N-formyl Loline Research Articles

Passive absorption across gastrointestinal tissues in vitro and postharvest distribution of loline alkaloid in lambs

Loline alkaloid has suggested antimicrobial and anthelmintic properties with very low mammalian toxicity. There are several known derivatives of loline; N-formyl loline (NFL), N-acetyl loline (NAL), N-acetyl norloline (NANL), N-methyl loline (NML), and loline base. However, these must reach the abomasum or intestine and then be absorbed to have any potential effect. Therefore, passive absorption in isolated gastrointestinal tissues and distribution in the tissue of lambs were determined. Experiment 1: Lamb (n = 6) isolated gastrointestinal tissues were removed and mounted in an Ussing chamber. Approximately, 1 034 µg/g of loline and 22.1 µg/mL of caffeine were added to the donor chamber to measure loline flux at 0, 0.5, 1, and 2 hours. Experiment 2: Two, 12-week-old lambs were dosed with 52.5 mg/kg BW loline twice and were slaughtered to determine the distribution in gastrointestinal, organs, and blood. Passive absorption was 5% in ileum, <2% in ruminal or abomasal tissues. Loline base and NFL were passively absorbed across all tissues, with NAL and NANL only crossing small intestine tissues. Surprisingly, no caffeine crossed any tissues. Loline base and small amounts of NFL were in blood plasma, and loline base was also found in liver and kidneys. Results indicate either the majority of loline is not passively absorbed or membrane integrity was affected as suggested by lack of caffeine absorption.

Antifeedant Effects and Repellent Activity of Loline Alkaloids from Endophyte-Infected Tall Fescue against Horn Flies, Haematobia irritans (Diptera: Muscidae).

Haematobia irritans is an obligate bloodsucking ectoparasite of cattle and is the global major pest of livestock production. Currently, H. irritans management is largely dependent upon broad-spectrum pesticides, which lately has led to the development of insecticide resistance. Thus, alternative control methods are necessary. Endophyte-infected grasses have been studied as an alternative due to their capability to biosynthesize alkaloids associated with anti-insect activities. Thus, the main aim of this study was to evaluate the antifeedant and repellent activity of lolines obtained from endophyte-infected tall fescue against H. irritans adults in laboratory conditions. The alkaloid extract (ALKE) was obtained by acid–base extraction. N-formyl loline (NFL) and N-acetyl loline (NAL) were isolated by preparative thin layer chromatography (pTLC) and column chromatography (CC), and the loline was prepared by acid hydrolysis of a NFL/NAL mixture. Loline identification was performed by gas chromatography coupled to mass spectrometry (GC/MS). Feeding behavior was evaluated by a non-choice test, and olfactory response was evaluated using a Y-tube olfactometer. Accordingly, all samples showed antifeedant activities. NFL was the most antifeedant compound at 0.5 µg/µL and 1.0 µg/µL, and it was statistically equal to NAL but different to loline; however, NAL was not statistically different to loline. NFL and NAL at 0.25 µg/µL were more active than loline. All samples except loline exhibited spatial repellency in the olfactometer. Thus, the little or non-adverse effects for cattle and beneficial activities of those lolines make them suitable candidates for horn fly management.

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species.

Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.

A QTL analysis of host plant effects on fungal endophyte biomass and alkaloid expression in perennial ryegrass.

