Genetic variation and relationships of constitutive and herbivore-induced glucosinolates,trypsin inhibitors,and herbivore resistance in Brassica rapa

We examined genetic variation in inducibility and in constitutive and herbivore-induced levels of glucosinolates, trypsin inhibitors, and resistance to herbivory in families of Brassica rapa originating from a wild population. We also examined phenotypic and genetic correlations among absolute levels of these traits in control and induced plants. We grew seedlings of 10 half-sib families in pairs in pots, and exposed one plant per pair to folivory by Trichoplusia ni larvae. Two days later, we sampled all plants for total glucosinolate and trypsin inhibitor levels and examined the preference and consumption by T. ni larvae of previously damaged (induced) and undamaged (control) plants. There was no significant variation among sire families in the induction of glucosinolates or trypsin inhibitors by T. ni feeding. Total glucosinolate levels in either control or induced plants did not vary by family. In contrast, trypsin inhibitor levels in both control and induced plants varied significantly by family. Trichoplusia ni fed less on induced plants than on control plants in the bioassay, but neither the induction of resistance by prior T. ni feeding nor absolute levels of damage done to control and induced plants varied significantly by sire family. Temporal blocking strongly affected trypsin inhibitor levels and the response of some families in the bioassays. There were no significant phenotypic or genetic correlations of levels of glucosinolates or trypsin inhibitors with each other or with damage in either control or induced plants. Overall, these results suggest that in the B. rapa population that we studied, both total glucosinolate content and biological resistance to herbivory by T. ni was nonvariable and almost universally inducible by prior T. ni feeding. In contrast, control and induced levels of trypsin inhibitors varied genetically and have the capacity to respond to future selection imposed by herbivores. However, the role of these defenses in constitutive or induced resistance to T. ni in this species remains unclear.     

The Herbivore-Induced Plant Volatile Methyl Salicylate Negatively Affects Attraction of the Parasitoid <Emphasis Type="Italic">Diadegma semiclausum</Emphasis>

The indirect defense mechanisms of plants comprise the production of herbivore-induced plant volatiles that can attract natural enemies of plant attackers. One of the often emitted compounds after herbivory is methyl salicylate (MeSA). Here, we studied the importance of this caterpillar-induced compound in the attraction of the parasitoid wasp Diadegma semiclausum by using a mutant Arabidopsis line. Pieris rapae infested AtBSMT1-KO mutant Arabidopsis plants, compromised in the biosynthesis of MeSA, were more attractive to parasitoids than infested wild-type plants. This suggests that the presence of MeSA has negative effects on parasitoid host-finding behavior when exposed to wild-type production of herbivore-induced Arabidopsis volatiles. Furthermore, in line with this, we recorded a positive correlation between MeSA dose and repellence of D. semiclausum when supplementing the headspace of caterpillar-infested AtBSMT1-KO plants with synthetic MeSA.     

Stratospheric ozone depletion and plantinsect interactions: Effects of UVB radiation on foliage quality ofCitrus jambhiri forTrichoplusia ni

Projected decreases in stratospheric ozone may result in increases in shortwave ultraviolet (UVB) irradiation at the earth's surface. Furanocoumarins, phototoxic compounds found inCitrus jambhiri foliage, increase in concentration when these plants are grown under enhanced UVB. Survivorship schedules ofTrichoplusia ni (Lepidoptera: Noctuidae) caterpillars reared on plants in the presence and absence of enhanced UVB regimes differ significantly; larvae develop more slowly in early life when reared on plants exposed to increased UVB. This same developmental pattern is observed whenT. ni larvae are reared on artificial diets amended with ecologically appropriate amounts of furanocoumarins. Thus, anthropogenically derived changes in stratospheric ozone and concomitant changes in UV light quality at the earth's surface may influence ecological interactions between insects and their host plants by altering secondary metabolism and hence foliage quality for herbivores.     

