A new sexual signal in rutting male red deer: Age related chemical scent constituents in the belly black spot

Rutting behaviour of red deer stags (Cervus elaphus) includes an extensive repertoire of visual and acoustic signals directed either to rival males or to females. As in other mammals, olfactory communication is expected to play a central role in these rutting interactions too, but this has rarely been investigated. Only during the rutting season, red deer males show a conspicuous black spot area throughout most of their underbelly produced by the impregnation of substances with a strong scent. Here, we examined the origin of these compounds and their potential role as chemical signals. By using gas chromatography–mass spectrometry (GC–MS), we identified 67 compounds in the hair from the belly black spot of red deer stags, mainly heterocyclic aromatic organic compounds, such as m-cresol, benzoic acid, cyclohexanecarboxylic acid and ethylphenol, but we also found steroids, such as cholesterol and androstane-3,17-dione, carboxylic acids and their esters between n-C₆ and n-C₂₂, alcohols, squalene and other minor compounds. Many of these compounds are found in the belly black spot but not in other hair areas, and may have originated from several sources, such as the urine or the sebaceous glands of the skin, which impregnated the belly. Moreover, we found differences in chemical profiles depending on age, with older males having higher proportions of benzoic acid and androstane-3,17-dione, but lower proportions of m-cresol. Because most of these compounds are strongly odoriferous, and appear related to male characteristics, our data indicate that scent from the hairs forming the black spot of the belly may be regarded as an overlooked new sexual chemical signal in red deer in the context of competition for mates during the rutting season.     

Metabolism of p-Cresol by the Fungus Aspergillus fumigatus

The fungus Aspergillus fumigatus ATCC 28282 was shown to grow on p-cresol as its sole source of carbon and energy. A pathway for metabolism of this compound was proposed. This has protocatechuate as the ring-fission substrate with cleavage and metabolism by an ortho-fission pathway. The protocatechuate was formed by two alternative routes, either by initial attack on the methyl group, which is oxidized to carboxyl, followed by ring-hydroxylation, or by ring-hydroxylation as the first step with subsequent oxidation of 4-methylcatechol to the acid. The pathway was elucidated from several pieces of evidence. A number of compounds, including 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, protocatechualdehyde, and 4-methylcatechol, appeared transiently in the medium during growth on p-cresol. These compounds were oxidized without lag by p-cresol-grown cells but not by succinate-grown cells. Enzyme activities for most of the proposed steps were demonstrated in cell extracts after growth on p-cresol, and the products of these activities were identified. None of the activities were found in succinate-grown cells.     

Smells like debauchery: The chemical composition of semen-like,sweat-like and faintly foetid floral odours in Xysmalobium (Apocynaceae: Asclepiadoideae)

Floral volatiles play an important role in plant communication with both pollinators and antagonists, but remain poorly explored for many plant groups. Asclepiads (Apocynaceae: Asclepiadoideae subtribe Asclepiadinae) represent a diverse group in South African grasslands, but the scents of most species remain unexplored and few genera are sufficiently sampled to allow comparisons between congeners. I used dynamic headspace extraction methods and coupled gas chromatography-mass spectrometry (GC–MS) to examine the scent chemistry of three unusually scented asclepiads in the genus Xysmalobium and then combined these data with previously published data to explore inter- and intraspecific variation in the genus. A total of 74 compounds (33–44 per species) from various compound classes were detected in the species examined here. The sweet but faintly foetid scent of Xysmalobium asperum was dominated by epoxy oxoisophorone in combination with various other terpenoids and aromatics, and small amounts of p-cresol. The sweat-like scent of Xysmalobium tysonianum was dominated by a few aromatics in combination with isovaleric acid and several aliphatic compounds normally associated with microbial degradation or fermentation. The semen-like scent of Xysmalobium parviflorum flowers examined here contained large relative amounts of 1-pyrroline, and comparison with previously published data for dung-scented flowers from a different population revealed clear divergence in the relative amounts of this compound and p-cresol. I also detected 25 compounds that were not shared between the two X. parviflorum populations. Comparison of scent data for eight Xysmalobium species revealed very distinct chemical profiles with limited overlap between species. These results are discussed in relation to the possible roles of these volatiles as pollinator attractants and the evolution of floral scents within the genus.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Simple high-performance liquid chromatographic analysis of phenol and p-cresol in urine and feces

Previous reports which present methods of analysis of phenol and p-cresol by HPLC are usually designed for the detection of these compounds in urine, can be complicated by the use of uncommon equipment or additional techniques such as steam distillation or derivatisation, or concentrate on the detection of phenol rather than p-cresol. In this paper we report a simple method suitable for the analysis of phenol and p-cresol in both urine and feces, based upon extraction into ether following acid hydrolysis and UV detection.     