The association between perennial ryegrass (Loliumperenne L.) and its Epichloë fungal endophyte symbiont, Epichloëfestucae var. lolii, supports the persistence of ryegrass-based pastures principally by producing bioactive alkaloid compounds that deter invertebrate herbivory. The host plant genotype affects endophyte trait expression, and elucidation of the underlying genetic mechanisms would enhance understanding of the symbiosis and support improvement of inplanta endophyte performance through plant breeding. Rapid metabolite profiling and enzyme-linked immunosorbent assay were used to quantify endophyte alkaloids and mycelial mass (MM) in leaves harvested, in consecutive autumns, from an F1 mapping population hosting standard toxic endophyte. Co-aligned quantitative trait loci (QTL) on linkage groups (LG)2, LG4 and LG7 for MM and concentrations of alkaloids peramine and ergovaline confirmed host plant effects on both MM and alkaloid level and inferred the effect on alkaloids was modulated through the quantity of endophyte present in the leaf tissue. For ergovaline, host regulation independent of endophyte concentration was also indicated, by the presence of MM-independent ergovaline QTL on LG4 and LG7. Partitioning of host genetic influence between MM-dependent and MM-independent mechanisms was also observed for the alkaloid N-formylloline (NFL), in a second mapping population harbouring a tall fescue-sourced endophyte. Single-marker analysis on repeated MM and NFL measures identified marker–trait associations at nine genome locations, four affecting both NFL and MM but five influencing NFL concentration alone. Co-occurrence of QTL on LG3, LG4 and LG7 in both mapping populations is evidence for host regulatory loci effective across genetic backgrounds and independent of endophyte variant. Variation at these loci may be exploited using marker-assisted breeding to improve endophyte trait expression in different host population × endophyte combinations.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0350-1) contains supplementary material, which is available to authorized users.

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.

Excretion of loline alkaloids in urine and faeces of sheep dosed with meadow fescue (Festuca pratensis) seed containing high concentrations of loline alkaloids

AIM: To determine the effect of oral dosing of sheep with loline alkaloids on their excretion in urine and faeces, and to monitor for any toxic effects. METHODS: In Experiment 1, six 9-month-old ewe lambs were given a single oral dose of loline alkaloids (52 mg/kg bodyweight (BW); acute exposure) as a suspension of ground meadow fescue (Festuca pratensis) seed in water. In Experiment 2, on six consecutive days, six ewe lambs were given three doses of loline (68 mg/kg BW/day; chronic exposure). Blood was collected at variable intervals up to 72 h in Experiment 1, and up to 8 days in Experiment 2, for haematology and measurement of alkaline phosphatase, aspartate aminotransaminase, creatine kinase and γ-glutamyl transferase in plasma. Urine and faecal samples were collected at similar times for measurement of creatinine in urine and loline alkaloid analysis. A post mortem with histopathology was carried out on two animals at the end of each experiment. RESULTS: The loline alkaloids, N-acetyl norloline, N-formyl loline, N-acetyl loline, N-methyl loline and loline base were detected in urine within 15 minutes after the single dosing. N-formyl loline and loline base were the predominant metabolites in urine in both experiments. The total quantity of lolines excreted in both urine and faeces was 10% and 4% of the amount dosed in Experiments 1 and 2, respectively. In both experiments, the clinical chemistry parameters in blood and urine were within normal ranges. Post-mortem and histopathological examination did not show any abnormalities. CONCLUSIONS: This is the first report of loline alkaloid profiles in both urine and faeces of sheep. The appearance of loline alkaloids and the loline base in urine within 15 minutes suggests rapid uptake, metabolism and excretion. Loline alkaloids were non-toxic to sheep at the concentrations they are exposed to under New Zealand grazing conditions. The low recovery of loline alkaloids in urine and faeces in the absence of toxicity signs suggests lolines are extensively metabolised; probably to forms other than N-formyl loline, N-methyl loline, N-acetyl loline, N-acetyl norloline, and loline base in the digestive tract of sheep prior to absorption, and/or in the liver or other tissues following absorption.

Seasonal distribution of loline alkaloid concentration in meadow fescue infected with Neotyphodium uncinatum

Loline alkaloids are present in meadow fescue containing the endophyte (Neotyphodium uncinatum Gams, Petrini and Schmidt) (Clavicipitacae). Root, crown and shoot loline alkaloid concentrations in 10 selected lines from meadow fescue ecotypes are reported for the first time, from a Canterbury farm during 2004–05. The concentrations of four loline alkaloid derivatives, N-formyl loline (NFL), N-acetyl loline (NAL), N-acetyl norloline (NANL) and N-methyl loline (NML), in these lines (each line represented by one genotype) were determined at four harvest dates during late spring, late summer, and early and late autumn. There were marked differences in loline alkaloid concentration between lines and seasons. Maximum shoot loline concentration was recorded in summer (up to 2860 µg/g in Fp408). Root loline alkaloid concentration was substantially higher in late autumn (up to 790 µg/g in Fp408) and the shoot concentration correspondingly lower than in spring, summer and early autumn suggesting loline alkaloid transportation from shoots to roots. In the lines tested at each of the four harvest dates in spring, summer, and autumn, the root, crown, and shoot alkaloid concentration with minor exceptions was NFL &gt; NAL &gt; NANL &gt; NML.

