J.J.A. van Loon

Role of Glucosinolates in Insect-Plant Relationships and Multitrophic Interactions

Glucosinolates present classical examples of plant compounds affecting insect-plant interactions. They are found mainly in the family Brassicaceae, which includes several important crops. More than 120 different glucosinolates are known. The enzyme myrosinase, which is stored in specialized plant cells, converts glucosinolates to the toxic isothiocyanates. Insect herbivores may reduce the toxicity of glucosinolates and their products by excretion, detoxification, or behavioral adaptations. Glucosinolates also affect higher trophic levels, via reduced host or prey quality or because specialist herbivores may sequester glucosinolates for their own defense. There is substantial quantitative and qualitative variation between plant genotypes, tissues, and ontogenetic stages, which poses specific challenges to insect herbivores. Even though glucosinolates are constitutive defenses, their levels are influenced by abiotic and biotic factors including insect damage. Plant breeders may use knowledge on glucosinolates to increase insect resistance in Brassica crops. State-of-the-art techniques, such as mutant analysis and metabolomics, are necessary to identify the exact role of glucosinolates.

ATTRACTION OF COLORADO POTATO BEETLE TO HERBIVORE-DAMAGED PLANTS DURING HERBIVORY AND AFTER ITS TERMINATION

Large, undamaged potato plants (>60 cm, 5–6 weeks old) attract the Colorado potato beetle (Leptinotarsa decemlineata), but small potato plants (15–25 cm high, 2–3 weeks old) do not. However, small plants become attractive to CPB when they are damaged. Mechanical damage inflicted with scissors results in short-term (lasting less than 15 min) attraction, while more severe damage with carborundum powder results in a longer lasting attraction (at least 1 hr). CPB adults are also attracted to small plants infested with CPB and Spodoptera exigua larvae. After the larvae had been removed for 50 min following a short duration (30 min) of feeding, CPB adults were no longer attracted to the plants. However, when CPB larvae had been removed after they had fed for 60–90 min, the plants were somewhat attractive to the beetles, although significantly less than they had been when the larvae were feeding. Attraction increased with time after feeding ceased. Furthermore, beetles were strongly attracted to plants 50 min after larvae were removed when the plants had been fed upon by larvae for 18–24 hr. Thus it appears that there are two stages of attraction, first, to volatiles released directly from the wound site, and second, to volatiles that are induced in response to herbivory. Chemical analyses of the headspace of infested potato plants show that infestation results in the emission of a mixture of chemicals that is qualitatively quite similar to that emitted by undamaged plants. The major components of the mixture are that emitted by undamaged plants. The major components of the mixture are terpenoids and fatty acid derivatives such as aldehydes and alcohols. The emission rate of some of these chemicals declines after removal of the beetles, while the emission rate of other chemicals increases with the duration of beetle feeding and remains at a high level even after removal of the beetles. Thus, the composition of the mixture changes temporally during and after herbivore feeding, which may explain the recorded behavior of the beetles.

Identification of olfactory stimulants for Anopheles gambiae from human sweat samples

The behavioral and electroantennogram (EAG) responses of female Anopheles gambiae mosquitoes to pooled samples of freshly collected human sweat and human sweat incubated for 42–52 hr were tested. No behavioral or EAG response was obtained to pooled fresh sweat samples, whereas incubated pooled sweat samples produced a behavioral as well as an EAG response. GC-MS analysis of the headspace composition of the fresh sweat revealed ethanol (15.1% of the total amount of volatiles trapped), acetic acid (10.9%), and 3-hydroxy-2-butanone (9.5%) as the most abundant compounds; a wide range of ethyl esters was present as well. None of the ethyl esters was detected in the headspace collections from incubated sweat, while the relative amounts of ethanol, acetic acid, and 3-hydroxy-2-butanone were strongly reduced. In the latter collections, indole (27.9%), 1-dodecanol (22.4%), and 3-methyl-1-butanol (10%) were present in high amounts, while they were absent or present in only minor amounts in the headspace collections from fresh sweat. Geranyl acetone (6%) and 6-methyl-5-hepten-2-one (1.9%) were relatively abundant in both the fresh and incubated headspace samples. EAG responses were observed in response to indole, 6-methyl-5-hepten-2-one, and geranyl acetone.