Responses of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> to Tomato Plants Previously Infected by ToMV or Damaged by <Emphasis Type="Italic">H. armigera</Emphasis>

We report the comparative inducing effects of a phytopathogen and a herbivorous arthropod on the performance of an herbivore. Tomato, Lycopersicon esculentum Mill., was used as the test plant, and tomato mosaic virus (ToMV) and corn earworm, Helicoverpa armigera Hübner, were used as the phytopathogen and herbivore, respectively. There were decreases in the efficiency of conversion of ingested food and efficiency of conversion of digested food when H. armigera was reared on tomato plants that had been previously inoculated with ToMV. However, virus inoculation did not affect feeding or oviposition preferences by H. armigera. In contrast, approximate digestibility, total consumption, relative growth rate, and relative consumption rate were lower for fourth-instar H. armigera that fed on plants previously damaged by the same herbivore. Feeding and oviposition were both deterred for H. armigera that fed on previously damaged plants. The duration of development of H. armigera was also prolonged under this treatment. Infection by ToMV and feeding damage by H. armigera increased the host plant’s peroxidase and polyphenol oxidase activity, respectively, suggesting that the performance of H. armigera may be affected by the induced phytochemistry of the host plant. Overall, this study indicated that, in general, insect damage has a stronger effect than ToMV infection on plant chemistry and, subsequently, on the performance of H. armigera.     

Acidic fog-induced changes in host-plant suitability

Phaseolus lunatus L. (Henderson Bush lima beans) were exposed to 2 hr acidic fogs with 2.51.0 (v/v) nitrogen-sulfur ratio typical of the west coast of the United States. Fogs with pH values of 2.0 (P < 0.01,t tests), 2.5 (P < 0.05), or 3.0 (P < 0.01) increased percent total nitrogen (dry weight) of foliage as compared to plants subjected to control fogs with a pH of 6.3–6.5. Fresh weight concentrations of soluble protein and certain free amino acid concentrations were increased by plant exposure to acidic fogs with a pH of 2.5 (t tests,P < 0.05). Concentrations of free amino acids considered essential for insect growth, as well as nonessential and total free amino acids were not significantly affected by any treatment (P > 0.05,t test). Water content (%) of foliage was not changed significantly (P > 0.05,t test) by exposure to any of the fogs.Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae ate significantly more foliage and gained significantly more weight on plants treated with 3.0 pH fogs (P < 0.01,t test). Several potential explanations are offered for the lack of significant weight gain by larvae on plants in which soluble protein levels, free amino acid concentrations, or percent total nitrogen contents were enhanced by acidic fogs with a pH of 2.5 and 2.0. No larval feeding preference was detected for foliage exposed to acidic versus control fogs, and no significant differences were detected in percent survival ofT. ni eggs exposed to acidic or control fogs. Some implications of acidic fogs for population dynamics ofT. ni are discussed.     

Host Suitability Affects Odor Association in <Emphasis Type="Italic">Cotesia marginiventris</Emphasis>: Implications in Generalist Parasitoid Host-Finding

Insect herbivores often induce plant volatile compounds that can attract natural enemies. Cotesia marginiventris (Hymenoptera: Braconidae) is a generalist parasitoid wasp of noctuid caterpillars and is highly attracted to Spodoptera exigua-induced plant volatiles. The plasticity of C. marginiventris associative learning to volatile blends of various stimuli, such as host presence, also has been shown, but little is known about how this generalist parasitoid distinguishes between host species of varying suitability. Spodoptera exigua is an excellent host that yields high parasitoid emergence, while Trichoplusia ni serves as a sub-optimal host species due to high pre-imaginal wasp mortality. We have found that S. exigua and T. ni induce different volatile blends while feeding on cotton. Here, wind tunnel flight assays were used to determine the importance of differentially induced volatiles in host-finding by C. marginiventris. We found that, while this generalist parasitoid wasp can distinguish between the two discrete volatile blends when presented concurrently, a positive oviposition experience on the preferred host species (S. exigua) is more important than host-specific volatile cues in eliciting flight behavior towards plants damaged by either host species. Furthermore, wasps with oviposition experience on both host species did not exhibit a deterioration in positive flight behavior, suggesting that oviposition in the sub-optimal host species (T. ni) does not cause aversive odor association.     