Seasonal changes in leaf and stem loline alkaloids in meadow fescue

Leaf and stem loline alkaloid concentration in 10 European meadow fescue (Festuca pratensis Huds.) lines grown in a field in Canterbury, New Zealand, were determined in samples collected six times between early spring 2004 and late autumn 2005. Significant differences in loline alkaloid concentrations were noted between lines and between harvest times. Higher total loline alkaloid concentrations (up to 4990 µg g–1) were found in stems compared to leaf (up to 1770 µg g–1). However, the seasonal accumulation pattern of different loline alkaloid concentrations in leaf and stem varied. In most lines, stem loline concentration peaked sharply in late spring and declined during early summer and autumn. The seasonal pattern of leaf loline alkaloid concentration followed the stem concentration except for a sharp decline in early summer followed by an increase in late summer. In most instances, the concentration of N-formyl loline was the highest &gt; N-acetyl loline &gt; N-acetyl norloline &gt; N-methyl loline. The possible role of stem and leaf loline alkaloids to deter pasture-feeding insects is briefly discussed.

Argentine stem weevil adults are affected by meadow fescue endophyte and its loline alkaloids

Responses of adult Argentine stem weevil (ASW) to meadow fescue infected with the endophyte Neotyphodium uncinatum and to two loline alkaloids produced by the fungus were investigated Endophyte in two different meadow fescue seedlines did not affect adult ASW feeding scores but reduced oviposition compared with endophytefree controls In a nochoice experiment adult ASW were given artificial diets containing Nformyl loline (NFL) or Nacetyl norloline (NANL) at three concentrations or a control diet with no lolines Mortality was significantly higher at the highest rate of NFL than for lower rates and the control For NANL mortality was increased at both the lowest and the highest concentrations Feeding scores were higher on control than loline diets but only after 4 weeks In a choice experiment NFL appeared to have no effect on ASW feeding preference whereas there was less feeding at the highest concentration of NANL compared with the control diet

Endophyteinfected meadow fescue and loline alkaloids affect Argentine stem weevil larvae

In a meadow fescue plant assay a lower percentage of tillers on endophyteinfected (E) than endophytefree (E) plants on which eggs had been laid were damaged by Argentine stem weevil (ASW) larvae There were indications of endophytemediated toxicity to young larvae The percentage of damaged tillers on E and E plants without ASW eggs was similar when they were planted alongside a plant with eggs but severity of damage was less on E plants In an artificial diet experiment eggs took 23 days longer to hatch on an alkaloidfree diet than on diets containing either of two loline alkaloids produced by E meadow fescue Growth and development of larvae was reduced by up to 36 at two higher concentrations of Nformyl loline (NFL) relative to the lowest concentration and to a lesser extent by Nacetyl norloline Larval survival declined with increasing concentrations of both alkaloids with the highest mortality occurring at the two higher concentrations of NFL

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, Δ lolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of Δ lolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

Mortality of Horn Fly Larvae (Diptera: Muscidae) in Bovine Dung Supplemented with Ergotamine andN-Formyl Loline

Dung-dwelling larvae of ectoparasites of livestock such as the horn fly, Hematobia irritans (L.), may be exposed to > or = 1 different alkaloid species in dung from animals ingesting herbage of the tall fescue (Festuca arundinacea Schreb.)--endophyte association (Neotyphodium coenophialum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.). First-instar horn flies were exposed to bovine dung supplemented with up to 50 microM each of N-formyl loline and ergotamine tartrate in factorial combination. In the absence of ergotamine tartrate, N-formyl loline caused a linear decline in the number of pupae recovered, and probit analysis indicated an LC50 of 36 microM. In the absence of N-formyl loline, significant quadratic responses of larvae to ergotamine tartrate were established, and probit analysis indicated a LC50 of 34 microM. An interaction (P < 0.001) was found between the 2 alkaloids for larval survival. This interaction showed that ergotamine tartrate moderated the toxicity of N-formyl loline and indicates that a membrane-bound receptor may be involved. There was no evidence of carryover of effects of alkaloids on subsequent stages of development or expressed as abnormalities of pupae or adults. Interactions between alkaloids probably are involved in other plant-herbivore relationships of endophyte-infected grasses.

