GENETICS OF PHYSIOLOGICAL AND BEHAVIORAL RESISTANCE TO HOST FURANOCOUMARINS IN THE PARSNIP WEBWORM

Depressaria pastinacella, the parsnip webworm, feeds almost exclusively on the flowers and fruits of Pastinaca sativa, the wild parsnip. Resistance to webworms in wild parsnip populations is largely attributable to genetically based variation in furanocoumarin chemistry; by differentially reducing fruit set among chemical phenotypes, parsnip webworms may act as selective agents on wild parsnip populations. To determine whether wild parsnip chemistry can act as a selective agent on webworm populations, it is necessary to establish that resistance mechanisms in the webworm to furanocoumarins are genetically based. In this study, we estimated the amount of genetic variation in behavioral and physiological responses of webworms to parsnip furanocoumarins. Virtually no variation was found among webworm families for feeding preferences for diets varying as much as fourfold in furanocoumarin content. Nor was significant variation found for mean furanocoumarin intake over the assay period, except in one case, in which maternal effects may account for differences among families. In contrast, substantial familial variation existed for cytochrome P450–mediated metabolism of bergapten and xanthotoxin, two host furanocoumarins. The presence of additive genetic variation in metabolism, and the absence of such variation in discriminative feeding behavior, suggests that adaptation to changes in furanocoumarin chemistry, resulting either from changes in the distribution of chemical phenotypes in parsnip populations or from shifts to new chemically different host plants, is likely to be facilitated by physiological rather than behavioral means.     

Patterns of furanocoumarin production and insect herbivory in a population of wild parsnip (Pastinaca sativa L.)

Summary Seasonal changes in the distribution and abundance of furanocoumarins in wild parsnip, Pastinaca sativa (Umbelliferae), were examined in a population of plants in Tompkins County, New York. Xanthotoxin, imperatorin and bergapten (linear furanocoumarins) occur in all above-ground parts of the plant; in addition, angelicin and sphondin (angular furanocoumarins) occur in umbels of some individuals. Total furanocoumarin content, as measured by percent dry weight, is greatest in reproductive parts, particularly buds and seeds; variation in concentrations between plants is greatest in vegetative structures (e.g., leaves).Within the plant, the distribution of furanocoumarins is significantly correlated with nitrogen, as opposed to biomass, allocation. In that nitrogen is often a factor limiting the plant growth, furanocoumarins appear to be allocated in proportion to plant tissue value; reproductive structures, obvious contributors to plant fitness, contain over ten times the amount of nitrogen and furanocoumarin contained in vegetative structures such as senescent leaves.Stepwise multiple regression analysis revealed that generalized insect herbivores tend to feed on plants or plant parts low in furanocoumarin content and, correspondingly, low in nitrogen content. Parsnip specialists, notably Depressaria pastinacella (Lepidoptera: Oecophoridae), feed exclusively on umbels, plant parts rich in nitrogen and furanocoumarins; furanocoumarin number and content in fact account for over 60% of the variance in number of umbel feeders. These patterns conform with previous determinations of the toxicological properties of furanocoumarins. Nitrogen is known to affect growth rate, fecundity, longevity and survivorship of insect herbivores; by tolerating or detoxifying furanocoumarins, D. pastinacella can consume plant tissues containing significantly greater amounts of nitrogen than tissues consumed by generalist feeders. That the presence of D. pastinacella on individual plants is correlated with the number of furanocoumarins present is consistent with the hypothesis that parsnip specialists use angular furanocoumarins as host recognition cues.     