Microbiological Degradation of Malodorous Substances of Swine Waste under Aerobic Conditions

Phenol, p-cresol, and volatile fatty acids (VFA; acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids) were used as odor indicators of swine waste. Aeration of the waste allowed the indigenous microorganisms to grow and degrade these malodorous substances. The time required for degradation of these substances varied according to the waste used, and it was not necessarily related to their concentrations. Using a minimal medium which contained one of the malodorous compounds as sole carbon source, we have selected from swine waste microorganisms that can grow in the medium. The majority of these microorganisms were able to degrade the same substrate when inoculated in sterilized swine waste but with an efficiency varying from one strain to the other. None of these strains was able to degrade all malodorous substances studied. Within 6 days of incubation these selected strains degraded the following: Acinetobacter calcoaceticus, phenol and all VFA; Alcaligenes faecalis, p-cresol and all VFA; Corynebacterium glutamicum and Micrococcus sp., phenol, p-cresol, and acetic and propionic acids; Arthrobacter flavescens, all VFA. On a laboratory scale, the massive inoculation of swine waste with C. glutamicum or Micrococcus sp. accelerated degradation of the malodorous substances. However, this effect was not observed with all of the various swine wastes tested. These results suggest that an efficient deodorization process of various swine wastes could be developed at the farm level based on the aerobic indigenous microflora of each waste.     

Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes.     

Use of Aromatic Compounds for Growth and Isolation of Zoogloea

Nine Zoogloea strains, were examined for their ability to utilize 35 aromatic compounds. Benzoate, m-toluate, and p-toluate, as well as phenol, o-cresol, m-cresol, and p-cresol, were utilized by eight strains. These strains exhibited meta cleavage of catechol and of methyl-substituted catechols. With the exception of L-tyrosine, none of the aromatic compounds tested supported growth of Z. ramigera ATCC 19623. A medium containing sodium m-toluate was used to isolate 37 zoogloea-forming bacteria from various polluted environments. The isolates were identified as strains of Zoogloea.     

Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in desulfobacterium cetonicum

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO₂ with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min⁻¹ (mg of protein)⁻¹. This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via β-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.     

Systemic Resistance Induced by Volatile Organic Compounds Emitted by Plant Growth-Promoting Fungi in Arabidopsis thaliana

Volatile organic compounds (VOC) were extracted and identified from plant growth-promoting fungi (PGPF), Phoma sp., Cladosporium sp. and Ampelomyces sp., using gas chromatography–mass spectrometry (GC-MS). Among the three VOC extracted, two VOC blends (emitted from Ampelomyces sp. and Cladosporium sp.) significantly reduced disease severity in Arabidopsis plants against Pseudomonas syringae pv. tomato DC3000 (Pst). Subsequently, m-cresol and methyl benzoate (MeBA) were identified as major active volatile compounds from Ampelomyces sp. and Cladosporium sp., respectively, and found to elicit induced systemic resistance (ISR) against the pathogen. Molecular signaling for disease suppression by the VOC were investigated by treating different mutants and transgenic Arabidopsis plants impaired in salicylic acid (SA) or Jasmonic acid (JA)/ethylene (ET) signaling pathways with m-cresol and MeBA followed by challenge inoculation with Pst. Results show that the level of protection was significantly lower when JA/ET-impaired mutants were treated with MeBA, and in SA-, and JA/ET-disrupted mutants after m-cresol treatment, indicating the involvement of these signal transduction pathways in the ISR primed by the volatiles. Analysis of defense-related genes by real-time qRT-PCR showed that both the SA-and JA-signaling pathways combine in the m-cresol signaling of ISR, whereas MeBA is mainly involved in the JA-signaling pathway with partial recruitment of SA-signals. The ET-signaling pathway was not employed in ISR by the volatiles. Therefore, this study identified two novel volatile components capable of eliciting ISR that may be promising candidates in biological control strategy to protect plants from diseases.     

Pungent odor of the adult skipper butterfly <Emphasis Type="Italic">Erynnis</Emphasis><Emphasis Type="Italic">montanus</Emphasis> (Lepidoptera: Hesperiidae)

Adults of some butterfly species have odors, several components of which are known to have pheromonal or defensive functions. Little is known about the odors of hesperiid butterflies, however. Erynnis montanus (Hesperiidae), especially male adults, emit a pungent odor that is detectable by the human nose. Chemical analysis has revealed that crude extracts of wild individuals contained 10 volatile substances, of which docosane and heneicosane were the main components. Because males contain a significantly larger amount of p-cresol than females, this aromatic compound is characterized as a strong male odor. At the end of the adult occurrence period, p-cresol decreased substantially in each male. Benzothiazole, identified for the first time in lepidopteran adults, was present in both sexes in almost equal amounts. Among the 10 volatile substances detected in males, biased distribution in the wings rather than the body was observed for benzothiazole, heptanal, and p-cresol. Male adults have androconial organs in the costal part of the forewing on which benzothiazole and p-cresol tended to concentrate. However, these compounds were detected not only in other parts of the forewing but also in the hindwing and body, suggesting the presence of undiscovered scent-producing organs.     

