Neotyphodium coenophialum Mycelial Protein and Herbage Mass Effects on Ergot Alkaloid Concentration in Tall Fescue

Livestock grazing endophyte-infected tall fescue (Festuca arundinacea Schreb.) pastures often suffer from ergot poisoning. The endophyte,Neotyphodium coenophialum (Morgan-Jones and Gams) Glenn, Bacon, and Hanlin, comb. nov., also provides drought-tolerant, insect-resistant, and disease-resistant qualities to the plant. Therefore, producers are faced with a biological dilemma of risking loss of pasture by using endophyte-free tall fescue pasture or animal losses with endophyte-infected tall fescue pasture. One potential solution is to breed endophyte-infected tall fescue with lower levels of alkaloids. However, breeding could select for plants that are antagonistic to the endophyte, resulting in reduced plant vigor as a consequence of disruption of the mutualistic association between the organisms. The objectives of this study were to determine the relationship between plant mass and endophyte mycelial proteins versus ergot alkaloid concentration. To examine the endophyte effect on mycelial mass, tissue culture regenerants from tall fescue genotype PDN2 were infected with endophyte isolates EDN11, EDN12, and EDN2 to eliminate confounding effects of multiple plant genotypes. Crosses with PDN11 as the maternal parent and plant genotypes DN2, DN12, and DN15 as paternal parents were used to produce a population of different plant genotypes, all containing the same endophyte. Fungal mycelial protein was extracted from lyophilized tall fescue leaf sheath tissue and immunochemically quantified with monoclonal antibodies specific toN. coenophialum proteins. Ergot alkaloid concentration was also immunochemically measured. Quantities of mycelial protein and ergot alkaloids were calculated by regressing experimental values against standards of each. There was no correlation between herbage mass and alkaloid concentration or fungal protein quantity and alkaloid concentration when different fungal isolates were inserted into the same plant genotype. Coefficients of determination (r ²) were low (0.31 and 0.17) between leaf sheath and leaf blade alkaloid concentrations and endophyte protein when different plant genotypes contained the same endophyte isolate. Likewise,r ² values were low between leaf sheath and leaf blade alkaloid concentrations and herbage mass. These data suggest that little or no antagonism occurred to the endophyte among plants low in alkaloid concentration.     

Does mowing height influence alkaloid production in endophytic tall fescue and perennial ryegrass?

The mutualistic symbiosis following infection of tall fescue, Festuca arundinacea, and perennial ryegrass, Lolium perenne, by fungal endophyte (Neotyphodium spp.) results in the production of alkaloids that are feeding deterrents or toxic to insects and livestock. If the levels of the alkaloids can be manipulated by cultural practices in the grasses that are used for home lawns and golf courses, this could alleviate the need for pesticide applications in urban environments. We evaluated the influence of mowing height on the levels of some alkaloids in a greenhouse experiment for two consecutive months. In tall fescue, levels of four of the nine alkaloids, including one presumptive alkaloid, showed increased levels with increasing the mowing height from 2.5 to 7.5 cm. The alkaloids were ergonovine, ergocryptine, perloline methyl ether, and an unidentified alkaloid designated as unknown C. In perennial ryegrass, three out of six alkaloids, perloline methyl ether, chanoclavine, and unknown A, showed similar increases. The alkaloid levels in perennial ryegrass showed more variability than those in tall fescue between the two sampling dates. It was clear in both grasses that the relative levels of the alkaloids varied with mowing height, as well as over time.     

Temporal and Spatial Variation in Alkaloid Levels in Achnatherum robustum, a Native Grass Infected with the Endophyte Neotyphodium