CONSTRAINTS ON CHEMICAL COEVOLUTION: WILD PARSNIPS AND THE PARSNIP WEBWORM

The parsnip webworm (Depressaria pastinacella) and the wild parsnip (Pastinaca sativa) together represent a potentially “coevolved” system in that throughout their ranges the plant has relatively few other herbivores and the insect has virtually no other hosts. Individual wild parsnip plants within a central Illinois population vary in their content and composition of furanocoumarins, secondary compounds with insecticidal properties. Half-sib and parent-offspring regression estimates of the heritability of furanocoumarins demonstrate that this variation is genetically based. Wild parsnip plants also vary in their resistance to damage by the parsnip webworm, which feeds on flowers and developing seeds. In an experimental garden, seed production in the primary umbel ranged from 0 to 1,664 seeds among individuals, and mean seed production of half-sib families ranged from 3.7 seeds to 446.0 seeds. Approximately 75% of the variation in resistance among half-sib families to D. pastinacella was attributable to four furanocoumarin characteristics—resistance is positively related to the proportion of bergapten and the amount of sphondin in seeds, and negatively related to the amount of bergapten and the proportion of sphondin in leaves. Each of the four resistance factors had significant heritability. Thus, resistance in wild parsnip to the parsnip webworm is to a large extent chemically based and genetically controlled. Genetic correlations among fitness and resistance characters, however, tend to limit coevolutionary responses between herbivore and plant. In greenhouse plants protected from herbivory, several of the resistance factors have negative genetic correlations with potential seed production. Ostensibly, highly resistant plants in the absence of herbivory would be at a competitive disadvantage in the field. The selective impact of the herbivore is also limited in this population by a negative genetic correlation among resistance factors. Selection to increase one resistance factor (e.g., the proportion of bergapten in the seed) would at the same time decrease the amount of a second resistance factor (e.g., the amount of sphondin in the seed). The wild parsnip and the parsnip webworm, then, appear to have reached an evolutionary “stalemate” in the coevolutionary arms race.     

Essential‐Oil Composition of the Fruits of Six Heracleum L. Species from Iran: Chemotaxonomic Significance

The fruit essential oils of Heracleum persicum, H. rechingeri, H. gorganicum, H. rawianum, H. pastinacifolium, and H. anisactis from Iran were obtained by hydrodistillation and characterized by GC‐FID and GC/MS analyses. The oils of the six species were compared to determine the similarities and differences among their compositions. Overall, 36 compounds were identified in the fruit oils, accounting for 92.40–96.74% of the total oil compositions. Aliphatic esters constituted the main fraction of the oils (86.61–94.31%), with octyl acetate and hexyl butyrate as the major components. The oil compositions of species belonging to section Pubescentia (H. persicum, H. gorganicum, and H. rechingeri) were discriminated by equally high contents of both octyl acetate (13.84–20.48%) and hexyl butyrate (17.73–38.36%). On the other hand, the oils of H. rawianum, H. pastinacifolium and H. anisactis, belonging to section Wendia, showed lower hexyl butyrate contents (3.62–6.6%) and higher octyl acetate contents (48.71–75.36%) than the former. Moreover, isoelemicin was identified at low amounts (0.10–2.51%) only in the oils of the latter species. The differences in the oil composition among the six species were investigated by hierarchical cluster and principal component analyses, which indicated that the oil composition confirmed well the taxonomical classification based on the morphological and botanical data, and, thus, may provide a reliable marker to discriminate Heracleum species at the intersectional level.     

Chemical barriers to adaptation by a specialist herbivore

Summary Depressaria pastinacella, the parsnip webworm (Lepidoptera: Oecophoridae), feeds throughout eastern North America on Pastinaca sativa (wild parsnip) and few other species. The assumption that specialist herbivores such as the parsnip webworm are adapted to hostplant chemistry, and are therefore unaffected by chemical variation in hostplants, was tested. Flower buds from plants grown first in the greenhouse and then in the field were fed to ultimate instar webworms. Plant phenotype had a significant effect on virtually all webworm food utilization parameters. While nutritional factors (i.e., nitrogen content) were correlated with approximate digestibility, two constituents of the flowers — bergapten and xanthotoxin, both linear furanocoumarins — independently accounted for a significant amount of variation in food utilization indicies. The physiological effects of these furanocoumarins were confirmed in artificial diet experiments. Despite the fact that the two most important furanocoumarins in parsnip flowers relative to webworm feeding and growth are isomers, differing only in the positioning of a methoxy substituent, they have different physiological effects; while xanthotoxin in general has no effect on growth, bergapten decreases growth and digestibility of the diet. These results underscore the need in studies of plant-animal interactions to examine individual chemical components rather than classes of compounds.     

