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.     

Effects on behavior ofApanteles melanoscelus females caused by modifications in extraction,storage, and presentation of Gypsy moth silk kairomone

Choice experiments were performed to investigate details of femaleApanteles melanoscelus (Ratzeburg) behavior when exposed to gypsy moth silk kairomone [Lymantria dispar (L.)] and to host larvae when kairomone is present. Female parasites only responded to the kairomone when it had been placed on thin strands such as cotton fibers. Both gypsy moth silk and silk glands contain the same or similar kairomones. Silk gland extracts were more active than head, alimentary canal, or hemolymph extracts of host larvae. Female responses decreased when low concentrations of silk gland extract or small numbers of treated fibers were presented to them. Silk gland extract was stable when frozen for 2 weeks, heated to 100 °C for 0.5 hr, freeze dried, or treated with 95% ethanol. The active component was nondialyzable. Silk deposited on the substrate increased host contacts and oviposition attempts, more so if wider areas were covered with silk, and even if the areas having silk were separated from the host. A theory of host selection inA. melanoscelus is proposed.Hymenoptera, Braconidae. Lymantria dispar L. (Lepidoptera: Lymantriidae).     

Role of diet in host selection ofHeliothis virescens by parasitoidCampoletis sonorensis (Hymenoptera: Ichneumonidae)

Gas chromatographic analysis revealed that chemicals attractive toCampoletis sonorensis (Cameron), previously identified in cotton, were not present in wheat germ diet-rearedHeliothis virescens (F.) larvae. Diet-reared larvae fed cotton obtained the chemicals from cotton, with the consequence of enhanced kairomonal activity of the larvae and their frass toC. sonorensis. Parasitoids, presented a choice between cotton, cotton plus hosts, hosts alone, and control in an olfactometer, responded non-randomly, with the greatest number of responses to cotton plus hosts, and three times as many responses to cotton alone as to larvae alone. The role of the plant in the parasitoid-host relationship is discussed.     

Investigation of oviposition deterrent in larval frass ofSpodoptera littoralis (Boisd.)

Previous experiments demonstrated an oviposition-deterring effect of larval frass in the Egyptian cotton leaf worm,Spodoptera littoralis (Boisd.). In this study, females were shown to perceive the oviposition-deterring substance(s) with their antennae. During dark, airtight, and cold (–10 °C) storage, the deterrent was persistent for at least 395 days. On the other hand, larval frass retained its activity for only two days when applied to cotton leaves. The deterrent activity of frass was independent of larval density. Frass of larvae reared at high densities deterred oviposition as well as frass of larvae feeding separately or in small groups. For significant oviposition deterrence, the minimum amount of frass was in the range of 5–10 mg frass per cotton leaf. An acetone extract of larval frass was highly deterrent, in contrast to extracts prepared with water, ethanol, chloroform, or pentane.     

Baldcypress allelochemics and the inhibition of silkworm enteric microorganisms Some Ecological Considerations

Exposure ofBombyx mori larvae to 2-furaldehyde, a major volatile component of baldcypress heartwood,Taxodium distichum, resulted in in vivo inhibition of enteric microorganisms at concentrations (1–47 ppm) that were released naturally from heartwood. The 7 bacterial and 2 fungal enteric isolates were also inhibited in vitro at the same concentrations. It is suggested that inhibition of leaf surface micro-organisms or in vivo inhibition of silkworm enteric microflora, as a result of indirect or direct action of 2-furaldehyde, exacerbates the growth-inhibitory effects of this compound on larvae by reducing the microbial nutritional contribution. The ecological significance of insect enteric microbial inhibition by plant allelochemics is discussed.     

Sex pheromone scent marking by females ofPectinophora gossypiella (Lepidoptera: Gelechiidae)

Females of the pink bollworm moth,Pectinophora gossypiella (Saunders), scent marked the substrate to which they were clinging with the extended pheromone gland during nocturnal pheromonereleasing periods. Scent marks were deposited on both natural (cotton leaf) and atypical (glass) substrates. In both situations, the scent marks elicited attraction responses from conspecific males.     