The native North American perennial grass Achnatherum robustum (Vasey) Barkworth [= Stipa robusta (Vasey) Scribn.] or sleepygrass is toxic and narcotic to livestock. The causative agents are alkaloidal mycotoxins produced from infections by a systemic and asexual Neotyphodium endophyte. Recent studies suggest that toxicity is limited across the range of sleepygrass in the Southwest USA. We sampled 17 populations of sleepygrass with varying distance from one focal population known for its high toxicity levels near Cloudcroft, NM, USA. For some, we sampled individual plants twice within the same growing season and over successive years (2001–2004). We also determined infection levels in each population. In general, all populations were highly infected, but infection levels were more variable near the focal population. Only infected plants within populations near the Cloudcroft area produced alkaloids. The ergot alkaloid, ergonovine, comprised the bulk of the alkaloids, with lesser amounts of lysergic and isolysergic acid amides and ergonovinine alkaloids. Levels of all alkaloids were positively correlated among individual plants within and between growing seasons. Infected plants that produced no alkaloids in 1 yr did not produce any alkaloids within the same growing season or in other years. Levels of alkaloids in sleepygrass populations declined with distance from the Cloudcroft population, although infection levels increased. Infected plants in populations in northern New Mexico and southern Colorado produced no alkaloids at all despite 100% infectivity. Our results suggest that only specific Neotyphodium haplotypes or specific Neotyphodium–grass combinations produce ergot alkaloids in sleepygrass. The Neotyphodium haplotype or host–endophyte combination that produces toxic levels of alkaloids appears restricted to one locality across the range of sleepygrass. Because of the wide variation in alkaloid levels among populations, interactions between the endophyte and host, and consequences for herbivores, competitors, and pathogens and other components of the community, are likely to vary widely across the geographic range of this native grass.     

Ergovaline and peramine production in endophyte-infected tall fescue: Independent regulation and effects of plant and endophyte genotype

Peramine and ergovaline have ecological and economical significance because they possess insect and/or mammalian toxicity properties. The relationship between these endophytically derived alkaloids in tall fescue (Festuca arundinacea Schreb.) is unknown. Seasonal and plant tissue effects on the concentration of peramine and ergovaline was investigated in field and greenhouse experiments. The relationship between the alkaloids and the regulatory effects of endophyte and plant on their content were investigated among progeny of reciprocal crosses between high- and low-ergovaline and peramine plant-endophyte combinations. Variation in peramine concentration ranged from 750 to 1742 µg/kg in greenhouse-grown plants, and there was no seasonal trend in peramine content. There was a correlation (r=0.69) between peramine and ergovaline content among leaf tissues of field-grown plants, but there was no correlation between the alkaloids in the culm (r=0.20) or panicle (r=0.17) tissues. Mean leaf ergovaline concentration of progeny derived from the low-ergovaline parent (163 µg/kg) was less than the midparent value (228 µg/kg), but mean of progeny from the high-ergovaline parent was not different from the mid-parent value. Ranges within each progeny set were approximately double their mean. Mean leaf peramine concentrations of the progeny sets were similar to the mid-parent value (3354 µg/kg) but ranges were from 1716 to 8753 µg/kg. There was no correlation between leaf peramine and ergovaline (r=0.01). These data suggest that endophytically produced alkaloids are independently regulated and are controlled by both plant and endophyte genotype.     

Does decreased mowing frequency enhance alkaloid production in endophytic tall fescue and perennial ryegrass?

Tall fescue, Festuca arundinacea, and perennial ryegrass, Lolium perenne, are widely infected with fungal endophytes (Neotyphodium spp.). The symbiosis between plant and fungus leads to synthesis of alkaloids that have been shown to be either toxic or act as feeding deterrents against insect pests. As cultural practices have the potential to regulate production of plant secondary metabolites, we evaluated the influence of mowing frequency on the levels of major alkaloids in tall fescue and perennial ryegrass in the greenhouse. Tall fescue and perennial ryegrass maintained in 15-cm-diam. pots were cut to 5-cm height weekly or biweekly. Samples were taken monthly and the alkaloids extracted and analyzed by reverse-phase LC-MS. In tall fescue, ergovaline, ergonovine, and ergocristine were identified, whereas only ergocristine was identified in perennial ryegrass samples. In tall fescue, we observed a trend showing higher levels in samples cut biweekly than in those cut weekly. A similar pattern was seen in some putative alkaloids that were not identified. In perennial ryegrass, ergocristine and two putative alkaloids followed a pattern similar to that of alkaloids in tall fescue. A survey of a few samples of perennial ryegrass using extractions specific to peramine and lolitrem B yielded evidence suggesting their presence as well as several other identified alkaloids. These data support the hypothesis that decreased mowing frequency enhances alkaloid production/accumulation in tall fescue and perennial ryegrass.     