Microwave-facilitated extraction of furanocoumarins onto paper substrates: an imaging technique to analyse spatial distribution and abundance in leaves

An impediment to studying the implications of fine-scale variation in the distribution of secondary compounds within plants has been an inability to map their distribution. Conventional approaches require fine-scale dissections, followed by extraction and analysis, a strategy which is often limited by the large number of extractions required and by the difficulty in quantifying compounds in very small samples. A method has been developed which allows the microwave-facilitated transfer of furanocoumarins from fresh leaves to two-dimensional substrates (phase separation paper). The fluorescence of these compounds under UV light is enhanced by treatment with base, which probably opens the lactone ring of the compounds. The developed prints are then photographed under UV light and the images analysed with commonly available software programs. Images so obtained for furanocoumarins in wild parsnip foliage revealed that the total fluorescence from extracted furanocoumarins is correlated with the amount of furanocoumarins remaining in the leaf. Thus, the images provide information not only on spatial distribution but also quantitative data.     

Physiological price of an induced chemical defense: photosynthesis, respiration, biosynthesis, and growth

A recurring theme in defense allocation theories is that defenses are costly. Most studies that attempt to quantify a cost of defense seek to establish a trade-off between a component of plant fitness and the level of a constitutive defense. Such estimates are ambiguous because they cannot discount the cost of traits that are correlated with defense but are not themselves defensive. We examined the effects of damage-induced synthesis of furanocoumarins, known defense compounds, on the growth of wild parsnip. Plants that had 2% of their leaf area removed accumulated 8.6% less total biomass and 14% less root biomass than intact plants over a 4-week period. We also found that this small amount of leaf damage significantly reduced net photosynthetic rates 0.5 h after damage; the effect was temporary, as photosynthetic rates were no longer significantly different after 48 h. Lastly, we found that increases in respiration rates associated with damage coincided spatially and temporally with increases in furanocoumarin production, and that respiration increases were phenotypically correlated with furanocoumarin production. When damage-induced changes in furanocoumarin content and respiration rates were expressed in glucose equivalents and compared, the energetic cost of furanocoumarin production (12.6 μg glucose cm⁻²) accounted for all of the increase in respiration (12.0 μg glucose cm⁻²). A comparison of other secondary compounds in damaged and intact leaflets revealed that myristicin, a furanocoumarin synergist, is the only other compound aside from furanocoumarins that is inducible. The inducible defense system of wild parsnip thus appears to involve a small subset of secondary compounds. Synthesis of these compounds is tightly linked to damage-induced rates of respiration. Because the negative impact that damage had on the rate of net photosynthesis was short-lived, the impact of damage on growth observed in this study was likely due to the cost of furanocoumarin synthesis elicited by damage rather than the loss of photosynthetic tissue caused by damage. Received: 4 April 1996 / Accepted: 29 August 1996     

Optimal defence, kin conflict and the distribution of furanocoumarins among offspring of wild parsnip