Altered Olfactory Receptor Neuron Responsiveness Is Correlated with a Shift in Behavioral Response in an Evolved Colony of the Cabbage Looper Moth, <Emphasis Type="Italic">Trichoplusia ni</Emphasis>

There is little understanding of how sex pheromone blends might change during speciation events. For the cabbage looper, Trichoplusia ni, there is a mutant laboratory strain that has exhibited characteristics of a shift to a new pheromone blend. Mutant females produce a blend that is significantly different from wild-type females in having a much higher proportion of a minor pheromone component and lower quantity of the major component. Males in this colony have changed over the years to become more broadly tuned and fly upwind equally well to both the wild-type and mutant female pheromone blends. They also exhibit reduced overall sensitivity to pheromone, flying upwind to either blend at a lower success rate than is typical when wild-type males respond to the wild-type blend. Using single-cell recordings, we examined the olfactory receptor neurons (ORNs) of males from evolved and wild-type colonies for evidence of changes in response characteristics that might explain the above-described behavioral evolution. We found that in evolved-colony males the ORNs tuned to the major sex pheromone component exhibited a somewhat lower responsiveness to that compound than the ORNs of wild-type males. In addition, the minor pheromone component, emitted at excessively high rates by mutant females, elicited a drastically reduced ORN responsiveness in evolved-colony males compared to wild-type males. This alteration in ORN responsiveness may be responsible for allowing evolved males to tolerate the excessive amounts of the minor pheromone component in the mutant female blend, which would normally antagonize the upwind flight of unevolved males. Thus, peripheral olfactory alterations have occurred in T. ni males that are correlated with the evolution of the more broadly tuned, but less sensitive, behavioral response profile.     

Effects of Phytic Acid and Xanthotoxin on Growth and Detoxification in Caterpillars

Phytic acid is abundant in the fruits and seeds of many plants and is found in foliage to a lesser extent. Among its several properties, phytic acid is a potent chelator of essential minerals and proteins; thus, the possibility exists that heme-based enzymes such as cytochrome P450 monooxygenases in herbivores are detrimentally affected by phytic acid via chelation of dietary iron. Mortality, growth performance, and P450-mediated metabolism of xanthotoxin, a plant allelochemical, were examined in the presence of phytic acid in three lepidopteran species: a polyphagous seed-feeding species (Heliothis virescens), a polyphagous foliage-feeding species (Trichoplusia ni), and a species oligophagous on immature reproductive structures of two genera of Apiaceae (Depressaria pastinacella). While first instar H. virescens experienced no increase in mortality after 120 hours on a diet containing 1% phytic acid compared to a control diet, both T. ni and D. pastinacella experienced virtually complete mortality over the same time period. Ultimate instars of all three species experienced reductions in relative growth rates (RGR) and relative consumption rates (RCR) in the presence of phytic acid, although the only species to experience reduced digestive efficiency (ECI) was H. virescens. Cytochrome P450-mediated metabolism of xanthotoxin was reduced 60% in the presence of phytic acid in D. pastinacella, although metabolism remained unaffected in the two noctuids. These studies suggest a defensive function of phytic acid in addition to its primary functions of phosphorus storage, energy storage, and cell wall precursor source.     

Effects of Ingested Secondary Metabolites on the Immune Response of a Polyphagous Caterpillar <Emphasis Type="Italic">Grammia incorrupta</Emphasis>

We considered the effects of plant secondary metabolites on the immune response, a key physiological defense of herbivores against pathogens and parasitoids. We tested the effect of host plant species and ingested iridoid glycosides on the immune response of the grazing, polyphagous caterpillar, Grammia incorrupta (Arctiidae). Individuals of G. incorrupta were fed either one of three plant diets with varying secondary metabolites, or an artificial diet with high or low concentrations of iridoid glycosides. An immune challenge was presented, followed by measurement of the encapsulation response. We failed to detect a significant difference in the immune response of G. incorrupta feeding on diets with varying concentrations of iridoid glycosides, or feeding on different host plants. However, the immune response was lower in caterpillars consuming the artificial diet compared to those consuming the plant diets. When caterpillar performance was measured, pupal weights were lower when caterpillars ingested high concentrations of iridoid glycosides due to a decrease in feeding efficiency. Overall, individuals of G. incorrupta that consumed different plant diets exhibited a high immune response with low variation. We conclude that the immune response of G. incorrupta is adapted to feeding on a variety of plants, which may contribute to the maintenance of this caterpillar’s polyphagous habit.     

