Diterpenoid Alkaloid Concentration in Tall Larkspur Plants Damaged by Larkspur Mirid

Tall larkspur (Delphinium barbeyi) is a serious poisonous plant threat to cattle on mountain rangelands. The larkspur mirid [Hopplomachus affiguratus] has been proposed as a biological tool to damage tall larkspur in an effort to deter grazing by cattle and thus prevent poisoning. Preliminary data suggested that it may also reduce toxic alkaloid levels. The objective of this study was to determine if damage caused by the larkspur mirid would reduce toxic alkaloid concentration. Larkspur mirids were collected in the field in 1992 and placed on potted plants in the greenhouse. The resulting mirid-damaged leaves were lower in toxic alkaloids than leaves from uninfested plants. In the 1995 field study, toxic and total norditerpenoid alkaloid concentrations were measured in two larkspur populations having established mirid populations and in two newly infested larkspur populations. In the 1996 field study, three widely separated larkspur populations infested with mirids were sampled. Mirid-damaged leaves were lower in toxic alkaloids in both years, but there were no differences in flowering heads. However, only at Yampa, Colorado, did mirids reduce toxic alkaloids to levels that would not pose a threat to cattle. There was no difference in toxic or total alkaloid concentration between larkspur populations with long-term mirid infestations compared to newly infested plants. The plant-to-plant variability in alkaloid concentration was greater than differences due to mirids.     

A Functional Explanation for Patterns of Norditerpenoid Alkaloid Levels in Tall Larkspur (Delphinium barbeyi)

Concentrations of norditerpenoid alkaloids vary among larkspur (Delphinium) species, locations, and years, but environmental stresses seem to have little effect on alkaloid levels. There is a need for a functional hypothesis of alkaloid synthesis and metabolism to explain the observed trends in concentration and to predict the toxicity of larkspur populations. This study was replicated at two locations over two years in the mountains of central Utah. Ten tall larkspur (D. barbeyi) plants were marked at each location, and a single stalk was harvested from each plant at weekly intervals throughout the growing season. Concentrations of toxic and total alkaloids were measured by Fourier-transformed infrared spectroscopy (FTIR), and alkaloid pools were calculated by multiplying the alkaloid concentration by the dry weight of the plant to determine the amount of alkaloids in the stalk. Alkaloid pools in the stalks increased for the first three weeks, leveled off, and then declined to low levels as the plants began to senesce. Concentrations of alkaloids declined through the season, as the alkaloids were diluted in the increasing biomass as the plants grew. These patterns will be used to predict potential toxicity of larkspur populations.     

Influence of alkaloid concentration on acceptability of tall larkspur (Delphinium spp.) to cattle and sheep