The factors influencing the allocation of chemical defences to plant offspring have largely been unexplored, conceptually and experimentally. Because evolutionary interactions between maternal plants and their progeny can affect resource allocation patterns among sibling offspring, we suggest that kin conflict as well as herbivore–plant interaction theories need to be considered to predict chemical defence allocation patterns. Optimal defence theory predicts that maternal plants should defend more heavily those offspring in which resources have been disproportionately invested. In contrast, kin conflict theory predicts that natural selection will favour genotypes that can compete successfully for maternal defences irrespective of their quality, even at the expense of the fitness of siblings and the maternal plant. Evidence for these defence patterns were evaluated by examining the allocation of furanocoumarins to seeds of the wild parsnip (Pastinaca sativa, Apiaceae). Furanocoumarins are toxins that are localized within the oil tubes of the maternal tissues of seeds. We evaluated the role of offspring investment (endosperm mass) and seed genotype on furanocoumarin allocation by mating an array of pollen donors with pollen recipients. Furanocoumarins were found to be positively correlated with endosperm mass on one side of the seed, a result consistent with optimal defence theory; however, on the other side of the seed, furanocoumarin content was influenced by seed genotype and was unrelated to endosperm mass. These effects varied with maternal plant. Further experiments demonstrated that nearly 80% of furanocoumarin production occurs after pollination, when fertilization products are active. Although the amount of furanocoumarin influenced by the seed genotype is small relative to the total quantity in the seed, these furanocoumarins are the first line of defence against important predators, such as the parsnip webworm, Depressaria pastinacella (Lepidoptera: Oecophoridae). We found that parsnip webworm larvae were able to discriminate among genotypes within an inflorescence. In line with previous studies, these results suggest that a genotype's ability to influence furanocoumarin defence may affect its probability of survival. We conclude that the distribution of defences among plant offspring in wild parsnip is probably influenced by competition among seed genotypes that conflicts with maternal optimal defence. This revised version was published online in July 2006 with corrections to the Cover Date.     

软儿梨果酒发酵过程中挥发性风味物质变化分析

软儿梨是西北地区特有的一种“冻果”产品,酸甜可口、果香浓郁,是酿造果酒的上乘原料。为明确果酒发酵过程中果香的保留情况,并探究发酵时间的延长对软儿梨果酒品质的影响,以青海省民和县软儿梨冻果为原料,基于带皮渣发酵工艺,先采用顶空固相微萃取（solid?phase microextraction, SPME）,再结合气相色谱?质谱联用技术（gas chromatography?mass spectrometry, GC?MS）对原汁（0 d）、前发酵（7、14、28 d）、后发酵（100 d）3个不同时期样品中挥发性风味物质进行了动态跟踪分析,并结合相对气味活度值（relative odor activity value,ROAV）和主成分分析（principal component analysis,PCA）法分别探讨了软儿梨果酒原果香味保留情况和后发酵时间的延长对果酒风味的影响。结果显示：整个发酵过程共检出88种挥发性化合物,其中酯类33种、醇类29种、酸类6种、萜烯类7种以及13种其他类化合物,且风味物质的总含量随发酵的进行呈先上升后略有下降的趋势。ROAV结果表明：软儿梨原汁关键风味物质共8种,分别是丁酸乙酯、己酸乙酯、2?甲基丁酸乙酯、正辛醇、大马士酮、芳樟醇、丁香酚和癸醛;其中果香物质己酸乙酯、正辛醇、芳樟醇、丁香酚和大马士酮在果酒发酵中得到了很好的保留,是决定软儿梨果酒风味的关键物质。主成分分析表明：发酵初期主要的香气贡献物质是具有水果香的丁酸乙酯、3?羟基丁酸乙酯、2?甲基丁酸乙酯;发酵100 d时,乙酸乙酯、α?松油醇、柠檬烯和芳樟醇对香气贡献较大,这些香气化合物共同赋予软儿梨果酒幽香清雅、馥香浓郁的独特风味品质。研究获得了软儿梨果酒关键风味物质及其在不同发酵阶段特征风味物质的变化规律,可为研发高品质软儿梨果酒产品、改进软儿梨果酒发酵工艺提供理论支撑和参考。     

Content and Fatty Acid Composition of Steryl and Wax Esters in Germinating Spores of Polytrichum commune