Does the Response of Insect Herbivores to Cadmium Depend on Their Feeding Strategy?

Phytoremediation has been proposed for the elimination of toxic metals in soil, yet little attention has been given to the performance of insects that feed on contaminant-tolerant plants. We tested the performance of two herbivores with different feeding behaviors, the cabbage looper, Trichoplusia ni, and the green peach aphid, Myzus persicae, reared on cadmium-tolerant Brassica juncea plants that contained different concentrations of cadmium. We also tested the performance of the aphid parasitoid Aphidius colemani developing in aphids reared on plants with different levels of cadmium. The hypothesis tested was that the chewing insect would be more negatively affected than the sucking insect, because of the localization of cadmium within the host plant, and that the aphid parasitoid would not be affected. We also compared the performance of T. ni on artificial diet with different levels of cadmium. Neither the phloem-feeding aphid nor its parasitoid was affected by cadmium in the host plant. The effects of cadmium on the foliage-feeding cabbage looper varied, with negative effects on development observed in experiments with artificial diet but not in those using natural host plants. These data, together with information available in the literature, support the idea that the effects of toxic metals present in a host plant may be influenced by a herbivore’s feeding strategy. However, a wide range of chewing and sucking species needs to be tested to confirm this hypothesis.     

Feeding Deterrence and Detrimental Effects of Pyrrolizidine Alkaloids Fed to Honey Bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Recent studies have shown the occurrence of plant derived pyrrolizidine alkaloids (PAs) in retail honeys and pollen loads, but little is known about how these compounds influence the fitness of foraging honey bees. In feeding experiments, we tested a mix of tertiary PAs and the corresponding N-oxides from Senecio vernalis, pure monocrotaline, and 1,2-dihydromonocrotaline in 50% (w/w) sucrose solutions. The bees were analyzed chemically to correlate the observed effects to the ingested amount of PAs. PA-N-oxides were deterrent at concentrations >0.2%. 1,2-Unsaturated tertiary PAs were toxic at high concentrations. The observed PAs mortality could be linked directly to the presence of the 1,2-double bond, a well established essential feature of PA cytotoxicity. In contrast, feeding experiments with 1,2-dihydromonocrotaline revealed no toxic effects. Levels of less than 50 μg 1,2-unsaturated tertiary PAs per individual adult bee were tolerated without negative effects. PA-N-oxides fed to bees were reduced partially to the corresponding tertiary PAs. Unlike some specialized insects, bees are not able to actively detoxify PAs through N-oxidation. To gain insight into how PAs are transmitted among bees, we tested for horizontal PA transfer (trophallaxis). Under laboratory conditions, up to 15% of an ingested PA diet was exchanged from bee to bee, disclosing a possible route for incorporation into the honey comb. In the absence of alternative nectar and pollen sources, PA-containing plants might exhibit a threat to vulnerable bee larvae, and this might affect the overall colony fitness.     

Influence of cabbage proteinase inhibitorsin situ on the growth of larvalTrichoplusia ni andPieris rapae

Trypsin and chymotrypsin inhibitors are proteins that are developmentally regulated in foliage of cabbage plants, appearing at high concentrations in young foliage on mature plants. This temporal and spacial regulation of foliar proteinase inhibitors is synchronized with the appearance and distribution of foliar feeding Lepidoptera. When insects were allowed to select their feeding sites, larvalPieris rapae fed on the young foliage of cabbage plants, while larvalTrichoplusia ni fed on the mature foliage on cabbage plants. LarvalP. rapae that fed on mature plants were significantly smaller than larvae feeding on young plants, while there was no significant difference between larvalT. ni feeding on mature plants and those feeding on young plants. Thus, there was a significant inverse correlation between the level of proteinase inhibitory activity in cabbage foliage and larval growth. WhenP. rapae andT. ni were provided with an artificial diet containing total protein (including significant levels of proteinase inhibitors) that was extracted from cabbage foliage, there was a significant reduction in growth and development of both species of Lepidoptera.     