Symbiont Regulation and Reducing Ergot Alkaloid Concentration by Breeding Endophyte-Infected Tall Fescue

Ergot alkaloids in endophyte-infected (E+) tall fescue (Festuca arundinacea) are responsible for livestock toxicosis. The host plant is capable of modifying the extent to which endophyte produces ergot alkaloids, possibly via endophyte nutrition by the host. Endophytes vary in alkaloid production. Maintaining the E+ tall fescue is essential for plant survival. Therefore, crop scientists are interested in reducing alkaloid concentration of E+ tall fescue. The objectives of this study were to determine maternal and paternal effects as an estimate of plant and endophyte genotype interactions for ergot alkaloid phenotype and the heritability of the ergot alkaloid trait in divergently selected E+ tall fescue populations. Diallel crosses were conducted among four tall fescue genotypes that varied in alkaloid concentration. Five of six crosses had progeny means less than the parental means when genotypes with lowest alkaloid were the female parents. Four of six crosses had progeny means similar to the parental means of crosses when high alkaloid genotypes were the female parents. Large ranges in progeny suggest an interaction between endophyte and plant affects alkaloid phenotype. In a second experiment, a tall fescue population was screened for alkaloid concentration. Low- and high-alkaloid plants were selected and poly-crossed within each class. Mean alkaloid concentrations of the high- and low-alkaloid progeny were higher or lower than the parental population. The trait was highly heritable and alkaloid concentration was reduced by 86% in the low-alkaloid population after two generations of selection.     

ParasiticMeloidogyne and mutualisticAcremonium increase chitinase in tall fescue

Tall fescue (Festuca arundinacea Schreb.) is a C-3 perennial grass noted for its persistence in harsh environments. Tall fescue persistence is enhanced byAcremonium coenophialum, a mutualistic fungal endophyte that increases resistance to drought, pathogens, and insects. This research was conducted to identify and elicit biochemical mechanism(s) that could account for tall fescue persistence. In initial studies, two cultivars known to differ in persistence were analyzed for chitinase, an antifungal hydrolase associated with disease resistance in other plants.Acremonium-infected Kentucky 31 (KY31), a persistent cultivar, and Johnstone, a nonpersistent cultivar, were inoculated with the parasitic nematode,Meloidogyne marylandi, grown for 50 days, and analyzed at 10-day intervals. Chitinase fluctuated throughout the 50-day period of seedling development, and activity was highest in the persistentAcremonium-infected KY31. In addition, chitinase was elicited by parasiticM. marylandi and expressed systemically. Subsequent studies were conducted to determine whether or not mutualisticAcremonium could increase chitinase activity. Genetically identical KY31, with and withoutAcremonium, were grown for 25 days and analyzed for chitinase at 5-day intervals. After 20 days,Acremonium-infected KY31 expressed more chitinase thanAcremonium-free KY31. We concluded that chitinase is related to tall fescue persistence; it was highest in the most persistent cultivar, increased under pathogen attack, and increased in the presence ofAcremonium, a symbiont known to enhance disease resistance.Contribution from University of Missouri Agricultural Experiment Station, Journal Series No. 11,472.     

Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.     

Alkaloid Production and Chinch Bug Resistance in Endophyte-Inoculated Chewings and Strong Creeping Red Fescues

Four Chewings fescue and two strong creeping red fescue selections that had been artificially inoculated and stably maintained with four different endophytes were evaluated in feeding trials with chinch bugs (Blissus leucopterus hirtus). Significant differences in survival were found between the endophyte-inoculated plants and their endophyte-free counterparts. After seven days, 54.2% of chinch bugs were alive on endophyte-free tillers versus only 7.4% of chinch bugs fed tillers from endophyte-inoculated plants. Some differences were also found among the various plant–endophyte combinations. In Petri dish preference trials, chinch bugs showed a preference for the CA endophyte (obtained from a Chewings fescue) over the RC endophyte (obtained from a strong creeping red fescue) in Chewings fescue selection C1117. Only the inoculated plants produced erogvaline, peramine, and lolitrem B; moreover, significant differences were found among the plant–endophyte combinations in the levels of these alkaloids. The Chewings selections C1117 and C1090 produced more ergovaline, and C1090 more lolitrem B, than other plants, regardless of endophyte source. In the presence of the RC endophyte, more ergovaline and lolitrem B was produced than in the presence of the CA endophyte regardless of plant genotype. Both host plant and endophyte, therefore, contributed factors that determined alkaloid production. No significant correlations between chinch bug survival and alkaloid levels were found, however, and overall, no one plant genotype or endophyte source proved to be significantly more toxic than another to chinch bugs. Nevertheless, the results clearly demonstrated that artificial inoculations of endophyte-free fescue genotypes can produce plants with increased toxicity to chinch bugs.     

