Larval Beetles Form a Defense from Recycled Host-Plant Chemicals Discharged as Fecal Wastes

Larvae of the leaf-feeding beetles Neolema sexpunctata and Lema trilinea carry feces on their backs that form shields. We used the generalist predatory ant, Formica subsericea, in a bioassay to determine whether shields were a physical barrier or functioned as a chemical defense. Fecal shields protected both species against ant attack. Larvae of both species reared on lettuce produced fecal shields that failed to deter ants. Commelina communis, N. sexpunctata's host, lacks noxious secondary compounds but is rich in phytol and fatty acids, metabolites of which become incorporated into the fecal defense. In contrast, the host plant of L. trilinea, Solanum dulcamara, contains steroidal glycoalkaloids and saponins, whose partially deglycosylated metabolites, together with fatty acids, appear in Lema feces. Both beetle species make modifications to host-derived precursors before incorporating the metabolites into shields. Synthetic chemicals identified as shield metabolites were deterrent when applied to baits. This study provides experimental evidence that herbivorous beetles form a chemical defense by the elimination of both primary and secondary host-derived compounds. The use of host-derived compounds in waste-based defenses may be a more widely employed strategy than was hitherto recognized, especially in instances where host plants lack elaborate secondary compounds.     

The Chlorophyll Catabolite,Pheophorbide <Emphasis Type="Italic">a</Emphasis>, Confers Predation Resistance in a Larval Tortoise Beetle Shield Defense

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 μg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 μg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 μg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 μg added Pha were comparable to intact shields, even though the Pha concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.     

Chemical Defense in Larval Tortoise Beetles: Essential Oil Composition of Fecal Shields of Eurypedus nigrosignata and Foliage of its Host Plant, Cordia curassavica

Larvae of the tortoise beetle Eurypedus nigrosignata construct fecal shields using cast skins and fecal strands. Survival of larvae with intact shields was higher in the field than for larvae with shields removed. In the laboratory, E. nigrosignata feculae had a deterrent effect on feeding in the ant Myrmica rubra as did an extract of the host plant, Cordia curassavica. Three chemical types were identified in the host-plant foliage and were named -terpinene, -pinene, and sabinene, depending on their mono- and sesquiterpene composition. This is the first report of lower terpenes (essential oils) in foliage of Cordia. Fecal shields of E. nigrosignata displayed the same terpene pattern as larval host-plant leaves. The absolute concentration of mono- and sesquiterpenes in the dorsal fecal shield depended on the plant chemical type and tended to decrease with larval age. No oxidation or detoxification products of ingested terpenes were detected in the larval fecula, indicating that the chemical composition of the larval fecal shield is influenced primarily by the host-plant secondary chemistry.     

Synergistic Effects of Three Piper Amides on Generalist and Specialist Herbivores

The tropical rainforest shrub Piper cenocladum, which is normally defended against herbivores by a mutualistic ant, contains three amides that have various defensive functions. While the ants are effective primarily against specialist herbivores, we hypothesized that these secondary compounds would be effective against a wider range of insects, thus providing a broad array of defenses against herbivores. We also tested whether a mixture of amides would be more effective against herbivores than individual amides. Diets spiked with amides were offered to five herbivores: a naïve generalist caterpillar (Spodoptera frugiperda), two caterpillar species that are monophagous on P. cenocladum (Eois spp.), leaf-cutting ants (Atta cephalotes), and an omnivorous ant (Paraponera clavata). Amides had negative effects on all insects, whether they were naïve, experienced, generalized, or specialized feeders. For Spodoptera, amide mixtures caused decreased pupal weights and survivorship and increased development times. Eois pupal weights, larval mass gain, and development times were affected by additions of individual amides, but increased parasitism and lower survivorship were caused only by the amide mixture. Amide mixtures also deterred feeding by the two ant species, and crude plant extracts were strongly deterrent to P. clavata. The mixture of all three amides had the most dramatic deterrent and toxic effects across experiments, with the effects usually surpassing expected additive responses, indicating that these compounds can act synergistically against a wide array of herbivores.     