Polytrichum commune spores contained 5.61 ± 0.52 mg steryl and wax esters, including volatile compounds, per 100 mg dry weight of spores. Volatile compounds were not found in 3-h-old sporelings. The content of the steryl and wax ester fraction, excluding the volatile compounds, is slightly increased during the first 6 h of germination. Thereafter, the content is decreased throughout the germination. Thus, 3-day-old sporelings contained 0.52 ± 0.05 mg steryl and wax esters per 100 mg dry weight of spores. In connection with protonema growth, steryl and wax esters were produced, and the 7-day-old cultures contained 5.09 ± 0.37 mg steryl and wax esters per 100 mg dry weight of spores. The main fatty acids of the steryl and wax ester fraction of dry spores and germinating spores as well as of protonemata were palmitic, oleic, linoleic and linolenic acids. Polyunsaturated C 20 acids were present only in trace or small amounts. Phytanic and phytenic acids were found in small amounts in dry spores, in 3- to 72-h-old sporelings, and in protonemata.     

白星花金龟对寄主植物挥发物的电生理反应

[目的]白星花金龟Protaetia brevitarsis是我国经济上重要的农业害虫.本研究拟鉴定苹果、葡萄和玉米3种果实在两种状态(健康与受白星花金龟成虫为害)下的挥发物中能引起白星花金龟雌成虫触角电生理反应的挥发物成分.[方法]采用动态顶空吸附法收集苹果、葡萄和玉米3种果实的挥发物,用气相色谱-触角电位联用技术(...     

Effects of florivory on floral volatile emissions and pollination success in the wild parsnip

In addition to reducing fitness by consuming reproductive structures, florivores may also reduce plant fitness by altering interactions with pollinators. To date, the effects of florivore activity on the volatile profile of flowers and subsequent attractiveness to pollinators have not been extensively investigated. In this study, we had three specific objectives: to determine the impact of florivory by the parsnip webworm Depressaria pastinacella on the floral volatile profile of the wild parsnip Pastinaca sativa, to ascertain the mechanisms by which florivory changes the volatile profile, and to estimate the consequences of florivory on visitation by pollinators and eventual seed set. An overall indirect effect of webworms on seed set, that is, the effect of infestation on pollination success, was not detected. However, this overall lack of indirect effect masks the heterogeneity among individual plants. For seven of 14 plants examined, pollination success was altered by webworms, and in four of these plants the alteration in pollination success was consistent with webworm-altered visitation. Webworms significantly altered floral fragrance, in particular causing disproportionate increases in the emissions of octyl esters. Additionally, volatiles from webworm frass, which contains large amounts of the octyl ester metabolite n-octanol, may alter the floral fragrance in ways that change attractiveness of flowers to pollinators. This study suggests that the effects of florivores on plant fitness are not limited to the removal of floral units but may also involve alterations in floral volatile composition, through damage-induced release and detoxification of particular constituents, that affect visitation and pollination success. Handling Editor: Steve Johnson. An erratum to this article can be found at     

Phenotype matching in wild parsnip and parsnip webworms: causes and consequences

According to the geographic mosaic theory of coevolution, selection intensity in interactions varies across a landscape, forming a selection mosaic; interaction traits match at coevolutionary hotspots where selection is reciprocal and mismatch at coldspots where reciprocity is not a factor. Chemical traits play an important role in the interaction between wild parsnip (Pastinaca sativa) and the parsnip webworm (Depressaria pastinacella). Furanocoumarins, produced as plant defenses, are detoxified by the webworms by cytochrome P450 monooxygenases; significant additive genetic variation exists for both furanocoumarin production in the plant and detoxification in the insect, making these traits available for selection. To test the hypothesis that differences in selection intensity affect the distribution of coevolutionary hotspots and coldspots in this interaction, we examined 20 populations of webworms and wild parsnips in Illinois and Wisconsin that varied in size, extent of infestation, proximity to woods (and potential vertebrate predators), and proximity to a chemically distinct alternate host plant, Heracleum lanatum (cow parsnip). Twelve of 20 populations displayed phenotype matching between plant defense and insect detoxification profiles. Of the eight mismatched populations, a logistic regression model related matching probability to two predictors: the presence of the alternate host and average content of xanthotoxin (one of the five furanocoumarins produced by P. sativa). The odds of mismatching were significantly increased by the presence of the alternate host (odds ratio = 15.4) and by increased xanthotoxin content (odds ratio = 6.053). Parsnips growing near cow parsnip displayed chemical phenotypes that were chemically intermediate between cow parsnip and parsnips growing in isolation. Rapid phenotype matching in this system is likely due in part to differential mortality every season; larvae transferred to a plant 30 m or more from the plant on which they developed tended to experience increased mortality over larvae transferred to another umbel on the same plant on which they had developed, and plant populations that mismatched in 2001 displayed a change in chemical phenotype distribution from the previous year. Trait mixing through gene flow is also a likely factor in determining mismatch frequency. Populations from which webworms were eradicated the previous year were all recolonized; in three of seven of these populations, infestation rates exceeded 90%. Our findings, consistent with the geographic mosaic theory, suggest that the presence of a chemically distinct alternate host plant can affect selection intensity in such a way as to reduce the likelihood of reciprocity in the coevolutionary interaction between wild parsnip and the parsnip webworm.     