Tall larkspur (Delphinium spp.) is a serious toxic plant problem on western U.S. ranges. The major toxins in tall larkspur are methyllycaconitine (MLA) and 14-deacetylnudicauline (14-DAN); the sum of both is termed the toxic alkaloid concentration. Toxic alkaloids comprise about 20–50% of the total alkaloid concentration in tall larkspur. Toxic and total alkaloid concentration generally declines with maturity, whereas cattle and sheep consumption of larkspur typically increases with plant maturity. We hypothesized that cattle and sheep consumption of tall larkspur was negatively related to higher concentrations of total or toxic alkaloid. We compared consumption of several collections of dried, ground larkspur and fresh larkspur in a series of trials. In another trial, a crude alkaloid fraction was extracted with ethanol, added to alfalfa hay, and consumption compared to untreated alfalfa hay, alcohol-treated hay, and the essentially alkaloid-free plant residue. In all cases we correlated amounts eaten with total and toxic alkaloid concentration. A grazing trial was also conducted to relate larkspur consumption over time to alkaloid concentrations. Total alkaloid concentrations in dried, whole-plant collections ranged from 9.3 to 38.8 mg/g of dry weight, whereas toxic alkaloid concentrations varied from 0.0 to 7.1 mg/g. In one pen trial, cattle preferred a larkspur collection (P<0.01) that contained no toxic alkaloids but had a high total alkaloid concentration (39 mg/g). There was no correlation (P>0.05), however, between concentrations of total or toxic alkaloids and amount of dry plant consumed in this or any other trial. Conversely, sheep consumption tended to be negatively influenced by total and toxic alkaloid concentration (P0.08). In the trials with extract, cattle preferred the alcohol-treated hay and rejected the alkaloid-free residue (P<0.01), whereas the alkaloid-treated hay was of intermediate acceptability. Cattle preferred the alkaloid-treated hay over the alkaloid-free residue, indicating that alkaloids did not deter consumption. Conversely, the alkaloid-treated hay was less preferred than either untreated or alcohol-treated hay, suggesting a negative effect on acceptability. There was no correlation between alkaloid concentration and amount of treated feed eaten. In field trials, the amount of composited, fresh leaves or flowers eaten by cattle was influenced by plant part (P=0.04), but was not related (P>0.05) to alkaloid concentration. Cattle preferred leaves over flowers when offered individual plants differing in phenological stage and/or amount of shade, but alkaloid concentration was not related to consumption. We conclude that knowledge of the concentration of total or toxic alkaloid in tall larkspur will give little or no indication of plant acceptability to cattle. Even though accurate predictions can be made about the potential toxicity of larkspur based on the concentration of toxic alkaloids, predictions about consumption must be based primarily on plant phenology.     

Distribution of Norditerpene Alkaloids in Tall Larkspur Plant Parts Through the Growing Season

Previous research showed that toxic and total alkaloid pools in tall larkspur (Delphinium barbeyi) increased during early growth, then declined precipitously during the late flower and pod stage of growth. The objective of this study was to measure the concentration and pools of toxic and total alkaloids in tall larkspur plant parts, including roots, and to evaluate the changes in these pools over the growing season as an estimate of diterpenoid alkaloid kinetics in tall larkspur. Twenty entire plants were harvested at each phenological stage: beginning of growth in the spring, early flower, early pod, late pod, and senescence. The plants were separated into their respective parts, freeze-dried, extracted, and analyzed for toxic and total alkaloid concentration, and alkaloid pools were calculated. Concentration of toxic and total alkaloids in leaves and stems declined as the plants matured, while concentration in flowers and pods increased (P < 0.004). Concentration of alkaloids in the root declined in the early growth, then increased at the end of the season (P = 0.002). Alkaloid pools in the root decreased during early growth, with a corresponding increase of pools in foliar parts. In the late flower and pod stage, alkaloid pools in the leaves and stems declined rapidly, while the pool in the crown and roots tended to increase.     

Influence of Defoliation on Toxic Alkaloid Concentration and Alkaloid Pools in Tall Larkspur

This study was replicated at two locations in the mountains of central Utah. In 1997, ten uniform plants of tall larkspur (Delphinium barbeyi) in the early bud stage (40 cm in height) were selected at each site and clipped at 5 cm above soil level. In 1998, one stalk from each plant was harvested on a weekly basis; in 1999, one stalk was harvested at four times during its phenological development. Toxic and total alkaloid concentrations were measured and alkaloid pools in the entire stalk were calculated. Clipping reduced stalk height to less than 50 cm in 1998 and 65 cm in 1999, compared to over 100 cm in unclipped control plants. Alkaloid concentration was similar to control plants, but toxic alkaloid pools were 70% lower than control plants, because of the reduction in biomass of the stalks. Clipping reduced subsequent vigor and the amount of toxic and total alkaloids in tall larkspur.     