Effects ofSolanum glycoalkaloids on chemosensilla in the Colorado potato beetle

Steroidal glycoalkaloids, found in species of the Solanaceae, elicit bursting activity in galeal and tarsal chemosensilla of adult Colorado potato beetles. The effect has an average latency of 6–12 sec, depending on the sensillum/alkaloid combination. A 20-sec alkaloid treatment is often suffficient to render galeal sensilla unresponsive to gamma-aminobutyric acid, normally an effective stimulant. The alkaloids have similar effects on galeal sensilla of larval Colorado potato beetles and on labellar chemosensilla of the blowfly. It is concluded that these compounds act independently of any specialized chemoreceptor in the Colorado potato beetle, and that association of the Colorado potato beetle with solanaceous plants has not led to evolution of a specific receptor forSolanum glycoalkaloids.     

Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.     

Chemical Deterrence of a Cyanobacterial Metabolite against Generalized and Specialized Grazers

Pitipeptolide A is the major secondary metabolite in a persistent population of the cyanobacterium Lyngbya majuscula from Guam. Because this cyanobacterium is of low preference to many sympatric consumers, we tested the hypothesis that this compound acted as a chemical defense. Pitipeptolide A was deterrent to urchins, two species of amphipods, and small herbivorous crabs, whereas it did not deter feeding by the sea hare Stylocheilus striatus, which readily consumes cyanobacteria. Although our comparison included various small invertebrates, which are often tolerant to algal chemical defenses, this cyanobacterial compound proved deterrent at natural concentrations to all but one of the consumer species tested. Thus, the production of defensive secondary metabolites is likely related to the common occurrence of L. majuscula on this coral reef despite the abundance of diverse grazers.     

Chemical Analysis of Squirt-gun Defense in Bursera and Counterdefense by Chrysomelid Beetles

The genus Bursera produces resin stored in canals in the leaf. When leaves are damaged, some, but not all, species release abundant resin. Species of Blepharida are specialized herbivores of Bursera, and they exhibit variation in their counterdefensive behavior. Species feeding on resin-releasing plants cut the leaf veins before feeding, which often makes them more prone to predation. They also adorn their backs with their feces and may regurgitate and release an anal secretion when attacked or disturbed by predators. Species that feed on Bursera species that release no fluids do not sever the leaf veins prior to feeding, and they do not carry their feces on their backs. Instead, they face their predators, raise their heads in a "boxing-like" display, and rapidly swing their abdomens from side to side. We performed a comparative chemical analysis of the compounds found in Bursera schlechtendalii, a species that releases abundant resins, and B. biflora, a species that does not. We also analyzed the frass, enteric discharges, and larvae of the two species of Blepharida that feed on each of these plants. The compounds found in the body, feces, and discharges of the Blepharida species that adorns itself with feces match the chemical mixture of its host plant, suggesting that this beetle species can compensate its higher risk of predation by using the compounds present in the plant for defense. The chemical mixture of B. biflora is more complex and does not match the compounds found in the body or frass of its beetle herbivore, suggesting that the defensive strategy of this insect is behavioral and does not rely on its host's constituents.     

Effect of Solanum Glycoalkaloids on Potato Aphid, Macrosiphum euphorbiae: Part II

We report the deterrent, toxic, and antireproductive effect of several Solanum glycoalkaloids on the potato aphid, Macrosiphum euphorbiae, and discuss the structure–activity relationship of the tested compounds. The results indicate a structure-dependent biological effect of the glycoalkaloids, suggesting that, while the structure of the aglycone defines a basal biological effect, the carbohydrate moiety is crucial for the overall biological effect.     

Host plant iridoid-based chemical defense of an aphid,Acyrthosiphon nipponicus,against ladybird beetles

A Rubiaceae-feeding aphid,Acyrthosiphon nipponicus, is seldom attacked by the ladybird beetle,Harmonia axyridis. A potent deterrent against the beetle was isolated from the aphid and identified as paederoside, an iridoid glycoside originating in the aphid's host,Paederia scandens. The iridoid content was as high as 2% of the intact body weight, and a large portion was found in the cornicle secretion.     