Behavioral and physiological responses of cabbage looper,Trichoplusia ni (Hübner), to steam distillates from resistant versus susceptible soybean plants

Soybean plant volatiles, extracted as steam distillates, significantly affected the behavior and biology of the cabbage looper,Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae). Distillates from the susceptible Davis variety attractedT. ni larvae and female adults, whereas those from resistant PI 227687 plants repelled them. When mixed in an artificial diet, steam distillates from PI 227687 plants caused mortality of first-instar larvae. Adults emerging from pupae topically treated with 5 g of such PI 227687 extractable showed developmental abnormalities. Larval feeding was significantly less on Davis leaves treated with PI 227687 volatiles as compared to solvent (acetone) or such Davis extractables. However, Davis volatiles on PI 227687 leaves did not increase larval feeding. HPLC analyses of steam distillates from susceptible Davis versus resistant PI 227687 indicated differences.     

Role of female-produced sex pheromone in behavioral reproductive isolation betweenTrichoplusia ni (Hübner) andPseudoplusia includens (Walker) (Lepidoptera: Noctuidae,plusiinae)

In laboratory flight tunnel bioassays, response rates of male cabbage looper,Trichoplusia ni (Hübner), to female soybean looper,Pseudoplusia includens (Walker), were similar to response rates of maleT. ni to conspecific females for plume tracking and source contact. Male soybean loopers, however, exhibited a greatly reduced response to female cabbage loopers compared to conspecific females. Similar differences were observed in male responses to extracts of female abdominal tips. Studies of flight tunnel responses of male soybean loopers to the different chemicals known to be components of the female cabbage looper sex pheromone indicated that the reduction in response was due to inhibitory effects of (Z)-5-dodecen-1-ol acetate and (Z)-9-tetradecen-1-ol acetate, when added singly to (Z)-7-dodecen-1-ol acetate (major component of both species) at release rates and at ratios close to those observed in female cabbage loopers.     

Pheromone-mediated responses of male cabbage looper moths,Trichoplusia ni,following various exposures to sex pheromone or (Z)-7-dodecenol

Prolonged preexposure (three days) of maleTrichoplusia ni to its six-component sex pheromone blend or its major pheromone component, (Z)-7-dodecenyl acetate, reduced subsequent upwind flight responses to a pheromone source. Preexposure to (Z)-7-dodecenol increased upwind flight responses to a pheromone source combined with (Z)-7-dodecenol. The impact of long-term preexposures was moderate when compared to the more immediate effects of background noise. When (Z)-7-dodecenyl acetate was presented as background noise, all maleT. ni failed to respond to a plume of the full pheromone blend. However, most moths succeeded in locking on to the pheromone plume and contacting the pheromone source in the presence of the five minor pheromone components as background noise. When (Z)-7-dodecenol was released as background noise the response rate to a pheromone source containing (Z)-7-dodecenol was increased dramatically. This indicates that males became adapted to (Z)-7-dodecenol while responding to the pheromone source. The results of this study indicate that both long-term preexposure treatments and immediate exposure to background noise can limit the ability of maleT. ni to respond to sex pheromone sources.     

Looplure efficacy and electrophysiological responses in three plusiinae species

Source concentration differences of (Z)-7-dodecen-1-ol acetate, or looplure, were evaluated for field trapping efficiency and electrophysiological responses with malePseudoplusia includens (Walker),Trichoplusia ni (Hubner) andRachiplusia ou (Guenné) (Lepidoptera: Noctuidae). Sticky traps baited with 1000 g of the lure captured a significantly greater (P < 0.05) number of maleP. includens andT. ni than any other concentration;R. ou males were caught at a greater rate in traps baited with 100 g of looplure, significantly more (P < 0.05) than with 1000 g. Electroantennogram (EAG) studies demonstrated that antennae of maleP. includens have a lower response threshold to looplure than eitherT. ni orR. ou antennae, the latter demonstrating the highest significant threshold of response. No differences in the stimulus-response functions of the three species were detected.Lepidoptera: Noctuidae.     