Defensive Role of Tomato Polyphenol Oxidases against Cotton Bollworm (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) and Beet Armyworm (<Emphasis Type="Italic">Spodoptera exigua</Emphasis>)

Tomato (Solanum lycopersicum) polyphenol oxidases (PPOs), enzymes that oxidize phenolics to quinones, have been implicated in plant resistance to insects. The role of PPO in resistance to cotton bollworm [Helicoverpa armigera (Hübner)] and beet armyworm [Spodoptera exigua (Hübner)] (Lepidoptera: Noctuidae) was evaluated. Consumption, weight gains, and mortality of larvae feeding on foliage of transgenic tomato lines overexpressing PPO (OP lines) and of larvae feeding on foliage of transgenic tomato lines with suppressed PPO (SP lines) were compared with consumption, weight gains, and mortality of larvae feeding on non-transformed (NT) plants. Increases in foliage consumption and weight gains were observed for cotton bollworms feeding on leaves of SP plants compared to NT and OP plants. PPO activity was negatively correlated with both weight gains and foliar consumption of cotton bollworm, substantiating the defensive role of PPO against this insect. Similarly, beet armyworm consumed less foliage (both young and old leaves) from OP plants than SP plants. Larvae feeding on OP leaves generally exhibited lower weight gains than those feeding on SP leaves. These results indicate that tomato PPO plays a role in resistance to both cotton bollworm and beet armyworm.     

Role of the Lipoxygenase/lyase Pathway of Host-food Plants in the Host Searching Behavior of Two Parasitoid Species, Cotesia glomerata and Cotesia plutellae

To elucidate the role of the plant lipoxygenase (LOX)/lyase pathway for host search behavior of two parasitic wasps attacking herbivorous larvae, an Arabidopsis mutant (all84) was isolated with a mutation somewhere in the LOX/lyase pathway. Detached leaves of the mutant were shown to release less (Z)-3-hexenal, a first green leaf volatile (GLV) product of the LOX/lyase pathway. The braconid larval parasitoids studied, Cotesia glomerata and Cotesia plutella, differ in their ability to discriminate among plant volatiles induced by feeding of lepidopteran hosts and nonhosts: C. plutella only responds to plant volatiles induced by hosts (Plutella larvae), whereas the response by the more generalist C. glomerata is not host specific. The Arabidopsis mutant all84 infested by Pieris larvae was less attractive to C. glomerata than Arabidopsis wild type (wt) infested by the host larvae. C. glomerata was attracted by two of the GLV biosynthesized through the LOX/lyase pathway, (E)-2-hexenal and (Z)-3-hexenyl acetate. However, attraction of C. plutellae to volatiles from Plutella-infested all84 plants did not differ from attraction to host-infested wt Arabidopsis. Both wasp species were arrested to the respective host-infested edge of the wt leaf by showing characteristic antennal searching behavior on the edge. In C. glomerata, the duration of this searching behavior at the infested leaf edge was significantly shorter on all84 plants than on wt plants. By contrast, the duration of the searching behavior of C. plutellae on the host-infested leaf edge of all84 was not significantly different from that on the wt leaf. These data suggest that the LOX/lyase pathway is directly involved in the production of attractants and arrestants important for host search behavior of the more generalist C. glomerata, but not for the specialist C. plutellae.     

Plant–herbivore–carnivore Interactions in Cotton, <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>: Linking Belowground and Aboveground

Most studies on plant–herbivore interactions focus on either root or shoot herbivory in isolation, but above- and belowground herbivores may interact on a shared host plant. Cotton (Gossypium spp.) produces gossypol and a variety of other gossypol-like terpenoids that exhibit toxicity to a wide range of herbivores and pathogens. Cotton plants also can emit herbivore-induced volatile compounds at the site of damage and systemically on all tissues above the site of damage. As these volatile compounds attract natural enemy species of the herbivore, they are thought to represent an indirect plant defense. Our study quantified gossypol and gossypol-like compounds in cotton plants with foliage feeding (Heliocoverpa zea), root feeding (Meloidogyne incognita), or their combination. Cotton plants with these treatments were studied also with respect to induced local and systemic volatile production and the attraction of the parasitic wasp Microplitis croceipes to those plants. We also evaluated whether foliage or root feeding affected foliar nitrogen levels in cotton. After 48 hr of leaf feeding and 5 wk of root feeding, local and systemic induction of volatiles (known to attract parasitoids such as M. croceipes) occurred with herbivore damage to leaves, and it increased in levels when root herbivory was added. Nevertheless, M. croceipes were equally attracted to plants with both leaf and root damage and leaf damage only. In contrast to previous studies in cotton, production of gossypol and gossypol-like compounds was not induced in leaf and root tissue following foliage or root herbivory, or their combination. We conclude that root feeding by M. incognita has little influence on direct and indirect defenses of Gossypium hirsutum against insect herbivory.     

