An electrophysiological analysis of the effect of phagostimulant mixtures on the responses of a deterrent-sensitive cell of gypsy moth larvae, Lymantria dispar (L.)

Gypsy moth larvae, Lymantria dispar (L.), are polyphagous feeders. The medial styloconic sensillum of this species bears a taste receptor cell that responds to alkaloids and another that responds to the sugar alcohol, inositol. The lateral styloconic sensillum bears a taste receptor cell that is sensitive to the sugar, sucrose. We tested the effect of two phagostimulants, namely sucrose and inositol, on the response of the deterrent-sensitive cell and found that both phagostimulants suppressed its response, equally, while their combination was significantly more effective. We also tested the effect of two alkaloids (i.e., strychnine and caffeine), which deter feeding in this species, on the response of the inositol- and sucrose-sensitive cells. Although both of these deterrents had no effect in suppressing the response of the sucrose-sensitive cell, they both had an effect in suppressing the inositol-sensitive cell. We also found that sucrose suppressed the response of the inositol-sensitive cell, whereas inositol had no significant effect on the response of the sucrose-sensitive cell. In this paper, we examined the effect of mixtures of these compounds to determine the nature of their interaction. In the context of host–plant interactions and, for example, host recognition, whereby host plant acceptability depends on the total sensory impression acquired from responses to multiple plant components rather than the presence or absence of single stimulant or deterrent compounds, this study could have a direct bearing in the development of natural compounds (i.e., alkaloids) for pest control and crop protection. It will also contribute to our understanding of the neural basis of the feeding behavior of this insect.     

Specialist deterrent chemoreceptors enable Pieris caterpillars to discriminate between chemically different deterrents

Pieris butterflies (Lepidoptera: Pieridae) are specialist herbivores of cruciferous plants. They exploit glucosinolates, secondary plant metabolites chemotaxonomically characteristic for this plant family, as token stimuli. In addition to particular glucosinolates, some genera of the Cruciferae contain cardenolides, steroidal allelochemicals that act as potent feeding and oviposition deterrents to several Pieris species. We investigated the sensory mechanisms by which these compounds are perceived in larvae. Pieris caterpillars and many other lepidopterous species are endowed with so-called generalist deterrent receptors, that respond to a broad spectrum of secondary plant substances. In Pieris caterpillars we found a second type of deterrent chemoreceptor in maxillary styloconic taste sensilla. This neuron is very sensitive to cardenolides (threshold 0.1–0.3 M). The generalist deterrent receptor also responds to these substances but its threshold lies at 50–100× higher concentrations. In behavioural preference experiments Pieris brassicae L. caterpillars preferred cardenolide-treated cabbage leaf discs when confronted with a choice between them and a deterrent substance that does not occur in the Brassicaceae. The cardenolides acted as potent deterrents when offered against untreated cabbage leaf discs. This demonstrates that the balance of activity elicited in the two types of deterrent chemoreceptors determines the behavioural decision.     

The role of an epipharyngeal sensillum in the perception of feeding deterrents by Leptinotarsa decemlineata larvae

An epipharyngeal taste sensillum in Leptinotarsa decemlineata larvae was studied. Electron microscopy showed that the sensillum is innervated by five neurons. Electrophysiological experiments showed that one of these cells responds to water, a second to sucrose and a third to two feeding deterrents that were also effective in a behavioural test. Receptor cells sensitive to feeding deterrents were not previously reported for L.␣decemlineata larvae or adults. The response of the sucrose-sensitive cell was strongly inhibited by one of the two feeding deterrents and only slightly by the other feeding deterrent. The relationship between the behavioural and electrophysiological results is discussed in order to elucidate the neural code of feeding deterrents in L. decemlineata larvae. We conclude that probably both the response of the deterrent cell and peripheral interactions exerted by feeding deterrents on the sucrose-sensitive cell determine the potency of feeding deterrents. The present results provide a physiological basis for the hypothesis that the presence or absence of feeding deterrents in potential food plants is a decisive cue in food plant selection by L. decemlineata larvae. Accepted: 25 March 1998     