Mortality of horn fly (Diptera: Muscidae) larvae in bovine dung supplemented with loline alkaloids from tall fescue.

Larvae of arthropod ectoparasites of livestock, such as the horn fly, Haematobia irritans (L.), may be exposed to acyl-loline alkaloids in dung of ruminant livestock ingesting herbage of the tall fescue (Festuca arundinacea Schreb.)-endophyte association [Neotyphodium coenophialum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.]. Biological activity of alkaloid-supplemented bovine dung was assayed by growth, development, and survival of 1st instars of horn fly. An extract from tall fescue seed, containing N-formyl loline (NFL), N-acetyl loline (NAL), and loline (59:21:20 by mass, respectively) caused 100% mortality of horn fly larvae when dung was supplemented at > or = 100 micrograms/g. Probit analysis of data corrected for natural mortality indicated a LD50 of 30 micrograms/g (95% fidicial limits: 20-49 micrograms/g). When horn fly larvae were introduced to dung supplemented with up to 50 microM of acyl-loline derivatives, mortality of larvae varied significantly between alkaloids (P < 0.0001). Probit analysis indicated that NFL [LD50: 34 microM (95% fidicial limits: 3-53 microM)] was more toxic than NAL [LD50: 46 microM (0-83 microM)], and that loline hydrochloride was not toxic.

Ammoniation to Reduce the Toxicity of Endophyte-Infected Tall Fescue Seed Fed to Rats

ABSTRACTTo assess the efficacy of ammoniation in the detoxification of endophyte-infected tall fescue (Festuca arundinacea Schreb.), 40 male Harlan Sprague-Dawley rats were randomly assigned to the following four treatments for 28 d: endophyte-free (E-), endophyte-infected (E+), ammoniated (2% dry matter basis, 7 d) endophyte-free (AE-), and ammoniated endophyte-infected (AE+) tall fescue seed. Total pyrrolizidine alkaloid (N-acetyl and N-formyl loline) and ergovaline contents of endophyte-infected fescue seed were reduced 24 and 54%, respectively, by ammoniation. Endophyte-infected treatment groups had lower (P < 0.01) daily feed intakes (DFI), daily weight gains (DWG), feed efficiencies, and primary serum hemagglutination titers to sheep red blood cell (SRBC) immunization than endophyte-free treatment groups. Performance parameters were higher (P < 0.01) for ammoniated diets in comparison to non-ammoniated dies; however, anti-SRBC titers were not significantly different. When compared to the E+ diet, the AIE+ diet increased (P < 0.01) DFI (24%), DWG (41%) and feed efficiency (13%).

Comparison of two Ammoniation Procedures to Reduce the Toxicity of Endophyte-Infected Tall Fescue Seed Fed to Rats

ABSTRACTTo determine the effect of extending the duration of ammonia (2% dry matter basis) treatment from 1 to 5 wk on the toxicity of endophyte-infected tall fescue seed, 60 male Harlan Sprague-Dawley rats were randomly assigned to the following six treatments during a 28-d trial: endophyte-free (E-), endophyte-infected (E+), 1 wk ammoniated endophyte-free (1AE-), 1 wk ammoniated endophyte-infected (1AE+), 5 wk ammoniated endophyte-free (5AE-), and 5 wk ammoniated endophyte-infected (5AE+) tall fescue seed. the concentration of total pyrrolizidine alkaloids (N-acetyl and N-formyl loline) of E+ fescue was reduced from 4203 äg/g to 3009 and 2533 ä;g/g by the 1AE+ and 5AE+ treatments, respectively. Ergovaline was lowered from 3.77 to 1.57 ä;g/g by 1AE+ and eliminated by 5AE+. Endophyte-infected treatment groups had depressed (P < 0.0001) daily feed intakes (DFI), daily weight gains (DWG), feed efficiencies (G/F), primary antibody responses, and T cell and B cell mitogenic responses than endophyte-free treatment groups. Ammoniation of endophyte-infected fescue seed improved DFI and DWG (P< 0.0001) and G/F (P< 0.05); however, there was no difference in performance criteria between the 1-wk and 5-wk ammoniation treatments. Endophyte-induced depressions in immune function were not alleviated by ammoniation.