Eucalyptus oils in larvae of gum emperor moth,Antheraea eucalypti

The ether-soluble portion of the foregut fluid from the larvae ofAntheraea eucalypti (Saturniidae) was found to contain traces (0.1–0.2%) of isoprenoids. The isoprenoids were those that were major components of the oils from the leaves of theEucalyptus species on which the larvae fed and were also soluble in the foregut fluid. This observation demonstrated that these larvae did not sequester the leaf oils in their foregut or use them for defense. The compositions of the oils, which were steam-distilled from the bodies and feces of the larvae, were identical with those of the oils from the leaves of the host trees. This fact implied that the volatile components of the leaf oils had not been metabolized and had no effect on the growth or feeding of the larvae, at least on the twoEucalyptus species examined. In order to explain the coevolution ofA. eucalypti andEucalyptus species, components of the leaf oils were suggested to serve the function of an ovipositional attractant to the female moths.     

Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores

The presence of amines and their derivatives in plant tissues is known to influence insect feeding and reproduction. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which is both a bioactive amine and a precursor of other indole derivatives. Transgenic poplar and tobacco plants ectopically expressing TDC1 accumulated elevated levels of tryptamine without affecting plant growth and development. This accumulation was consistently associated with adverse effects on feeding behavior and physiology of Malacosoma disstria Hub. (forest tent caterpillar, FTC) and Manduca sexta L. (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that acceptability of the leaf tissue to larvae was inversely related to foliar tryptamine levels. Physiological studies with FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is deleterious to larvae growth, apparently due to a postingestive mechanism. Thus, ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to suppress significantly the growth of insect pests that normally feed on these plants.     

Synergistic Interactions Between Cry1Ac and Natural Cotton Defenses Limit Survival of Cry1Ac-resistant Helicoverpa Zea (Lepidoptera: Noctuidae) on Bt Cotton

Larvae of the bollworm Helicoverpa zea (Boddie) show some tolerance to Bacillus thuringiensis (Bt) Cry1Ac, and can survive on Cry1Ac-expressing Bt cotton, which should increase resistance development concerns. However, field-evolved resistance has not yet been observed. In a previous study, a population of H. zea was selected for stable resistance to Cry1Ac toxin. In the present study, we determined in laboratory bioassays if larvae of the Cry1Ac toxin-resistant H. zea population show higher survival rates on field-cultivated Bt cotton squares (= flower buds) collected prebloom—bloom than susceptible H. zea. Our results show that Cry1Ac toxin-resistant H. zea cannot complete larval development on Cry1Ac-expressing Bt cotton, despite being more than 150-fold resistant to Cry1Ac toxin and able to survive until pupation on Cry1Ac toxin concentrations greater than present in Bt cotton squares. Since mortality observed for Cry1Ac-resistant H. zea on Bt cotton was higher than expected, we investigated whether Cry1Ac interacts with gossypol and or other compounds offered with cotton powder in artificial diet. Diet incorporation bioassays were conducted with Cry1Ac toxin alone, and with gossypol and 4% cotton powder in the presence and absence of Cry1Ac. Cry1Ac toxin was significantly more lethal to susceptible H. zea than to resistant H. zea, but no difference in susceptibility to gossypol was observed between strains. However, combinations of Cry1Ac with gossypol or cotton powder were synergistic against resistant, but not against susceptible H. zea. Gossypol concentrations in individual larvae showed no significant differences between insect strains, or between larvae fed gossypol alone vs. those fed gossypol plus Cry1Ac. These results may help explain the inability of Cry1Ac-resistant H. zea to complete development on Bt cotton, and the absence of field-evolved resistance to Bt cotton by this pest.     