Feeding behaviour of the grass grub Costelytra zealandica (White) (Coleoptera: Melolonthinae)

The feeding response of 3rd‐instar Costelytra zealandica larvae to 20 amino acids tested individually at 2 concentrations was assessed. Six of these compounds had a phagostimulatory effect; in particular, the ubiquitous amino acids L‐aspartic acid, L‐glutamic acid, and L‐serine induced considerable feeding by the grubs. Ascorbic acid was also a strong feeding stimulant and, in combination with sucrose, evoked an intense behavioural response. A synergistic effect of ‘Salt Mix W on the larval response to sucrose was demonstrated.     

Cross habituation to feeding deterrents and acceptance of a marginal host plant byPieris rapae larvae

Sensitivity of the cabbage butterfly,Pieris rapae L. to feeding deterrents was compared for larvae reared on different food sources under laboratory conditions. Since cabbage-reared larvae normally reject nasturtium,Tropaeolum majus L., the effects of previous exposure to allelochemicals on larval acceptance or rejection of this plant were also examined. When compared with cabbage-reared larvae, nasturtium-reared larvae were less sensitive to feeding deterrents including cymarin, erysimoside and 2-O-β-d-glucosyl cucurbitacin E. Nasturtium-reared larvae were insensitive to chlorogenic acid, which was deterrent to cabbage-reared larvae. Feeding by larvae reared on a wheat germ diet was not deterred by these compounds. The results indicate that dietary experience can extensively affect larval sensitivity to feeding deterrents and that cross habituation of larvae to deterrents occurs in response to certain chemical constituents of nasturtium and wheat germ diet. Digitoxin, however, proved to be an exception. Larvae reared on either nasturtium or wheat germ diet were as sensitive to digitoxin as those reared on cabbage. Previous results have shown that rejectionof nasturtium by cabbage-reared larvae is due to the presence of strong feeding deterrents in this plant. However, more than 50% of 2nd instar larvae reared from neonate on cabbage leaves treated with strophanthidin, cymarin, erysimoside, digitoxigenin and digitoxin accepted nasturtium as a food source. 2-O-β-d-glucosyl cucurbitacin E, 2-O-β-d-glucosyl cucurbitacin I and rutin were also active in causing larvae to feed on nasturtium. Thus dietary exposure to unrelated plant chemicals can profoundly affect insect acceptance of a plant that contains feeding deterrents.     

Effects of pyrrolizidine alkaloids and sesquiterpenes on snail feeding

Summary We determined in the laboratory the feeding response of two populations of the generalist herbivorous snail Arianta arbustorum (Helicidae) towards the composite Adenostyles alliariae and towards various allelochemicals. These were: a pyrrolizidine alkaloid (PA) extract of Adenostyles leaves; senecionine (a PA present in Adenostyles); retrorsine (a PA not present in Adenostyles) and two sesquiterpene (ST) fractions from Adenostyles: a mixture of the STs adenostylone and neoadenostylone, and deacyladenostylone. Tertiary PAs and PA N-oxides were tested separately. For each allelochemical, we tested whether it was deterrent or whether it induced changes of feeding behaviour (i.e. whether it had pre- or postingestive effects), and whether the effects were more pronounced with younger (smaller) snails. The tertiary PA extract from Adenostyles was deterrent, especially for young snails, but did not induce changes of feeding behaviour. Tertiary PA senecionine was deterrent for young snails only and induced changes of feeding behaviour. Also, consumption of untreated Petasites was higher after this treatment. Tertiary PA retrorsine was not deterrent, but induced changes of feeding behaviour. The PA N-oxides showed no activity against the snails. The mixture of adenostylone and neoadenostylone was deterrent and induced feeding aversions. Deacyladenostylone was highly deterrent, but did not induce changes of feeding behaviour. At the Jura site, PA content of Adenostyles was lower than at the Black Forest site. The snails from Jura consumed much less Adenostyles than the snails from Black Forest, and also ate a little less of the treated leaf discs. The PAs which are encountered by the snails in their natural food plants (PA extract and senecionine) were more deterrent than retrorsine (a novel compound). This suggests that the snails have mechanisms for the rejection of allelochemicals which they encounter in their natural food plants, but not for novel allelochemicals. The results suggest two hypotheses regarding the function of the allelochemicals in Adenostyles: (1) The allelochemicals act mainly on very young snails. (2) PAs render Adenostyles toxic, while STs act as feeding deterrents.     