Influence of natural antioxidants on the formation of trans‐fatty‐acid isomers during heat treatment of sunflower oil

The intake of foods containing trans fatty acids (TFAs) can have deleterious effects on human health, mainly on the cardiovascular system. Thus, it is important to consider the processes that form TFAs in foods, and the alternatives to minimise their formation. The influence of two added natural antioxidants on TFA formation during heat treatment (120 h at 180°C) of sunflower vegetable oil were examined: rosemary extract (Rosmarinus officinalis L.) (1 g per kg oil) and lutein (0.1 g per kg oil). Changes in FA composition were determined using Ag‐ion SPE and gas–liquid chromatography, with total polar compounds determined using dielectric constant measurements and the index of atherogenicity was calculated. Total TFAs with ≥1 trans double bond increased from 0.91 to 1.71% in control samples; this increase was significantly less with both rosemary extract (1.55%) and lutein (1.43%) additions. Among the individual TFAs, significant increases were seen for C18:1,t‐9, C18:2,t‐9,t‐12 and C18:2,c‐9,t‐12/9‐t,12‐c. Polar compounds also increased, with the highest concentrations in control samples, and significantly less with both rosemary extract and lutein additions. According to results of our study, we can summarize that addition of lutein have greater effect on reduction of TFA formation than rosemary extract. Practical applications: Antioxidants, particularly from plants, are widely used in the food industry. They can provide benefits in food preparation, including improving colour, odour and stability, acting as acid regulators and natural preservatives. They have also become accepted by customers and consumers, and so indirectly they have had effects on consumer perception. Addition of natural antioxidants such as rosemary extract is usually limited by the sensory characteristics of the food, with one study showing that addition of rosemary extract at 1–3 g per kg vegetable oil is recommended. The effects of antioxidants on the formation of TFAs in vegetable oils has not been well studied in the literature. Among the already known benefits, the use of such antioxidants as functional ingredients in lipid technologies might reduce the formation of TFAs during thermal treatment.     

Mutation breeding of nuclease p1 production in <Emphasis Type="Italic">Penicillium citrinum</Emphasis> by low-energy ion beam implantation

Nuclease p1 is an important enzyme in the nucleotide industry that is used to hydrolyze nucleic acid into nucleotides. To improve enzyme activity, Penicillium citrinum, a nuclease p1 producing strain, was mutated by low-energy Nitrogen ion beam implantation at an energy level of 15 keV and a dose ranging from 1×10¹⁵–1×10¹⁶ ions/cm². The mutant strain designated as N409 was obtained with a high yield of nuclease p1. The activity of nuclease p1 was 421 U/mL from the mutant strain N409, which was increased by 86% compared with the control. The fermentation kinetics of nuclease p1 by the mutant strain N409 was studied in a 30 L external airlifting bioreactor. A model was proposed using the logistic equation for microbial growth, the Luedeking-Piret equation for product formation and a Luedeking-Piret-like equation for substrate uptake. The results predicted from the model were in good agreement with the experimental observations.     

Root-secreted Allelochemical in the Noxious Weed <Emphasis Type="Italic">Phragmites Australis</Emphasis> Deploys a Reactive Oxygen Species Response and Microtubule Assembly Disruption to Execute Rhizotoxicity

Phragmites australis is considered the most invasive plant in marsh and wetland communities in the eastern United States. Although allelopathy has been considered as a possible displacing mechanism in P. australis, there has been minimal success in characterizing the responsible allelochemical. We tested the occurrence of root-derived allelopathy in the invasiveness of P. australis. To this end, root exudates of two P. australis genotypes, BB (native) and P38 (an exotic) were tested for phytotoxicity on different plant species. The treatment of the susceptible plants with P. australis root exudates resulted in acute rhizotoxicity. It is interesting to note that the root exudates of P38 were more effective in causing root death in susceptible plants compared to the native BB exudates. The active ingredient in the P. australis exudates was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). We tested the phytotoxic efficacy of gallic acid on various plant systems, including the model plant Arabidopsis thaliana. Most tested plants succumbed to the gallic acid treatment with the exception of P. australis itself. Mechanistically, gallic acid treatment generated elevated levels of reactive oxygen species (ROS) in the treated plant roots. Furthermore, the triggered ROS mediated the disruption of the root architecture of the susceptible plants by damaging the microtubule assembly. The study also highlights the persistence of the exuded gallic acid in P. australis’s rhizosphere and its inhibitory effects against A. thaliana in the soil. In addition, gallic acid demonstrated an inhibitory effect on Spartina alterniflora, one of the salt marsh species it successfully invades. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.