Responses of two hymenopteran predators to surface Chemistry of their prey: Significance for an alkaloid-sequestering caterpillar

Larvae ofUresiphita reversalis (Guenée) (Lepidoptera: Pyralidae) sequester quinolizidine alkaloids from their leguminous hosts and store them primarily in the cuticle. Stored alkaloids are lost with the last larval molt. Extracts of late-instar larvae and of pupae were applied to larvae of the potato tuber moth,Phthorimaea operculella (Zeller) (Gelichiidae), which are normally palatable to two hymenopteran predators, the Argentine ant,Iridomyrmex humilis (Mayr) (Formicidae), and the paper wasp,Mischocyttarus flavitarsus (Sauss.) (Vespidae). Larvae ofP. operculella treated with alkaloid extracts ofU. reversalis larval exuviae, or with surface extracts of whole larvae, were deterrent to both predators, compared to untreated prey. Extracts of pupal exuviae added toP. operculella, however, were not deterrent.P. operculella larvae treated with the authentic alkaloids sparteine and cytisine were also deterrent to these hymenopteran predators. Storage of small but concentrated amounts of plant secondary compounds in the cuticle appears to be an efficacious means of defense against at least two common predators of lepidopteran larvae.     

Effect of Solanum Glycosides on the Aphid Schizaphis graminum

Common Solanum glycosides were tested against a non-Solanum pest, the greenbug, Schizaphis graminum, fed by artificial diet supplemented with the tested compounds. In contrast with the potato aphid Macrosiphum euphorbiae, S. graminum is sensitive to both potato and tomato glycoalkaloids, as well as to all the different Solanum steroidal glycosides tested. S. graminum was less sensitive to glycoalkaloids than to the steroidal glycosides. Laxumin A (LC₅₀ 4.3 M) and laxumin B (LC₅₀ 6.1 M), isolated from Solanum laxum, were the most active of the tested compounds. The different sensitivity of both aphids against Solanum glycosides could be related to a possible adaptation of the insects to their host plants secondary metabolites.     

Evaluation of the Evolution of Increased Competitive Ability (EICA) Hypothesis: Loss of Defense Against Generalist but not Specialist Herbivores

The evolution of increased competitive ability (EICA) hypothesis predicts that invasive plant species may escape their specialized natural enemies in their introduced range and subsequently evolve with a decrease in investment in anti-herbivore chemical defenses relative to native conspecifics. We compared the chemical profile of 10 populations of US native and 20 populations of European invasive Solidago gigantea. To test for differences in inducibility between native and invasive populations, we measured secondary chemistry in both damaged and undamaged plants. We also performed bioassays with three specialist and two generalist insect herbivores from four different feeding guilds. There was no evidence that invasive populations had reduced concentrations of sesquiterpenes, diterpenes, or short-chain hydrocarbons (SCH), although significant variation among populations was detected. Sesquiterpene and diterpene concentrations were not influenced by damage to the host plant, whereas SCH concentrations were decreased by damage for both native and invasive plants. Performance of the three specialist insects was not affected by the continental origin of the host plant. However, larval mass of the generalist caterpillar Spodoptera exigua was 37% lower on native plants compared to invasive plants. The other generalist insect, a xylem-tapping spittlebug that occurs on both continents, performed equally well on native and invasive plants. These results offer partial support for the defense predictions of the EICA hypothesis: the better growth of Spodoptera caterpillars on European plants shows that some defenses have been lost in the introduced range, even though our measures of secondary chemistry did not detect differences between continents. Our results show significant variation in chemical defenses and herbivore performance across populations on both continents and emphasize the need for analysis across a broad spatial scale and the use of multiple herbivores.     