Comparative growth inhibitory and antifeedant effects of plant extracts and pure allelochemicals on four phytophagous insect species

Antifeedant and growth inhibitory effects of crude plant extracts (Melia volkensii and Origanum vulgare) and pure allelochemicals (digitoxin, cymarin, xanthotoxin, toosendanin, thymol and trans‐anethole) were investigated in the cabbage looper (Trichoplusia ni), and in the armyworm (Pseudaletia unipuncta) using different bioassays. Antifeedant effects of M. volkensii, O. vulgare and thymol were investigated in larvae of the diamondback moth (Plutella xylostella), and of O. vulgare and thymol in the Mexican bean beetle (Epilachna varivestis), using leaf disc choice bioassays. M. volkensii was the most potent growth inhibitor for T. ni and P. unipuncta (dietary EC₅₀ = 7.6 and 12.5 p.p.m., respectively) of all the test substances. Cymarin was the second most potent growth inhibitor (EC₅₀ = 132.0 p.p.m.) for T. ni. The most effective feeding deterrents for third instar T. ni larvae were xanthotoxin andM. volkensii (DC₅₀ = 0.9 and 8.3 μg/cm², respectively). M. volkensii was also the most potent feeding deterrent for third instar P. unipuncta, P. xylostella and adult E. varivestis (DC₅₀ = 10.5, 20.7 and 2.3 μg/cm², respectively). Because of interspecific differences in response to feeding deterrents and the lack of a strong relationship between EC₅₀ and DC₅₀ values, we recommend testing a battery of bioassay species with candidate compounds and the use of more than one bioassay. Based on their growth inhibitory and feeding deterrent properties, some of these plant extracts and pure allelochemicals have potential for use as alternative crop protectants against a number of pest species.     

Food mixing by generalist grasshoppers: Plant secondary compounds structure the pattern of feeding

Plant secondary compounds can prevent feeding by phytophagous insects or, if ingested, can be poisonous to them. Less attention has been paid to the additional effects they have on feeding behavior when they are only weakly deterrent or not deterrent at all. Experiments were carried out on the generalist grass-hopperSchistocerca americana. Individuals were presented either with two cakes of high-quality artificial food with a single deterrent compound added or with two cakes, each with a different added deterrent compound. The deterrents consisted of single plant secondary compounds that were either marginally or strongly deterrent. There were profound differences in feeding behavior between those individuals given identical and those given different cake types, including longer feeding bouts on single cakes when a choice of different cake types was available. The behavioral effects demonstrate that the presence of secondary compounds in one food can influence the patterns of feeding on other available foods and suggest that such chemicals could impact foraging activities in a complex manner.     

The role of phenolic compounds and other plant constituents in feeding by Canada geese in a coastal marsh

Summary We have assessed the relative importance of phenolic compounds, other secondary metabolites, and gross nutrient levels as feeding cues to Canada geese. Phenolic content was the most significant constituent influencing feeding selection by geese. Nutrient content had little or no effect on feeding selection.Correlative data showing the negative influence of plant phenolics on food choices by wild geese were supported by feeding preference tests. Extracts of unpalatable plants inhibited feeding by captive geese relative to extracts of palatable plants. In high phenolic plants, the phenolic containing methanol extract was more inhibitory than extractions made with petroleum ether. In a relatively low phenolic, unpalatable plant, an inhibitory factor was extractable in petroleum ether, indicating that for this species, another class of deterrents was involved. Preference tests with individual secondary metabolites showed that tannic acid and quebracho tannin were very effective in inhibiting feeding by geese and phenolic acids were slightly inhibitory, but a sesquiterpene lactone was not deterrent. These results point out the primacy of some secondary metabolites in determining food choices by geese.     