Alkaloid Quantities in Endophyte-Infected Tall Fescue are Affected by the Plant-Fungus Combination and Environment

Many grass species are symbiotic with systemic, vertically-transmitted, asymptomatic Epichloë endophytic fungi. These fungi often produce alkaloids that defend the host against herbivores. We studied how environmental variables affect alkaloids in endophyte-infected tall fescue (Schedonorus phoenix) from three Northern European wild origins and the widely planted US cultivar ‘Kentucky-31’ (KY31). The plants were grown in identical common garden experiments in Finland and Kentucky for two growing seasons. Plants were left as controls (C) or given water (W), nutrient (N) or water and nutrient (WN) treatments. For 8–10 replications of each plant origin and treatment combination in both experiments, we analyzed ergot alkaloids, lysergic acid, and lolines. In Finland, tall fescue plants produced 50 % more ergot alkaloids compared to plants of the same origin and treatments in Kentucky. Origin of the plants affected the ergot alkaloid concentration at both study sites: the wild origin plants produced 2–4 times more ergot alkaloids than KY31, but the ergot alkaloid concentration of KY31 plants was the same at both locations. Overall lysergic acid content was 60 % higher in plants grown in Kentucky than in those grown in Finland. Nutrient treatments (N, WN) significantly increased ergot alkaloid concentrations in plants from Finland but not in plants from Kentucky. These results suggest that the success of KY31 in US is not due to selection for high ergot alkaloid production but rather other traits associated with the endophyte. In addition, the environmental effects causing variation in alkaloid production of grass-endophyte combinations should be taken into account when using endophyte-infected grasses agriculturally.     

Relationship Between the Endophyte <Emphasis Type="Italic">Embellisia</Emphasis> spp. and the Toxic Alkaloid Swainsonine in Major Locoweed Species (<Emphasis Type="Italic">Astragalus</Emphasis> and <Emphasis Type="Italic">Oxytropis)</Emphasis>

Locoweeds (Astragalus and Oxytropis spp. that contain the toxic alkaloid swainsonine) cause widespread poisoning of livestock on western rangelands. There are 354 species of Astragalus and 22 species of Oxytropis in the US and Canada. Recently, a fungal endophyte, Embellisia spp., was isolated from Astragalus and Oxytropis spp. and shown to produce swainsonine. We conducted a survey of the major locoweeds from areas where locoweed poisoning has occurred to verify the presence of the endophyte and to relate endophyte infection with swainsonine concentrations. Species found to contain the fungal endophyte and produce substantial amounts of swainsonine were A. wootoni, A. pubentissimus, A. mollissimus, A. lentiginosus, and O. sericea. Astragalus species generally had higher concentrations of swainsonine than Oxytropis. Swainsonine was not detected in A. alpinus, A. cibarius, A. coltonii, A. filipes, or O. campestris. The endophyte could not be cultured from A. mollissimus var. thompsonii or A. amphioxys, but was detected by polymerase chain reaction, and only 30% of these samples contained trace levels of swainsonine. Further research is necessary to determine if the endophyte is able to colonize these and other species of Astragalus and Oxytropis and determine environmental influences on its growth and synthesis of swainsonine.     