Herbivore-induced responses in alfalfa (Medicago sativa)

The herbivore-induced response of alfalfa (Medicago sativa) was examined through assays with Spodoptera littoralislarvae and analyses of important secondary substances. In food preference experiments, larvae preferred young undamaged alfalfa plants over plants that had been damaged by feeding larvae 5 and 7 days earlier, while no difference in feeding preferences could be detected 1, 9, and 14 days after damage. This suggests a peak in the herbivore induced resistance of alfalfa approximately one week after initial damage. The induced resistance in young plants was also shown to be systemic, while older flowering plants failed to show increased resistance after defoliation. Larvae gained weight slower and had lower pupal mass when fed damaged alfalfa than when fed undamaged alfalfa. Levels of total saponins were increased in foliage of damaged alfalfa, and detailed analyses of specific saponin components revealed doubled concentrations of 3GlcA,28AraRhaXyl medicagenate (medicagenic acid bidesmoside) and 3GlcAGalRha soyasapogenol B (soyasaponin I). Levels of the flavonoid apigenin (as free aglycone) also were increased in herbivore damaged plants. The herbivore-induced response of alfalfa was significantly weaker than that of cotton: S. littoralis larvae given a choice of undamaged cotton and undamaged alfalfa preferred to feed on cotton, whereas preferences shifted towards alfalfa when plants were damaged.     

Effects of Drought Stressed Cotton, Gossypium hirsutum L., on Beet Armyworm, Spodoptera exigua (Hübner) Oviposition, and Larval Feeding Preferences and Growth

The beet armyworm, Spodoptera exigua (Hübner), has been anecdotally reported to oviposit more on drought stressed than on nonstressed cotton plants. Using potted cotton plants in cages, this study demonstrated that beet armyworms deposited 3.3, 4.6, and 2.3 times more (P 0.05) eggs on cotton plants that were grown on 1500, 1000, and 750 ml water/wk, respectively, than on cotton plants grown in well watered (4000 ml water/wk) soil. Third instars, however, showed no preference for stressed cotton foliage over nonstressed foliage. Third instar beet armyworms raised on well watered cotton plants were 1.5, 2.3, and 2.6 times heavier than those reared on cotton grown in the 1500, 1000, and 750 ml water/wk plants (P 0.05), respectively. Physiochemical analyses showed that drought stressed leaves had significantly greater accumulations of free amino acids that are essential for insect growth and development. Soluble protein and soluble carbohydrates were also more abundant in stressed leaves compared to nonstressed leaves. Despite the apparent increase in nutritional quality in drought stressed plants, larval survival was reduced, probably because the limiting factor became water. Greater amounts of cotton leaf area were consumed from drought stressed leaves (P 0.05) than from nonstressed leaves, probably because the larvae had to metabolize greater portions of assimilated energy to supplement body water with metabolic water derived from respiration. The association of greater host plant nutritional quality to oviposition preference, and conversely, to reduced survivorship, is discussed.     

Species-specific composition of free amino acids on the leaf surface of fourSenecio species

The specificity of the chemical composition of the leaf surface of fourSenecio species was studied in order to understand the host selection ofTyria jacobaeae, a monophagous moth that hasSenecio jacobaea as its principal host plant. Pyrrolizidine alkaloids characteristic forSenecio species were not detected in water and sulfuric acid extracts of the leaf surface ofSenecio jacobaea. Water extracts of the leaf surfaces ofS. jacobaea, S. vulgaris, S. viscosus, andS. sylvaticus contained low concentrations of free amino acids. The proportions of these amino acids were very different from those inside the leaf tissues. In a discriminant analysis, the four species could be completely separated on the basis of the relative proportions of amino acids on the leaf surface.     

