotton can be characterized by accumulations of certain FAAs. The study also demonstrates how some FAA levels can indicate degrees of cotton stress resulting from weed competition and from kaolin particle film application. Porometry and leaf water potential measurements assisted in corroborating some findings of the study.     

Spodoptera exigua Oviposition and Larval Feeding Preferences for Pigweed, Amaranthus hybridus, over Squaring Cotton, Gossypium hirsutum, and a Comparison of Free Amino Acids in Each Host Plant

The beet armyworm, Spodoptera exigua (Hübner), can utilize a number of different host plants for oviposition and larval development, but some host plants are preferred over others. This study, using cage choice tests and olfactometer assays, demonstrates the beet armyworm's preference for pigweed, Amaranthus hybridus L., over cotton, Gossypium hirsutum L. Cage and olfactometer choice assays indicated that olfaction plays an important role in host selection by ovipositing females. First instars exhibited no feeding preference, but the more mobile third instars showed a significant feeding preference for excised pigweed leaves. The higher quantities and more diverse accumulations of free amino acids in pigweed might, in conjunction with other physiochemical and mechanical attractant and deterrent factors in the two-plant species system, play roles in the tendency of the beet armyworm to select pigweed over cotton and in providing a superior array of easily absorbed amino acids as compared to cotton.     

Induction of Plant Synomones by Oviposition of a Phytophagous Insect

Earlier investigations of host habitat location in the egg parasitoid Oomyzus gallerucae have shown that oviposition of the elm leaf beetle (Xanthogaleruca luteola) induces the field elm (Ulmus minor) to emit volatiles that attract the egg parasitoid. In this study we investigated the mechanism of this induction by testing the effects of differently treated elm leaves on O. gallerucae in a four-arm olfactometer. First we investigated which sequence of the herbivore oviposition behavior is necessary for the synomone induction. The following major sequences were observed: (1) Prior oviposition, the gravid female gnawed shallow grooves into the leaf surface. (2) After gnawing upon the leaf surface, the female attached about 20–30 eggs with oviduct secretion in the grooves. We experimentally mimicked the shallow grooves on the leaf surface by scratching the leaf surface with a scalpel (= scratched leaves). Volatiles from such scratched leaves did not attract the egg parasitoid. However, as soon as eggs with oviduct secretion, or only oviduct secretion, was applied to these scratched leaves, they emitted attractive volatiles. Application of oviduct secretion and eggs on undamaged leaves did not elicit release of attractive synomones. Thus, an elicitor is located in the oviduct secretion, but becomes active only when the leaf surface is damaged. Jasmonic acid is known as a mediator of plant responses induced by feeding of herbivorous arthropods, and we demonstrate that it mediates production of elm synomones that attract O. gallerucae. The plant's reaction to oviposition was systemic, and leaves without eggs near leaves with eggs emitted attractants.     

Chemical Stimulants of Leaf-Trenching by Cabbage Loopers: Natural Products, Neurotransmitters, Insecticides, and Drugs

Larvae of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae), often transect leaves with a narrow trench before eating the distal section. The trench reduces larval exposure to exudates, such as latex, during feeding. Plant species that do not emit exudate, such as Plantago lanceolata, are not trenched. However, if exudate is applied to a looper's mouth during feeding on P. lanceolata, the larva will often stop and cut a trench. Dissolved chemicals can be similarly applied and tested for effectiveness at triggering trenching. With this assay, I have documented that lactucin from lettuce latex (Lactuca sativa), myristicin from parsley oil (Petroselinum crispum), and lobeline from cardinal flower (Lobelia cardinalis) elicit trenching. These compounds are the first trenching stimulants reported. Several other constituents of lettuce and parsley, including some phenylpropanoids, monoterpenes, and furanocoumarins had little or no activity. Cucurbitacin E glycoside found in cucurbits, another plant family trenched by cabbage loopers, also was inactive. Lactucin, myristicin, and lobeline all affect the nervous system of mammals, with lobeline acting specifically as an antagonist of nicotinic acetylcholine receptors. To determine if cabbage loopers respond selectively to compounds active at acetylcholine synapses, I tested several neurotransmitters, insecticides, and drugs with known neurological activity, many of which triggered trenching. Active compounds included dopamine, serotonin, the insecticide imidacloprid, and various drugs such as ipratropium, apomorphine, buspirone, and metoclopramide. These results document that noxious plant chemicals trigger trenching, that loopers respond to different trenching stimulants in different plants, that diverse neuroactive chemicals elicit the behavior, and that feeding deterrents are not all trenching stimulants. The trenching assay offers a novel approach for identifying defensive plant compounds with potential uses in agriculture or medicine. Cabbage loopers in the lab and field routinely trench and feed on plants in the Asteraceae and Apiaceae. However, first and third instar larvae enclosed on Lobelia cardinalis (Campanulaceae) failed to develop, even though the third instar larvae attempted to trench. Trenching ability does not guarantee effective feeding on plants with canal-borne exudates. Cabbage loopers must not only recognize and respond to trenching stimulants, they must also tolerate exudates during the trenching procedure to disable canalicular defenses.