The Biogeographical Distribution of Duncecap Larkspur (Delphinium occidentale) Chemotypes and Their Potential Toxicity

Larkspurs (Delphinium spp.) are poisonous plants found on rangelands in western North America. Larkspur’s toxicity has been attributed to the norditerpenoid alkaloids, which are divided into two main structural groups: the highly toxic (N-methylsuccinimido) anthranoyllycoctonine type (MSAL type) and the less toxic 7,8-methylenedioxylycoctonine type (MDL type). Plants high in the MSAL-type alkaloids are thought to be the most toxic to cattle, and the concentrations of these alkaloids have been used as a predictor of plant toxicity. Duncecap larkspur, Delphinium occidentale, occurs throughout much of the Intermountain West and Northwestern United States. Specimens from field collections and herbaria deposits were evaluated taxonomically and chemically. Two distinct alkaloid profiles were identified: one that contains the MSAL-type alkaloids and one that contains little, if any, MSAL-type alkaloids. Thus, plants with these two alkaloid profiles should differ in their toxic potential. Each profile was unique in its geographical distribution. These findings have important implications in grazing management decisions on D. occidentale-infested rangelands, and they demonstrate that botanical classification alone is not a good indicator to determine the toxic risk of D. occidentale.     

Predicting toxicity of tall larkspur (Delphinium barbeyi): measurement of the variation in alkaloid concentration among plants and among years

Tall larkspur (Delphinium barbeyi) is the principal mountain larkspur responsible for the majority of cattle deaths on mountain rangelands in western Colorado and central and southern Utah in the United States. Ten plants in each of two tall larkspur populations in the mountains near Ferron and Salina, Utah, were marked, and single stalks were harvested periodically through the growing season for 4 yr. Toxic alkaloid concentration [alkaloids containing the N-(methylsuccimimido)-anthranilik ester group] was determined by Fourier transform infrared (FTIR) spectroscopy. Individual larkspur plants varied in alkaloid concentrations, especially in early growth (14–38 mg/g). As the concentration declined over the growing season, variation among plants also declined. There were yearly differences in alkaloid concentration among individual plants (P < 0.01) and populations (P < 0.001), even after accounting for differences in phenological growth between years. Variables such as precipitation, temperature, days since snow melt, growing degree days (sum of mean temperature each day from snow melt), and plant height and weight were all considered in a Mallows Cp multiple regression selection procedure to predict alkaloid concentration. The mixed model procedure in SAS adjusted the regression equation for locations and years. Growing degree days was the best single predictor of alkaloid levels: ln y = (3.581 – 0.00423 GDD), R ² = 0.85. Internal validation of this equation within individual years and locations from which the equation was developed, produced correlations between observed versus predicted values ranging from r = 0.73 to 0.93. External validations on nine other larkspur populations produced correlations ranging from r = 0.76 to 0.99. This predictive equation can provide a tool for ranchers and land managers to make management decisions of when to graze cattle in larkspur areas.     

Short-term induction of alkaloid production in lupines Differences between N2-fixing and nitrogen-limited plants

We used N₂-fixing and nonfixing lupines to examine the effects of plant nutrition on short-term alkaloid production in damaged leaves. Three different treatments were used: damaged leaves from N₂-fixing plants; undamaged leaves from these damaged, N₂-fixing plants; and damaged leaves on nitrogen-limited, nonfixing plants. Relative to controls, alkaloids increased in concentration more quickly in the N₂-fixing than in the nitrogen-limited plants. The magnitude of this increase in alkaloids was correlated with the initial alkaloid concentration. These results suggest that nitrogen-rich plants may benefit from faster and higher alkaloid induction than nitrogen-limited plants. In addition, the detailed dynamics of individual alkaloids are consistent with earlier proposals for the mechanism of lupine alkaloid induction.     

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.     