Ammoniation to Reduce the Toxicity of Endophyte-Infected Tall Fescue Seed fed to Rats

ABSTRACTTo assess the efficacy of ammoniation in the detoxification of endophyte-infected tall fescue (Festuca arundinacea Schreb.), 40 male Harlan Sprague-Dawley rats were randomly assigned to the following four treatments for 28 d: endophyte-free (E-), endophyte-infected (E+), ammoniated (2% dry matter basis, 7 d) endophyte-free (AE-), and ammoniated endophyte-infected (AE+) tall fescue seed. Total pyrrolizidine alkaloid (N-acetyl and N-formyl loline) and ergovaline contents of endophyte-infected fescue seed were reduced 24 and 54%, respectively, by ammoniation. Endophyte-infected treatment groups had lower (P < 0.01) daily feed intakes (DFI), daily weight gains (DWG), feed efficiencies, and primary serum hemagglutination titers to sheep red blood cell (SRBC) immunization than endophyte-free treatment groups. Performance parameters were higher (P < 0.01) for ammoniated diets in comparison to non-ammoniated diets; however, anti-SRBC titers were not significantly different. When compared to the E+ diet, the AE+ diet increased (P < 0.01) DFI (24%), DWG (41%) and feed efficiency (13%).

Effect of a Fungal Endophyte on the Growth and Survival of Two Euplectrus parasitoids

Presence of the endophytic fungus, Acremonium coenophialum, within tall fescue (Festuca arundinacea) deters many vertebrate and invertebrate herbivores, apparently because of alkaloids produced by the grass-fungus symbiotum. In one set of experiments we tested if A. coenophialum alters the growth and survival of two parasitoids of an insect herbivore feeding on tall fescue. We fed fall armyworm [Spodoptera fruigiperda (Lepidoptera: Noctuidae)] larvae tall fescue either infected or lacking its endophytic fungus and then allowed the parasitoids, Euplectrus comstockii and Euplectrus plathypenae (Hymenoptera: Eulophidae), to parasitize larvae. We found the presence of endophyte-infected plants in the diet of fall armyworm had a negative impact on the pupal mass of parasitoids. In contrast, developmental rate of the parasitoids was generally not affected by the fungal endophyte. Survival of neither parasitoid to the adult stage was reduced by the presence of endophyte-infected grass in the diet of fall armyworm. We also tested if effects of the fungus on E. comstockii were due to the specific alkaloids, N-acetyl and N-formyl loline, that are produced by the grass-fungus symbiotum. When added to artificial diets of fall armyworm, both lolines resulted in reduced survival of parasitoids. Overall, we found the fungus had a moderate negative impact on two natural enemies that mirrors some of the effects the fungus has on insect herbivores and that may be mediated, at least in part, by alkaloids associated with the fungus. We discuss implications of our work for the hypothesis that endophytes defend their host grasses from herbivores.

In Vitro and In Vivo Ruminal and Physiological Responses to Endophyte-Infected Tall Fescue