Sequestration of Furostanol Saponins by <Emphasis Type="Italic">Monophadnus</Emphasis> Sawfly Larvae

Sawfly larvae of the tribe Phymatocerini (Hymenoptera: Tenthredinidae), which are specialized on toxic plants in the orders Liliales and Ranunculales, exude a droplet of deterrent hemolymph upon attack by a predator. We investigated whether secondary plant metabolites from Ranunculaceae leaves are sequestered by phymatocerine Monophadnus species, i.e., Monophadnus alpicola feeding upon Pulsatilla alpina and Monophadnus monticola feeding upon Ranunculus lanuginosus. Moreover, two undescribed Monophadnus species were studied: species A collected from Helleborus foetidus and species B collected from Helleborus viridis. Comparative high-performance liquid chromatographic–photodiode array detection–electrospray ionization–mass spectrometric analyses of plant leaf and insect hemolymph extracts revealed the presence of furostanol saponins in all samples. Larvae of species A and B actively sequestered (25R)-26-[(α-l-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→3)-O-[6-acetyl-β-d-glucopyranosyl-(1→3)]-O-β-d-glucopyranoside (compound 1). This compound occurred at a 65- to 200-fold higher concentration in the hemolymph of the two species (1.6 and 17.5 μmol/g FW, respectively) than in their host plant (0.008 and 0.268 μmol/g FW, respectively). In M. monticola, compound 1 was found at a concentration (1.2 μmol/g FW) similar to that in the host plant (1.36 μmol/g FW). The compound could not be detected consistently in M. alpicola larvae where, however, a related saponin may be present. Additional furostanol saponins were found in H. foetidus and H. viridis, but not in the two Monophadnus species feeding on them, indicating that sequestration of compound 1 is a highly specific process. In laboratory bioassays, crude hemolymph of three Monophadnus species showed a significant feeding deterrent activity against a potential predator, Myrmica rubra ant workers. Isolated furostanol saponins were also active against the ants, at a concentration range similar to that found in the hemolymph. Thus, these compounds seem to play a major role for chemical defense of Monophadnus larvae, although other plant secondary metabolites (glycosylated ecdysteroids) were also detected in their hemolymph. Physiological and ecological implications of the sequestered furostanol saponins are discussed. Dedicated to the memory of Professor Ivano Morelli (1940–2005)     

Deterrent and insecticidal effects of extracts of pithraj,Aphanamixis polystachya (Meliaceae), againstTribolium castaneum in storage

Laboratory experiments were carried out to investigate the efficacy of seed extracts of pithraj,Aphanamixis polystachya (Wall & Parker), a locally grown plant in Bangladesh, against the red flour beetle,Tribolium castaneum (Herbst.). Results of three different bioassays showed that crude extracts of pithraj seeds have strong repellent effects and moderate feeding deterrent and insecticidal (direct-contact) effects on adultTribolium castaneum.     

Bioassays of segregating plants

Solanum chacoense is a wild potato species resistant to the Colorado potato beetle,Leptinotarsa decemlineata. Most genotypes ofS. chacoense synthesize the glycoalkaloids solanine (sol) and chaconine (chac) and are hosts of the beetle. A few rare genotypes have a gene(s) for acetylation of carbon-23 of the steroid aglycone of sol and chac. Laboratory bioassays and replicated field tests of clones differing in the presence or absence of the acetyl moiety showed that acetylation of sol and chac markedly affects the response of both adults and larvae to the foliage. Adult feeding deterrency conferred by acetylated forms of sol and chac (leptines) in leaf-disk preference tests was consistent with the degree of antixenosis measured in the field. Development of larvae on foliage of clones with leptines was also inhibited. The studies support the validity of using laboratory bioassays of plants segregating for levels of a suspected defense compound to determine the role the compound has in defending the plant from attack by an insect predator in the field.     