Damage-induced alkaloids in tobacco: Pot-bound plants are not inducible

Field-grown wild tobacco plants (Nicotiana sylvestris) were subjected to a defoliation regime designed to mimic the rate and amount of leaf mass removed by one tobacco hornworm per plant. Undamaged leaves on these plants undergo a dramatic (457% for leaf position 5, 410% for leaf position 8) increase in total leaf alkaloids compared to same-age and positioned control leaves on undamaged control plants. However, potted greenhouse-grown plants fail to exhibit the same damage-induced increase in alkaloid content. The greenhouse environment differs from the field environment in factors known to influence leaf alkaloid content, particularly soil N, P, K, near-UV radiation, and relative humidity. However, altering these environmental factors does not make potted plants able to increase their leaf alkaloid levels in response to defoliation. Transplanting plants into larger pots with more soil does allow the plants to respond to defoliation. Thirty days after transplanting, the plants are again unresponsive to damage, probably as a result of becoming pot-bound. This result suggests a mechanism for the induction response, specifically that leaf damage triggers synthesis of these alkaloids in the roots, and offers a potentially valuable experimental tool for the study of induced-plant defenses in tobacco and other plants that synthesize alkaloids in their root tissues.     

Ingestion of tall larkspur by cattle

Tall larkspur (Delphinium spp.) is a palatable but toxic poisonous plant in the western United States. The toxins in tall larkspur are diterpenoid alkaloids. We examined the influences of food flavor and postingestive consequences on consumption of a 33% larkspur pellet during 30-min feeding periods for five days using esophageally fistulated cattle that were sham-fed larkspur pellets. Consumption by the sham-fed group was compared to a control group fed alfalfa pellets, and a larkspur group fed only larkspur pellets. Sham-fed cattle did not decrease (P > 0.1) feed consumption compared to controls, indicating no significant difference in food flavor. The larkspur group decreased (P < 0.05) feed consumption by 41% relative to controls and by 31% relative to sham-fed animals (P = 0.08). This reduction in feed consumption indicates the adverse postingestive consequences of tall larkspur ingestion, as the larkspur group apparently developed a conditioned taste aversion to the larkspur pellet. Even though these animals were averted to the pellets, they showed none of the classical signs of intoxication from ingestion of tall larkspur.This research was supported in part by the Utah Agricultural Experiment Station, Utah State University, Logan, Utah 84322. Approved as journal article No. 3846.     

Importance of quinolizidine alkaloids in the relationship between larvae ofUresiphita reversalis (Lepidoptera: Pyralidae) and a host plant,Genista monspessulana

Larvae ofUresiphita reversalis feed almost exclusively on legumes in the tribe Genisteae, which characteristically contain a variety of quinolizidine alkaloids. The larvae are aposematic, and onGenista monspessulana, a major host in California, they feed on the youngest leaves, at the periphery of the plant. These leaves, which were preferred over older foliage in choice tests, contained four to five times the level of alkaloid found in older leaves. The major alkaloids detected in these plants were dehydroaphylline andN-methylcytisine, together accounting for 74% of the total. Preliminary analyses showed the alkaloid profile of exuviae from larvae feeding on these plants was very similar to that of the plants. Two alkaloids, sparteine and cytisine, which are known components of some hosts ofU. reversalis, were phagostimulants for fifth-instar larvae when added to sucrose-impregnated glass-fiber disks. In addition, when sparteine was added to foliage ofG. monspessulana, effectively doubling the percent dry weight of alkaloid, the growth rate of late-instar larvae was positively affected. Cytisine added to plants had no discernible effect on growth of larvae. Alkaloid levels in larvae and in their frass were proportional to levels in the plants on which they fed. Although the majority of alkaloid was excreted, that which was sequestered by the insect was found entirely in the integument, possibly confering some protection from predators.     

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.     