Formation of mercapturic acids in rats after the administration of aralkyl esters

1. Benzylmercapturic acid and hippuric acid were isolated from the urine of rats that had been injected subcutaneously with benzyl acetate. 2. 1-Menaphthylmercapturic acid and 1-naphthoic acid were isolated from the urine of rats after the subcutaneous injection of each of the following compounds: 1-menaphthyl alcohol and its acetate, propionate, butyrate and benzoate esters. 3. A quantitative method for determining 1-menaphthylmercapturic acid in urine was developed and used to measure the excretion of this compound in the urine of rats in the 4-day period after the subcutaneous injection of 1-menaphthyl alcohol and its acetate, propionate, butyrate and benzoate esters. 4. Chromatographic evidence was obtained for the presence of S-(1-menaphthyl)glutathione and S-(1-menaphthyl)-l-cysteine in bile collected from rats with cannulated bile ducts after the animals had been injected subcutaneously with each of the following compounds: S-(1-menaphthyl)glutathione, 1-menaphthyl acetate, propionate and butyrate. 5. Benzylmercapturic acid and 1-menaphthylmercapturic acid were isolated from the urine of rats that had been injected with sodium benzyl sulphate and sodium 1-menaphthyl sulphate respectively.     

Metabolism of myristicin by Depressaria pastinacella CYP6AB3v2 and inhibition by its metabolite

Although methylenedioxyphenyl (MDP) compounds, such as myristicin, are useful in the management of insecticide-resistant insects, the molecular mechanisms for their action in mammals and insects have not been elucidated. In this study, GC-MS analyses of methanol extracts of foliage of wild parsnip (Pastinaca sativa) have identified myristicin as a substrate for CYP6AB3v2, an imperatorin-metabolizing cytochrome P450 monooxygenase from Depressaria pastinacella (parsnip webworm). In contrast with its strong inhibitory effects on many mammalian P450s, myristicin is effectively metabolized by CYP6AB3v2 (V(max) and K(m) of 97.9 pmol/min/pmol P450 and 17.9 microM, respectively) at a rate exceeding that recorded previously for imperatorin, the only other known substrate for this highly specialized enzyme. The myristicin metabolite of CYP6AB3v2 is 1-(3',4'-methylenedioxy-5'-methoxyphenyl)-2,3-epoxypropane. Molecular dockings have indicated that, unlike other epoxide metabolites of furanocoumarins, this epoxide metabolite is likely to remain in the CYP6AB3v2 catalytic site due to its low binding energy (-31.0 kcal/mol). Inhibition assays indicate that myristicin acts as a mixed inhibitor of this insect P450 and suggest that the epoxide metabolite may be an intermediate involved in the formation of P450-methylenedioxyphenyl complexes.     

Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters

The adhesion and germination of conidia of nine fungal species were assessed on polycarbonate membranes or on the skin of apple fruit in sealed glass bottles injected or not injected with acetate esters. Adhesion was determined after dislodging conidia from surfaces using a sonication probe. Adhesion and germination of conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium citrinum, Penicillium claviforme, or Trichoderma sp. on membranes after 48 h were not increased in a 1.84 microg mL(-1) headspace of butyl acetate (BA), ethyl acetate, hexyl acetate, 2-methylbutyl acetate, pentyl acetate, or propyl acetate. Adhesion and germination of Botrytis cinerea, Penicillium expansum, and Penicillium roquefortii conidia were stimulated by all esters. Only conidia of B. cinerea and P. expansum exhibited increased adhesion and germination on the skin of apple fruit in bottles exposed to 0.92 microg mL(-1) of BA. Only conidia of B. cinerea and P. expansum produced decay in inoculated puncture wounds on fruit. Freshly made puncture wounds or 24-h-old puncture wounds in fruit were more adhesive than the unpunctured skin of fruit to conidia of B. cinerea or P. expansum. Fresh wounds were more adhesive to both fungi than 24-h-old puncture wounds. The skin and wounds of fruit were as adhesive to B. cinerea conidia as they were to P. expansum conidia. A 4-h exposure to 1.43 microg mL(-1) of BA increased adhesion of B. cinerea and P. expansum conidia in 24-h-old wounds. Results suggest that acetate-ester stimulation most likely is not a rare phenomenon in the fungi. For nutrient-dependent decay pathogens of apple fruit, acetate esters may be an alternative chemical cue used to maintain adhesion of conidia to wound surfaces.     

Identification of the Volatile Compounds Produced in Sterile Fish Muscle (Sebastes melanops) by Pseudomonas fragi

Volatile compounds produced by Pseudomonas fragi strain 18 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. The fruity odor that developed in fish muscle during incipient spoilage was attributed to a synergistic flavor interaction involving the ethyl esters of acetate, butyrate, and hexanoate.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

The use of octyl beta-D-glucoside as detergent for hog kidney brush border membrane

Octyl beta-D-glucoside was synthetized from alpha-acetobromoglucose with an improved method yielding a very pure product with a sharp melting point (108-109 degrees C) and free of intermediate products as judged by IR and NMR spectra. The yield of the synthesis is 66% when referred to alpha-acetobromoglucose. The potency of this compound as a detergent on hog kidney brush border membranes was compared to the action of Triton X-100. Octyl glucoside preferentially extracts aminopeptidase M and gamma-glutamyltranspeptidase in a concentration-dependent manner. The more deeply imbedded membrane enzyme, alkaline phosphatase, was relatively resistent to the action of octyl glucoside. In contrast, Triton X-100 extracted all membrane proteins to about the same extent. Additionally it was found that octyl glucoside can be removed from membrane extracts by Biobead SM 2. The capacity of the beads is about 170 mg detergent/g of dry Biobead SM 2. Thus octyl glucoside seems to be a useful tool for solubilization and purification of brush border membranes proteins.     

Reciprocal Isomerization of Butyrate and Isobutyrate by the Strictly Anaerobic Bacterium Strain WoG13 and Methanogenic Isobutyrate Degradation by a Defined Triculture

Isomerization of butyrate and isobutyrate was investigated with the recently isolated strictly anaerobic bacterium strain WoG13 which ferments glutarate to butyrate, isobutyrate, CO₂, and small amounts of acetate. Dense cell suspensions converted butyrate to isobutyrate and isobutyrate to butyrate. ¹³C-nuclear magnetic resonance experiments proved that this isomerization was accomplished by migration of the carboxyl group to the adjacent carbon atom. In cell extracts, both butyrate and isobutyrate were activated to their coenzyme A (CoA) esters by acyl-CoA:acetate CoA-transferases. The reciprocal rearrangement of butyryl-CoA and isobutyryl-CoA was catalyzed by a butyryl-CoA:isobutyryl-CoA mutase which depended strictly on the presence of coenzyme B₁₂. Isobutyrate was completely degraded via butyrate to acetate and methane by a defined triculture of strain WoG13, Syntrophomonas wolfei, and Methanospirillum hungatei.