Habituation in Frankliniella occidentalis to deterrent plant compounds and their blends

Feeding and oviposition deterrence of three secondary plant compounds and their 1:1 blends to adult female Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) and the potential for habituation of the thrips to the pure compounds and the 1:1 blends at various concentrations were investigated. In choice assays, we tested dose‐dependent feeding and oviposition deterrence of the two fatty acid derivatives methyl jasmonate and cis‐jasmone, the phenylpropanoid allylanisole, and their blends when directly applied to bean leaf discs. The concentration required to reduce the feeding damage by 50% relative to the control treatment (FDC₅₀) was lowest for cis‐jasmone and highest for allylanisole. The feeding deterrent effect of both jasmonates was increased when blended with allylanisole. Feeding deterrence and oviposition deterrence were strongly correlated. In no‐choice assays conducted over four consecutive days, we discovered that dilutions at low concentrations (FDC₁₅) applied to bean leaves resulted in habituation to the deterrents, whereas no habituation occurred at higher concentrations (FDC₅₀). We observed a tendency that the 1:1 blends reduce the probability that thrips habituate to the deterrent compounds. Our results may be useful in the development of integrated crop protection strategies with the implementation of allelochemicals as pest behaviour‐modifying agents.     

Continuous exposure to the deterrents cis‐jasmone and methyl jasmonate does not alter the behavioural responses of Frankliniella occidentalis

Behavioural responses of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), a generalist, cell sap‐feeding insect species with piercing‐sucking mouthparts, after continuous exposure to two deterrent secondary plant compounds are investigated. We compared in choice assays on bean leaf discs, the settling, feeding, and oviposition preferences of F. occidentalis females that had no experience with the two fatty acid derivatives methyl jasmonate and cis‐jasmone before testing (naïve thrips) vs. females that had been exposed to the deterrent compounds before testing (experienced thrips). The thrips were exposed to the deterrents at low or high concentrations for varied time periods and subsequently tested on bean leaf discs treated with the respective deterrent at either a low or a high concentration. Frankliniella occidentalis females avoided settling on the deterrent‐treated bean leaf discs for an observation period of 6 h, independent of their previous experience. Our results demonstrate that feeding and oviposition deterrence of the jasmonates to the thrips were not altered by continuous exposure of the thrips to the jasmonates. Habituation was not induced, neither by exposure to the low concentration of the deterrents nor by exposure to the high concentration. These results indicate that the risk of habituation to two volatile deterrent compounds after repeated exposure is not evident in F. occidentalis. This makes the two compounds potential candidates to be integrated in pest management strategies.     

Multiple mode of action of the feeding deterrent,toosendanin, on the sense of taste in Pieris brassicae larvae

Toosendanin, a tetranortriterpenoid isolated from the bark of Melia toosendan, is a feeding deterrent for larvae of Pieris brassicae. By using electrophysiological techniques, it was found that toosendanin stimulates a deterrent receptor cell located in the medial maxillary sensillum styloconicum. Toosendanin also inhibits responses of both the sugar and glucosinolate receptor cell, which are localized in the lateral sensillum styloconicum. The degree of inhibition of the sugar receptor increases with increasing sucrose concentration. The glucosinolate receptor cell shows a reversed reaction: inhibition by toosendanin decreases with increasing sinigrin concentration. Inhibitory effects occur at a toosendanin concentration as low as 10^–9 M and are dose dependent. The taste neurons that respond to amino acids or deterrents in the lateral sensillum, however, are not affected by toosendanin. It is concluded that the sensory code underlying feeding behaviour is modulated by toosendanin via several different peripheral sensory mechanisms.     