Mechanism of damage-induced alkaloid production in wild tobacco

Greenhouse-grown tobacco plants of the speciesNicotiana sylvestris (Solanaceae) subjected to leaf damage show a fourfold increase in the alkaloid content of their undamaged leaves. This increase in nicotine and nornicotine concentrations begins 19 hr after the end of the damage regime, reaches a maximum at nine days, and wanes to control levels 14 days after the start of leaf damage. The increase in leaf alkaloid content in damaged plants is largely due to a 10-fold increase in the alkaloid concentration of the xylem fluid entering leaves, which, in turn, suggests that increased synthesis of alkaloids is occurring in the roots. This research distinguishes between positive and negative cues affecting the change in xylem fluid alkaloid concentrations. A negative cue, such as auxin, when lost or diminished as a result of leaf damage could signal the alkaloidal response. Indeed, exogenous applications of auxin to damaged leaves inhibit the alkaloidal response. However, attempts to block endogenous auxin transport by steam girdling or applying an auxin transport inhibitor fail to mimic the effect of leaf damage on leaf alkaloid concentrations. The damage cue appears to be a positive cue that is related to the timing and the amount of leaf damage rather than to the amount of leaf mass lost. Moreover, when performed proximally to leaf damage, steam girdling truncates the alkaloidal response. This induced alkaloidal response appears to be triggered by a phloem-borne cue that allows the plant to distinguish between different types of leaf damage. The physiological and ecological consequences of the mechanism of this damage-induced alkaloidal response are further explored.     

Visualizing a Plant Defense and Insect Counterploy: Alkaloid Distribution in <Emphasis Type="Italic">Lobelia</Emphasis> Leaves Trenched by a Plusiine Caterpillar

Insects that feed on plants protected by latex canals often sever leaf veins or cut trenches across leaves before feeding distal to the cuts. The insects thereby depressurize the canals and reduce latex exudation at their prospective feeding site. How the cuts affect the distribution and concentration of latex chemicals was not known. We modified a microwave-assisted extraction technique to analyze the spatial distribution of alkaloids in leaves of Lobelia cardinalis (Campanulaceae) that have been trenched by a plusiine caterpillar, Enigmogramma basigera (Lepidoptera: Noctuidae). We produced sharp two dimensional maps of alkaloid distribution by microwaving leaves to transfer alkaloids to TLC plates that were then sprayed with Dragendorff’s reagent to visualize the alkaloids. The leaf prints were photographed and analyzed with image processing software for quantifying alkaloid levels. A comparison of control and trenched leaves documented that trenching reduces alkaloid levels by approximately 50% both distal and proximal to the trench. The trench becomes greatly enriched in alkaloids due to latex draining from surrounding areas. Measurements of exudation from trenched leaves demonstrate that latex pressures are rapidly restored proximal, but not distal to the trench. Thus, the trench serves not only to drain latex with alkaloids from the caterpillar’s prospective feeding site, but also to isolate this section, thereby preventing an influx of latex from an extensive area that likely extends beyond the leaf. Microwave-assisted extraction of leaves has potential for diverse applications that include visualizing the impact of pathogens, leaf miners, sap-sucking insects, and other herbivores on the distribution and abundance of alkaloids and other important defensive compounds.     

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.     

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.     

Sequestration ofVeratrum alkaloids by specialistRhadinoceraea nodicornis konow (Hymenoptera,Tenthredinidae) and its ecoethological implications

The larvae of the specialist sawflyRhadinoceraea nodicornis Konow (Hymenoptera, Tenthredinidae) store in their hemolymph ceveratrum alkaloids originating from the host plantVeratrum album L. (Liliales, Melanthiaceae). The major alkaloid found in the hemolymph is 3-acetyl-zygadenine. Qualitative and quantitative data showed that the plant alkaloid 3-angeloylzygadenine is most probably metabolized in the larval gut to zygadenine and then acetylated. A still unidentified alkaloid with a molecular weight of 591 Da was detected in plant leaves as well as in the gut, hemolymph, and excrement of larvae. Protoveratrine A and B, on the other hand, seem to be degraded by the larvae. These findings indicate that the pathway of ceveratrum alkaloids inR. nodicornis larvae is fourfold: direct sequestration, metabolism followed by sequestration, excretion of intact alkaloids, and degradation. In contrast, no ceveratrum alkaloids were detected in the hemolymph and excrement of larvae of the generalist sawflyAglaostigma sp. fed withV. album leaves. Bioassays with the antMyrmica rubra L. proved that the hemolymph ofR. nodicornis larvae is highly deterrent and toxic. In bioassays evaluating defensive efficiency against predators (ants, spiders, and bushcrickets), no larvae were eaten. Ceveratrum alkaloids were also detected in the hibernating prepupae ofR. nodicornis. In feeding bioassays, the shrewCrocidura russula Hermann rarely fed upon prepupae, suggesting that this stage is also protected from predation to some degree. In field surveys, the only parasitoids recorded were two ichneumonid species that are believed to be specialized onR. nodicornis. Bioassays and field observations enable us to suppose thatR. nodicornis and its enemies produce a food web of ion connectance.     