Subterranean Herbivore-induced Volatiles Released by Citrus Roots upon Feeding by <Emphasis Type="Italic">Diaprepes abbreviatus</Emphasis> Recruit Entomopathogenic Nematodes

Herbivore-induced volatile emissions benefit plant hosts by recruiting natural enemies of herbivorous insects. Such tritrophic interactions have been examined thoroughly in the above-ground terrestrial environment. Recently, similar signals have also been described in the subterranean environment, which may be of equal importance for indirect plant defense. The larvae of the root weevil, Diaprepes abbreviates, are a serious pest of citrus. Infestations can be controlled by the use of entomopathogenic nematodes, yet the interactions between the plant, insect and nematode are poorly understood and remain unpredictable. In bioassays that used a root zone six-arm olfactometer, citrus roots (‘Swingle citrumelo’ rootstock) recruited significantly more entomopathogenic nematodes (Steinernema diaprepesi) when infested with root weevil larvae than non-infested roots. Infested plants were more attractive to nematodes than larvae alone. Roots damaged by weevil larvae attracted more nematodes than mechanically damaged roots and sand controls. By dynamic in situ collection and GC-MS analysis of volatiles from soil, we determined that four major terpene compounds were produced by infested plant roots that were not found in samples from non-infested roots or soil that contained only larvae. Solvent extracts of weevil-infested roots attracted more nematodes than extracts of non-infested roots in a two choice sand-column bioassay. These findings suggest that Swingle citrus roots release induced volatiles as an indirect defense in response to herbivore feeding, and that some of these induced volatiles function as attractants for entomopathogenic nematodes.     

Peramine alkaloid variation in Neotyphodium-infected Arizona fescue: effects of endophyte and host genotype and environment

We determined concentrations of peramine, the only alkaloid produced by Neotyphodium-infected (E+) Arizona fescue plants (of the four major types typically assayed in infected grasses), in a long-term field experiment. Four plant genotypes with (E+) and without (experimentally removed, E–) their respective haplotypes (two haplotypes in two plant genotypes) of Neotyphodium were grown in the field under manipulated soil moisture and nutrients. Peramine production required the presence of the endophyte; plants without their endophytes did not contain peramine. Whereas the endophyte was necessary for peramine production, levels of peramine did not vary by Neotyphodium haplotype within plant genotypes. Furthermore, peramine levels did not differ among soil moisture and nutrient treatments, and growth and reproductive measures of the host grass explained little of variation in peramine levels. Instead, peramine levels differed significantly between plant genotypes harboring the same endophyte haplotype, suggesting that plant genotype, rather than endophyte haplotype or environment, largely determines levels of peramine in Arizona fescue. The results suggest that whereas the endophyte is required for peramine production, the plant genotypic background in which the endophyte is embedded, rather than endophyte haplotype or environmental factors, mostly influences peramine levels within this population of Arizona fescue.     

Quantitative bioassay for chemotaxis with plant parasitic nematodes

A simple, flexible, and quantifiable bioassay for the attraction or repulsion of plant parasitic nematodes to or from root fractions or pure substances is described. Accurate gradients of volatile and nonvolatile substances can be measured and established. The method entails placing the nematodes in narrow agarose tracks such that their movement is essentially linear and the distance a population has traveled away or toward a given substance can be monitored with time. Plastic plates, each containing 10 such tracks, are described. The method is illustrated with second-stage larvae ofMeloidogyne incognita and a volatile attractant and nonvolatile repellent fraction obtained from cucumber roots.     

α-Tomatine and resistance of tomato cultivars toward the nematode,Meloidogyne incognita

Susceptible and resistant tomato cultivars were assayed for root tomatine concentrations at different developmental times. No correlation between high tomatine levels and resistance towardMeloidogyne incognita was apparent. In plants infected with nematodes, tomatine in the roots was the same as in uninfected controls. Tomatine concentration, as measured by bulk analysis, does not appear to play an important role in resistance toward the nematode studied.     

Effects of Root Isoquinoline Alkaloids from <Emphasis Type="Italic">Hydrastis canadensis</Emphasis> on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> Isolated from <Emphasis Type="Italic">Hydrastis</Emphasis> Root Tissue

Goldenseal (Hydrastis canadensis L.) is a popular medicinal plant distributed widely in North America. The rhizome, rootlets, and root hairs produce medicinally active alkaloids. Berberine, one of the Hydrastis alkaloids, has shown antifungal activity. The influence of a combination of the major Hydrastis alkaloids on the plant rhizosphere fungal ecology has not been investigated. A bioassay was developed to study the effect of goldenseal isoquinoline alkaloids on three Fusarium isolates, including the two species isolated from Hydrastis rhizosphere. The findings suggest that the Hydrastis root extract influences macroconidia germination, but that only the combined alkaloids—berberine, canadine, and hydrastine—appear to synergistically stimulate production of the mycotoxin zearalenone in the Fusarium oxysporum isolate. The Hydrastis root rhizosphere effect provided a selective advantage to the Fusarium isolates closely associated with the root tissue in comparison with the Fusarium isolate that had never been exposed to Hydrastis.     