A method to test compounds for feeding deterrence towards redlegged earth mite (Acarina: Penthaleidae)

A bioassay, based on a membrane sachet technique, has been developed to identify antifeeding compounds affecting the redlegged earth mite, Halotydeus destructor (Acari: Penthaleidae). The method consists of counting H. destructor numbers on membrane sachets in choice experiments, which was quicker and more efficient than weighing the mites. Five per cent aqueous glucose solution was used as a feeding stimulant, with Tween 80® at 5% concentration as a solubilising agent for water-insoluble compounds. (+)-Catechin, rutin, biochanin A, formononetin, chlorogenic acid, and gramine acted as feeding deterrents at 1% concentration. Quercetin (1 %) and azadirachtin (100 ppm) had no significant effect. At lower concentrations (0.01%), compounds showed antifeeding (gramine), phagostimulating (quercetin and chlorogenic acid), or no effects on mite numbers. Dose-dependent deterrent effects of plant extracts were demonstrated with the bioassay, which could be used for other mites.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

Whitebellied sunbirds (<Emphasis Type="Italic">Nectarinia talatala</Emphasis>, Nectariniidae) do not prefer artificial nectar containing amino acids

Amino acids are the most abundant class of compounds in nectar after sugars. Like its sugar concentration, the amino acid concentration of nectar has been linked to pollinator type, and it has been suggested that amino acid concentrations are high in the floral nectars of plant species pollinated by passerine birds compared to those pollinated by hummingbirds. We investigated the feeding response of whitebellied sunbirds (Nectarinia talatala) to the inclusion of amino acids in artificial nectar (0.63 M sucrose solution). The response to asparagine, glutamine, phenylalanine, proline, serine and valine, amino acids commonly found in floral nectars, was tested individually and using a mixture of all six amino acids, at two different concentrations (2 and 15 mM). Sunbirds showed no significant preference for amino acids in nectar, or avoided them, especially at the higher concentration. We discuss these findings in the light of the nitrogen requirements of nectarivorous birds and data on amino acids in floral nectars.     

Food discrimination and induction of preference for artificial diets in the tobacco hornworm, Manduca sexta

ABSTRACT. Feeding discrimination of Manduca larvae reared on leaves and a variety of artificial media was tested against leaves, various media, solvent fractions of leaves and media, and media components, including pure chemicals. Field-collected animals showed the same preferences as did animals cultured in the laboratory for many generations. Rearing larvae on a leaf species, an artificial diet, or homogenized leaves added to artificial diet induced a preference for that food in subsequent choice tests. Extracts of these foods using organic solvents (particularly hexane) elicited feeding choices resembling those evoked by the foods themselves: water extracts were effective as stimulants or deterrents, but responses to them differed considerably from responses to the foods. Animals raised on a defined artificial diet showed an increased preference (or decreased deterrence) for the lipid component (linolenic acid) of that diet in behavioural tests. Thus induction of preference was shown to be influenced by a specific nutrient compound. Fifteen artificial diets were tested; three were successful, including a completely defined medium. Various components of the diets were tested for feeding preferences, both as omissions from the main diet and as pure compounds. Some were stimulatory, most were neutral or slightly deterrent; a few were strongly deterrent. With the exception of linolenic acid, preferences for pure compounds were not significantly correlated with the food on which the animals were reared. The data suggest that food discrimination depends on the perception of a complex chemical message comprised of both polar and non-polar compounds, with the latter being of somewhat greater importance. The results are consistent with the explanation of the induction of feeding preferences being due to a reduced responsiveness to deterrents, to an increased preference for feeding stimulants or to both.     