Tall Larkspur Ingestion: Can Cattle Regulate Intake Below Toxic Levels?

Tall larkspur (Delphinium barbeyi) is a toxic forb often consumed by cattle on mountain rangelands, with annual fatalities averaging about 5%. This study examined the relationship between food ingestion and toxicity in cattle. Two grazing studies suggested that larkspur consumption above 25–30% of cattle diets for one or two days led to reduced larkspur consumption on subsequent days. We subsequently hypothesized that cattle can generally limit intake of larkspur to sublethal levels. This hypothesis was tested by feeding a 27% larkspur pellet in experiment 1. Cattle given a 27% larkspur pellet ad libitum showed distinct cyclic patterns of intake, where increased larkspur consumption on one or two days was followed by reduced (P < 0.025) consumption on the following day. The amount of larkspur (mean 2007 g/day; 17.8 mg toxic alkaloid/kg body wt) consumed was just below a level that would produce overt signs of toxicity. Experiment 2 was conducted to examine cattle response to a toxin dose that varied with food intake. Lithium chloride (LiCl) paired with corn ingestion was used as a model toxin, and we hypothesized that if increased (decreased) consumption was followed by a stronger (weaker) dose of LiCl, cattle would show a transient reduction (increase) in corn intake. There was no difference (P > 0.05) between controls and treatment animals at the 20 or 40 mg LiCl/kg dose in the percentage of corn consumed, but the 80 mg LiCl/kg dose induced a cyclic response (mean 46%) compared to intake by controls (mean 96%) (P < 0.001). At the 80 mg/kg dose, LiCl induced an aversion to corn; when corn intake decreased on subsequent days and LiCl dose also decreased, cattle responded by increasing corn intake and apparently extinguishing the transient food aversion. Experiment 3 was similar to the LiCl trial, except that tall larkspur was the toxin. Cattle responded to oral gavage of ground larkspur with distinct cycles; days of higher corn consumption were followed by one to three days of reduced consumption. Corn intake for controls was higher (P < 0.01) than for larkspur-treated animals (means 84 and 52%, respectively; day × treatment interaction P < 0.01). The threshold for toxic effects on corn intake was 14 mg toxic alkaloid/kg body weight. In conclusion, cattle apparently limit ingestion of some toxins so that periods of high consumption are followed by periods of reduced consumption to allow for detoxification. Cyclic consumption generally enables cattle to regulate tall larkspur consumption below a toxic threshold and allows cattle the opportunity to safely use an otherwise nutritious, but toxic, plant.     

Environmental and Genotypic Influences on Isoquinoline Alkaloid Content in Sanguinaria canadensis

In a common garden, we investigated genetic and environmental influences on alkaloid production using Sanguinaria canadensis as a model. Nutrient and shade regimes were applied to replicated clones over one growing season, and induction of alkaloid production in bloodroot was tested on a whole-plant basis using jasmonic acid as an elicitor. Alkaloid concentrations increased with decreasing light intensity and fertilizer levels. Induction was not achieved by foliar application of jasmonic acid. Genetic influences represented by clone effects may be indicated by variation in alkaloid concentration by clone, but this experimental design did not allow us to distinguish genetic from pre-experiment environmental influences on the rhizomes.