Metabolic and Transcriptomic Changes Induced in Arabidopsis by the Rhizobacterium Pseudomonas fluorescens SS101

Systemic resistance induced in plants by nonpathogenic rhizobacteria is typically effective against multiple pathogens. Here, we show that root-colonizing Pseudomonas fluorescens strain SS101 (Pf.SS101) enhanced resistance in Arabidopsis (Arabidopsis thaliana) against several bacterial pathogens, including Pseudomonas syringae pv tomato (Pst) and the insect pest Spodoptera exigua. Transcriptomic analysis and bioassays with specific Arabidopsis mutants revealed that, unlike many other rhizobacteria, the Pf.SS101-induced resistance response to Pst is dependent on salicylic acid signaling and not on jasmonic acid and ethylene signaling. Genome-wide transcriptomic and untargeted metabolomic analyses showed that in roots and leaves of Arabidopsis plants treated with Pf.SS101, approximately 1,910 genes and 50 metabolites were differentially regulated relative to untreated plants. Integration of both sets of “omics” data pointed to a prominent role of camalexin and glucosinolates in the Pf.SS101-induced resistance response. Subsequent bioassays with seven Arabidopsis mutants (myb51, cyp79B2cyp79B3, cyp81F2, pen2, cyp71A12, cyp71A13, and myb28myb29) disrupted in the biosynthesis pathways for these plant secondary metabolites showed that camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf.SS101. Also for the insect S. exigua, the indolic glucosinolates appeared to play a role in the Pf.SS101-induced resistance response. This study provides, to our knowledge for the first time, insight into the substantial biochemical and temporal transcriptional changes in Arabidopsis associated with the salicylic acid-dependent resistance response induced by specific rhizobacteria.

Toxicity and repellence of African plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus

In a search for botanical products to control the main insect pest of stored cowpea, Callosobruchus maculatus, 33 traditionally used African plants were tested in the laboratory for their toxic and repellent effects against this beetle. Toxicity was evaluated measuring life history parameters in a no-choice situation. Powders of Nicotiana tabacum, Tephrosia vogelii and Securidaca longepedunculata significantly reduced the number of progeny. Repellence was evaluated by observing the behaviour of female beetles exposed to treated and untreated beans in a linear olfactometer. Clausena anisata, Dracaena arborea, T. vogelii, Momordica charantia and Blumea aurita were repellent to the beetle, whereas Chamaecrista nigricans, Azadirachta indica and Hyptis suaveolens were attractive. Our results indicate that botanical products may provide effective control of C. maculatus in cowpea.

Behavioural and electrophysiological responses of the female malaria mosquito Anopheles gambiae (Diptera: Culicidae) to Limburger cheese volatiles

Limburger cheese, previously shown to attract female Anopheles gambiae Giles, was solvent extracted and chemically fractionated into acid and non-acid fractions. The extracts and aliquots of headspace odour of the cheese were analysed by gas chromatography and electron impact mass spectrometry. Nineteen saturated and unsaturated aliphatic fatty acids, ranging in carbon chain length from C 2 to C 18 , were detected. The most abundant acids (>1 mg/g of cheese) identified in the acid extract were ethanoic, propanoic, butanoic, hexadecanoic and 9-octadecenoic acid. The same compounds were identified in analyses of headspace samples but only trace quantities of the less volatile acids (C 10 to C 16 ) were present, whilst C 18 acids were absent. Behavioural responses of female A. gambiae towards a range of dilutions of the acid extract (in diethyl ether) were recorded in a windtunnel bioassay. The undiluted extract was found to be repellent, but became highly attractive ( P «0.001) at lower doses, and was still significantly attractive ( P 6 times. A synthetic mixture of 12 of the more abundant aliphatic acids identified in the acid extract was found to be significantly attractive ( P 8 times. Electroantennographic (EAG) studies showed significant and reproducible responses to (saturated) Limburger cheese headspace. At doses higher than 0.1%, the synthetic mixture of 12 acids elicited significantly higher EAG amplitudes than the solvent control (paraffin oil). EAG responses were recorded from mosquitoes stimulated with C 5 to C 8 acids, that were characterized by significant dose-dependencies. Weaker, though significant EAG responses were obtained with the less volatile acids (C 9 to C 14 ). Only hexadecanoic acid did not elicit a detectable response. The electrophysiological and behavioural responses obtained with fatty acids isolated from Limburger cheese suggests that together they could act as a kairomone for female A. gambiae . The implications of this are discussed together with the occurrence and bacterial production of these compounds on human skin.

Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.

Olfactory receptors on the antennae of the malaria mosquito Anopheles gambiae are sensitive to ammonia and other sweat-borne components

Electrophysiological studies on female An. gambiae s.s. mosquitoes revealed a receptor neuron within a subpopulation of the antennal grooved-peg sensilla sensitive to the odour of incubated sweat, but not responding to fresh sweat. This receptor neuron was sensitive to ammonia as well, a sweat-borne component which attracts female An. gambiae in a windtunnel bioassay. Neurons innervating a different subpopulation of grooved-peg sensilla did not show a response to incubated sweat. In the latter sensilla, however, one type of neuron responded to water or water containing solutions, while another receptor neuron was inhibited when stimulated with dry air, ether or ethanol. Neurons innervating sensilla trichodea, a more abundant antennal type of olfactory sensillum, did not respond to fresh or incubated sweat at the doses offered. However, receptor neurons within the sensilla trichodea responded with excitation to several sweat-borne components. A subpopulation of the sensilla trichodea was innervated by neurons sensitive to geranyl acetone. A second subpopulation housed receptor neurons sensitive to indole. 3-Methyl-1-butanol and 6-methyl-5-hepten-2-one evoked excitation of receptor neurons within both subpopulations of sensilla trichodea. Neurons were most sensitive to indole and geranyl acetone with a threshold of 0.01%. These findings are discussed in the context of host-seeking behaviour.

Herbivore-induced plant responses in Brassica oleracea prevail over effects of constitutive resistance and result in enhanced herbivore attack

1. Plant responses to herbivore attack may have community‐wide effects on the composition of the plant‐associated insect community. Thereby, plant responses to an early‐season herbivore may have profound consequences for the amount and type of future attack.

Comparative headspace analysis of cabbage plants damaged by two species of Pieris caterpillars: consequences for in-flight host location by Cotesia parasitoids

Headspace composition, collected from intact cabbage plants and cabbage plants infested with either Pieris brassicae L. or P. rapae L. (Lepidoptera: Pieridae) first instar larvae, was determined by GC‐MS. Twenty‐one volatiles were identified in the headspace of intact plants. Twenty‐two volatiles were identified in the headspace of plants infested by P. brassicae larvae, 2 of which, Z‐3‐hexenyl butyrate and Z‐3‐hexenyl isovalerate, were not detected in the headspace of either intact or P. rapae damaged plants. In the headspace of the latter, 21 compounds were identified, all of which were also produced by intact plants. No significant quantitative differences were found between headspace composition of the plants damaged by one or the other caterpillar species. Major differences between intact and caterpillar‐damaged plants in contribution to the headspace profile were revealed for hexyl acetate, Z‐3‐hexenyl acetate, myrcene, sabinene and 1, 8‐cineole. The larval endoparasitoid Cotesia glomerata L. was attracted by the volatiles emanating from B. oleracea damaged by P. brassicae first instar larvae. C. rubecula L., a specialized larval endoparasitoids of P. rapae, was attracted by the volatiles released from the B. oleracea ‐ P. rapae plant‐host complex. This shows that cabbage plants kept under the conditions of headspace collection produce attractive volatiles for both parasitoids.