Chemical requirements of feeding in rainbow trout, Oncorhynchus mykiss (Walbaum); palatability studies on amino acids, amides, amines, alcohols, aldehydes, saccharides, and other compounds

Past studies on palatability perception and the chemical requirements of feeding in rainbow trout have focused on the amino acids. The stimulatory effectiveness of other classes of compounds remains largely unknown. In the present study representative members from several chemical classes were tested for their palatability to rainbow trout. Three heretofore untested amino acids (L-norvaline, L-norleucine and c-aminocaproic acid), three amides (acetamide, valeramide and caproic amide), two alcohols (hexanol and octanol) and four sugars (o-ribose, o-glucose, D-fructose and sucrose) were all mildly to highly palatable substances at their highest concentration tested. All other compounds tested within these classes did not evoke a positive behavioural response, demonstrating that rainbow trout are selective in thosecompounds deemed as palatable. Moreover, all of the aldehydes, trimethylamine, pyrrolidine and all of the amines except the alcoholic derivative, ethanolamine, were without effect. A provisional scheme for relating chemical structure versus the perception of palatability in rainbow trout is presented.     

Electrophysiological responses of taste cells to nutrient mixtures in the polyphagous caterpillar of Grammia geneura

In a normally feeding insect, the taste receptors are exposed to complex mixtures of chemicals, not single compounds. We investigate the responses of neurons in the galeal sensilla of the caterpillar of Grammia geneura to mixtures of nutrient compounds at concentrations occurring in plants. Compounds that stimulated the same neuron were generally additive in their effects in binary mixtures. Amino acids that did not stimulate usually had no effect in mixtures with a stimulating compound, but glutamic acid reduced the response to serine in the medial sensillum. Nutrient compounds that stimulated different cells in a sensillum acted independently of each other. Complex mixtures of amino acids resembling samples of free amino acids from three host plants were less stimulating than expected from their molar concentrations. In host plant selection, the response from the medial sensillum is probably dominated by sucrose; unless sucrose levels are low, amino acids will contribute little to sensory input because they stimulate the same cell as sucrose. In the lateral sensillum, amino acids act independently of sugars. The limited contact chemosensory array of caterpillars seems inadequate to allow them to make fine distinctions between plants on the basis of their free amino acids.     

Stimulatory effectiveness of amino acids on the olfactory response in an algivorous marine teleost, the rabbitfish Siganus fuscescens Houttuyn

The detection of threshold concentrations and relative stimulatory effectiveness (RSE) for 19 amino acids were studied by olfactory bulbar electroencephalogram (EEG) in the algivorous rabbitfish. The threshold concentrations for 19 amino acids ranged from 10⁻¹⁰ to 10⁻⁵ M. l -Alanine was the most effective amino acid, and the threshold concentration was estimated to be from 10⁻¹⁰ to 10⁻⁹M. These results were compared with the olfactory response in herbivorous and carnivorous fishes previously reported. The comparison of RSE for rabbitfish with those for other fishes showed correlation coefficients ranging from r = 0.97 to r = 0.50, but high similarities did not correspond with difference in feeding habits for herbivores and carnivores. The role of amino acids in fish chemosensory behaviour and the olfactory response of the rabbitfish was compared with the gustatory response reported previously. It was found that l -serine was the most potent stimulant in both chemoreceptors, l -proline and l -glutaminic acid were the most potent gustatory stimulants, and l -alanine, l -glutamine, l -arginine and l -lysine were the most potent olfactory stimulants.     

Activation of chemical defenses in the tropical green algae Halimeda spp.

Halimeda spp. are among the most common seaweeds on tropical reefs where herbivory is intense. These calcified seaweeds produce diterpenoid feeding deterrents; the major metabolites are halimedatetraacetate and halimedatrial. We found that most species of Halimeda on Guam immediately convert the less-deterrent secondary metabolite halimedatetraacetate to the more potent feeding deterrent halimedatrial when plants are injured by grinding or crushing. This conversion would therefore occur when fishes bite or chew Halimeda plants. We term this process of rapid conversion “activation”. Extracts from injured plants contained higher amounts of halimedatrial and were more deterrent toward herbivorous fishes than extracts from control plants. Herbivore-activated defenses are common in many families of terrestrial plants: however, this is the first example of an activated defense in a marine plant.