Relationships between alkaloid compounds in endophyte-infected tall fescue and ruminal metabolism were studied in two experiments. In the first experiment, different combinations of the pyrrolizidine alkaloids, N-formyl and N-acetyl loline, were incubated with ruminal fluid for 0, 24, or 48h. Rate of disappearance of N-formyl and N-acetyl loline increased over time. After 48h, disappearance of N-formyl loline and combinations of N-formyl and N-acetyl loline was greater than N-acetyl loline. Significant amounts of N-formyl and N-acetyl loline were metabolized and converted to loline. In the second experiment, abomasally cannulated sheep were fed increasing amounts of endophyte-infected feed to compare diet digestibility, alkaloid metabolism, and physiological responses. Total tract DM digestibility was greatest for the endophyte-free diet, as were ruminal and total tract ADF, ruminal NDF, and total tract CP digestibilities. N-Formyl and N-acetyl loline recoveries averaged 5% from abomasal contents and 0% in feces. Sixty-eight percent of the pyrrolizidine alkaloids recovered in abomasal contents had been metabolizable to loline. Ergot alkaloids administered in the diet were recovered (50 to 60%) in the abomasal contents, but recovery was only 5% in fecal collections. No significant differences occurred in the physiological parameters measured. Results indicate that response to endophyte-infected tall fescue may be influenced by ruminal metabolism.

The effect of alkaloids and seed extracts of endophyte-infected tall fescue on prolactin secretion in an in vitro rat pituitary perfusion system.

The objective of this research was to measure the effects of endophyte-infected tall fescue seed extract and various alkaloids associated with the endophyte on in vitro prolactin secretion by rat hemipituitaries. Rat anterior pituitaries (AP) were dissected into halves and placed in temperature-controlled culture chambers (37 degrees C). The tissue was perfused with culture media at a flow rate of 12 mL/h. After perfusion for at least 90 min with control media, AP halves were exposed to their respective treatments for 15 min before they were returned to the control media. The treatments for Exp. 1 were .01 micrograms of alpha-ergocryptine/mL of culture medium, .01 microgram of ergonovine/mL of culture medium, .01 gram-equivalents of endophyte-infected tall fescue seed/mL of culture medium, and .01 gram-equivalents of endophyte free tall fescue seed/mL of culture medium. Treatments for Exp. 2 consisted of 10(-4), 10(-6), and 10(-8) M concentrations of perloline, N-formyl loline, N-acetyl loline, N-methyl loline, and alpha-ergocryptine. alpha-Ergocryptine suppressed (P less than .10) prolactin secretion in both experiments. Ergonovine and perloline both stimulated (P less than .10) prolactin secretion. The loline alkaloids (N-formyl loline, N-acetyl loline, N-methyl loline) had no effect on prolactin secretion. The endophyte-infected seed extract treatment suppressed (P less than .10) prolactin secretion. The endophyte-free seed extract treatment had no effect on prolactin secretion. In Exp. 2, prolactin secretion from AP responded to alpha-ergocryptine treatment in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Turfgrass Species and Tall Fescue Endophyte on Feeding Ecology of Japanese Beetle and Southern Masked Chafer Grubs (Coleoptera: Scarabaeidae)

Suitability of six cool-season turfgrasses, including tall fescue, festuca arundinacea Schreb., infected or not infected by the endophyte Acremonium coenophialum Morgan-Jones & Gams, was studied for root-feeding grubs of the Japanese beetle, Popillia japonica Newman, and the southern masked chafer, Cyclocephala lurida Bland. Larval growth, survival, food use, effects of larval diet on adult weight and fecundity, and natural incidence of grubs were investigated in laboratory, greenhouse, and field assays. Hard fescue, Festuca ovina var. duriuscula L., endophyte-free tall fescue, and perennial ryegrass, Lolium perenne L., were generally more favorable than creeping bentgrass, Agrostis palustris (Huds.), for growth of C. lurida, whereas Kentucky bluegrass, Poa pratensis L., was a relatively poor host for both grub species. Variation in nitrogen content of roots is apparently too small to explain these differences. Roots of endophyte-infected tall fescue contained 93 micrograms/g N-formyl loline, a level previously shown to deter grub feeding on artificial medium. However, this study suggests that antixenosis may not occur in the presence of the stimulus complex in tall fescue roots. Survival or growth or both of neonate first instars were lower on endophyte-infected than on endophyte-free tall fescue. However, response of older instars was variable, and there was no measurable effect of endophyte on density or weight of grubs in field plots or on fecundity of P. japonica adults that emerged from endophyte-infected turf. Variation in susceptibility of cool-season turfgrasses to white grubs is probably affected more by differences in their ability to tolerate the feeding damage than by their inherent suitability as food.