Detrimental effects ofCinchona leaf alkaloids on larvae of the polyphagous insectSpodoptera exigua

YoungCinchona ledgeriana plants contain two types of alkaloid: indole alkaloids in the leaves and quinoline alkaloids in the root. FromCinchona leaves, a crude alkaloid extract was made, containing the cinchophylline type of indole alkaloids and a small amount of 5-methoxytryptamine. The leaf alkaloid extract exerted a strong detrimental effect on the growth of larvae of the polyphagous beet armyworm,Spodoptera exigua (Lepidoptera). Feeding of larvae on an artificial diet containing the leaf alkaloids at the same concentrations as those found in the plant resulted in significant growth reduction, retardation in development, and mortality of the larvae. Cinchophyllines are composed of 5-methoxytryptamine coupled to a corynantheal unit. When incorporated into the artificial diet, 5-methoxytryptamine alone had no effect on the 5.exigua larvae. Corynantheal, however, had a strong detrimental effect on growth of the larvae, its effect being comparable to that of the leaf alkaloid extract. In contrast to the indole-type leaf alkaloids, the quinolinetypeCinchona root alkaloids did not affect growth and development of the larvae. These results suggest that the indole-type alkaloids, which inCinchona plants are present at the highest concentrations in the young, vulnerable leaflets, are involved in the chemical defense of the plant against herbivorous insects.     

Characterization and partial purification of attractants for nematodeOrrina phyllobia from foliage ofSolanum elaeagnifolium

An unknown attractant for the nematodeOrrina phyllobia was extracted with water from foliage ofSolanum elaeagnifolium. Stability, solubility, ionic character, and Chromatographie purification were investigated using a bioassay based on nematode aggregation in agar. Activity was nonvolatile, dialyzable, heat stable below 60 °C, and partially lost within 30 min at 100 °C. Activity was unchanged from pH 5 to 12, but was entirely lost at pH 2. Loss of activity at low pH did not appear to result from direct effects of pH on nematode behavior and was partially recovered by readjustment to pH 7. The attractive factor was most soluble in water and appeared to be cationically but not anionically exchangeable. Activity appeared to Chromatograph as several compounds by high-performance liquid chromatography employing reverse phase C₁₈ and amine-bonded columns. Various known compounds that are common toSolanum spp. or that attract other nematodes were unattractive. Extraction ofS. elaeagnifolium foliage specifically for solanaceous glycoalkaloids using methods developed forS. tuberosum did not yield an attractive product.     

Dihydropyrrolizine attractants for arctiid moths that visit plants containing pyrrolizidine alkaloids

Adults of three species of arctiid moths (Cisseps fulvicollis, Ctenucha virginia, andHalysidota tessellaris) are attracted to plants that contain pyrrolizidine alkaloids (PAs). The moths use olfactory cues to locate these plants, then feed on leaves, flowers, and roots with the proboscis. To investigate the chemical basis of attraction, sticky traps were baited with roots of a PA-containing plant,Eupatorium maculatum, alkaloids ofE. maculatum, and several derivatives of these alkaloids. Volatile derivatives of the bicyclic pyrrolizidine skeleton attracted all three arctiid species. The dihydropyrrolizines, (S)-(+)-hydroxydanaidal and (R)-(–)-hydroxydanaidal, proved to be the most attractive compounds tested, accounting for over 70% of the moths captured. Different alkaloid derivatives attracted different proportions of male and femaleCisseps. Both (S)-(+)-hydroxydanaidal (52% male) and (R)-(–)-hydroxydanaidal (71% male) attracted a significantly lower percentage ofCisseps males thanE. maculatum roots (87% male).Cisseps males possess eversible scent organs (coremata) that are displayed during courtship. Analysis of corematal extracts revealed the presence of hydroxydanaidal.Cisseps moths thus resemble danaine and ithomiine butterflies, both in their attraction to PA sources and in the presence of PA derivatives in the male scent organs.