Insect feeding and oviposition deterrents from western red cedar foliage

The feeding deterrent activity of fractions from the foliage of western red cedar,Thujaplicata Donn, was studied in laboratory bioassays using the white pine weevil,Pissodes strobi Peck, as a test insect. The most active fraction was the volatile mixture that comprises the leaf oil of this tree species. Further fractionation of the leaf oil indicated feeding deterrent activity in the monoterpene hydrocarbon, thujone, and terpene alcohol fractions. When tested alone, both (–)-3-isothujone and (+)-3-thujone, which made up 75–88% and 5–10% of the leaf oil, respectively, deterred feeding by the weevils. Western red cedar leaf oil also showed antifeedant activity with the alder flea beetle,Altica ambiens (Le Conte), and served as an oviposition deterrent for the onion root maggot,Hylemya antiqua Meigen. The leaf oil, however, had no inhibitory effect on the feeding of the leaf roller,Epinotia solandriana L., and the red-backed sawfly,Eriocampa ovata L.Research supported by the Natural Science and Engineering Research Council, Canada, Co-op Grant No. A0243 and Operating Grant Nos. A3881 and A3706, and by the British Columbia Ministry of Labour Youth Employment Program.     

Effects ofneo-clerodane diterpenes fromTeucrium on feeding behavior of colorado potato beetle larvae

Treatment of potato leaf disks with fourneo-clerodane diterpenes fromTeucrium (eriocephalin, teucrin-A, teucvin, and teuscorolide) significantly reduced feeding by larvae ofLeptinotarsa decemlineata (Colorado potato beetle). Choice and no-choice tests suggest that teuscorolide acts as a feeding deterrent, whereas the antifeedant activity of teucrin-A, teucvin, and eriocephalin is likely associated with a toxic mode of action. Nutritional tests, antifeedant simulation assays, and posttreatment studies confirmed that teucrin-A can be categorized by its mode of action as a toxin, rather than as a feeding deterrent.     

Structure–Activity Relationship of Inhibition of Fish Feeding by Sponge-derived and Synthetic Pyrrole–Imidazole Alkaloids

We investigated the relationship between the structures of pyrrole-containing alkaloids from marine sponges of the genus Agelas and their capacity to deter feeding by the omnivorous Caribbean reef fish, Thalassoma bifasciatum. Seven natural products were assayed at volumetric concentrations of 1, 5, and 10 mg/ml: dispacamide A, keramadine, oroidin, midpacamide, 4,5-dibromopyrrole-2-carboxylic acid, 4,5-dibromopyrrole-2carboxamide, and racemic longamide A. We also assayed 14 structural analogs obtained mostly by chemical synthesis. Of the seven natural products, only rac-longamide A was not significantly deterrent at any of the assay concentrations. The pyrrole moiety was required for feeding inhibition activity, while the addition of the imidazole group enhanced this activity. Variously functionalized imidazoles lacking the pyrrole moiety were not deterrent. Combinations of the natural products appeared to have an additive effect on feeding inhibition; there was no evidence of synergy. Given their high concentrations in sponge tissue, dispacamide A and oroidin most probably serve as the primary chemical defenses of many Agelas sp., while minor compounds such as keramadine are not present in high enough concentrations to contribute much to chemical defense.     

Chemical Defense in Harvestmen (Arachnida,Opiliones): Do Benzoquinone Secretions Deter Invertebrate and Vertebrate Predators?

Two alkylated 1,4-benzoquinones were identified from the defensive secretion produced by the neotropical harvestman Goniosoma longipes (Gonyleptidae). They were characterized as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. We tested the effectiveness of these benzoquinone secretions against several predator types, including invertebrates and vertebrates. Different predators were exposed to the harvestmen's gland secretion or to distilled water in laboratory bioassays. Our results indicate that secretions containing the 1,4-benzoquinones released by G. longipes can be an effective defense against predation, and that the effectiveness of the secretion is dependent on the predator type. The scent gland secretion repelled seven ant species, two species of large wandering spiders, and one frog species, but was not an effective defense against an opossum. Our study also demonstrates that the scent gland secretion of G. longipes can work as a chemical shield preventing the approach of three large predatory ants for at least 10 min. The chemical shield may protect the harvestman against successive attacks of the same ant worker and also allow the harvestman to flee before massive ant recruitment. Our data support the suggestion that chemical defenses may increase survival with some but not all potential predators. This variation in defense effectiveness may result from many interacting factors, including the attack strategy, size, learning ability, and physiology of the predators, as well as the chemical nature of the defensive compounds, type of emission, and amount of effluent released by the prey.