Sequestration of distasteful compounds by some pharmacophagous insects

Several pharmacophagous insects have been shown to sequester specific kairomonal substances or their derivatives in their body tissues. Turnip sawflies,Athalia rosae, visit a plant,Clerodendron trichototmum (Verbenaceae), and feed voraciously on the leaf surface. Clerodendrins were characterized as the potent phagostimulants forA. rosae adults. The insect sequesters some of the analogs and becomes extremely bitter on its body surface. Some chrysomelid leaf beetles associated with cucurbitacins were found to store high concentrations of these bitter principles in their body. South American polyphagous beetles,Diabrotica speciosa andCerotoma arcuata, are strongly arrested by root components from the cucurbit plant,Ceratosanthes hilariana, and selectively accumulate 23,24-dihydrocucurbitacin D, effectively gaining bitterness. Similarly, four species of Asian pumpkin leaf beetles belonging to the genusAulacophora were shown to sequester the same compound in body tissue as the major bitter principle. Three phenylpropanoids closely related to methyl eugenol were found to accumulate in the rectal glands of the male Oriental fruit fly,Dacus dorsalis. One of the rectal gland components, 2-allyl-4,5-dimethoxyphenol was shown to be released in the air during courtship. In all of these cases, selectively sequestered compounds strongly deterred feeding by some predators, thus serving as allomones in this context. Kairomonal and pheromonal functions linked with allomonal sequestration by pharmacophagous feeding has also been suggested.     

Effects ofHymenaea courbaril leaf resin on the generalist herbivoreSpodoptera exigua (beet armyworm)

The hypothesis that leaf resin ofHymenaea courbaril (Leguminosae) functions as a defence against herbivory was tested in a series of experiments with the generalist herbivore, beet armyworm (Spodoptera exigua Hübn). Pure leaf resin obtained by steam distillation was incorporated into an artificial diet at 0.0%, 0.16%, 0.50%, and 1.60% (dry weight) in one experiment and 0.0%, 1.0%, and 3.2% (dry weight) in a second experiment;S. exigua larvae were reared on these diets. In four palatability experiments the amount of leaf area eaten was compared between pairs of different leaf material. Mortality due to viral infection of larvae in the first feeding experiment showed a dose-response to leaf resin concentration. In the second experiment larvae showed a dose-response in the reduction of pupal weight and delay of time to pupation. These are interpreted as a dose-related stress onS. exigua which occurs with increased resin concentration in the diet. In the palatability tests,S. exigua strongly preferred untreated to resintreated bean leaf disks, bean disks toH. courbaril immature leaf disks,H. courbaril immature leaf toH. courbaril mature leaf, and bean leaf disks toH. courbaril newly emerged leaf disks. From these results, we conclude thatH. courbaril leaf resins possess toxic and feeding-deterrent properties which make them effective as an herbivore defense.     

Electroantennogram responses ofCampoletis sonorensis (hymenoptera: Ichneumonidae) to chemicals in cotton (Gossypium hirsutum L.)

Combined gas chromatography-electroantennogram (GC-EAG) recording ofCampoletis sonorensis (Cameron) responses to cotton plant volatile chemicals was performed.C. sonorensis antennal olfactory receptors respond differentially to green leaf, mono-, and sesquiterpene chemicals that have been identified previously in cotton. EAG depolarizations to green leaf chemicals were greater than to terpenes.     

Host-plant protein and phenolic resin effects on larval growth and survival of a butterfly

Euphydryas chalcedona prediapause larvae were reared on fertilized and control shrubs of the host plant,Diplacus aurantiacus. Larval growth was enhanced by high leaf nitrogen content and inhibited by high leaf phenolic resin content. Larvae fed less on leaves near the branch tip which contained a higher leaf resin content. The results agree with prior laboratory investigation that the dietary content of nitrogen andD. aurantiacus leaf resin are major determinants ofE. chalcedona larval growth and suggest that the phenolic leaf resin ofDiplacus may both deter and inhibit leaf herbivores.     

Insect antifeedant activity of clerodane diterpenoids against larvae ofSpodoptera Littoralis (Boisd.) (Lepidoptera)

Antifeedant activities of nine clerodane diterpenoids, isolated in this laboratory from different species ofAjuga plants, have been studied against larvae of Egyptian cotton leafwormSpodoptera littoralis (Boisd.) (Lepidoptera) by application of the leaf disk method. Evaluation of activity was carried out by calculating, at different time intervals, the feeding ratio (FR) from the relationship between the consumed areas of treated disks (CTD) and control disks (CCD); for comparison purposes a FR₅₀ defined as the FR at a CCD of 50% was established. Some compounds exhibited activity at a 0.01 g/cm² dose (0.3 ppm). Structure-activity relationships are discussed