The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc^–/+ cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc^–/+ cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc^–/+ cells with 0.8 mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2 mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc^–/+ and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8 mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their “respiratory reserve.” At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes.     

Constituents of Cane Molasses

By gas chromatography the following eight phenolic compounds and benzoic acid were identified from a sample of cane final molasses using both polar and non-polar stationary phases: anisole, phenetole, phenol, m-cresol, salicylic acid, resorcinol, vanillic acid, and syringic acid. The peaks corresponding to p-coumaric acid and vanillin were also found using non-polar phase. The structures of four or five unidentified components were inferred from the relation between retention temperature and functional group number of the phenolic compounds.     

Simultaneous removal of 2-chlorophenol, phenol, p-cresol and p-hydroxybenzaldehyde under nitrifying conditions: kinetic study

The kinetic behavior of a stable nitrifying consortium exposed to 2-chlorophenol (2-CP), phenol, p-cresol and p-hydroxybenzaldehyde (p-OHB) was evaluated in batch assays. Phenolic compounds were evaluated either individually or in mixture. In individual assays, 2-CP inhibited stronger the nitrification, diminishing the ammonium consumption efficiency (16%) and the nitrate production rate (at 91%). Nonetheless, the consumption efficiencies for all phenolics were of 100%. On the other hand, in mixture, the inhibitory effect of 2-CP diminished significantly, since ammonium consumption efficiency and nitrate production rate were improved. Consumption efficiencies for most of the phenolic compounds were high. Furthermore, the kinetic of 2-CP oxidation was 2.4-fold-faster than the individual assays. Finally, the experimental results showed the potential of nitrifying consortium for removing 2-CP, phenol, p-cresol and p-OHB. This is the first work showing the simultaneous removal of these pollutants and also this information might be useful for treating wastewaters of chemical complexity.     

Influence of p-cresol on the proteome of the autotrophic nitrifying bacterium Nitrosomonas eutropha C91

In this study, the effect of the organic micropollutant and known inhibitor of nitrification, p-cresol, was investigated on the metabolism of the ammonia oxidizing bacteria (AOB) Nitrosomonas eutropha C91 using MS-based quantitative proteomics. Several studies have demonstrated that AOB are capable of biotransforming a wide variety of aromatic compounds making them suitable candidates for bioremediation, yet the underlying molecular mechanisms are poorly described. The effect of two different concentrations of the aromatic micropollutant p-cresol (1 and 10 mg L^?1) on the metabolism of N. eutropha C91, relative to a p-cresol absent control, was investigated. Though the rate of nitrification in N. eutropha C91 appeared essentially unaffected at both concentrations of p-cresol relative to the control, the expressional pattern of the proteins of N. eutropha C91 changed significantly. The presence of p-cresol resulted in the repressed expression of several key proteins related to N-metabolism, seemingly impairing energy production in N. eutropha C91, contradicting the observed unaltered rates of nitrification. However, the expression of proteins of the TCA cycle and proteins related to xenobiotic degradation, including a p-cresol dehydrogenase, was found to be stimulated by the presence of p-cresol. This indicates that N. eutropha C91 is capable of degrading p-cresol and that it assimilates degradation intermediates into the TCA cycle. The results reveal a pathway for p-cresol degradation and subsequent entry point in the TCA cycle in N. eutropha C91. The obtained data indicate that mixotrophy, rather than cometabolism, is the major mechanism behind p-cresol degradation in N. eutropha C91.     

Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l^?1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g^?1 dry weight (DW), and between 50.23 and 81.56 mg 100 g^?1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g^?1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g^?1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g^?1 DW) and syringic acid (41.20 mg 100 g^?1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g^?1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g^?1 DW) were predominant.     

1-Phenyl-2-pentanone and methyl salicylate: new defense allomone components and their content shift during ontogenetic development of the millipede <Emphasis Type="Italic">Nedyopus tambanus mangaesinus</Emphasis> (Polydesmida: Paradoxosomatidae)

Twelve components were identified in hexane extracts of the polydesmid millipede Nedyopus tambanus mangaesinus (Attems, 1909) and their contents were examined at all stadia of ontogenetic development including two adult conditions (before and after hibernation). Two compounds, 1-phenyl-2-pentanone and methyl salicylate, were newly identified as components of the millipede together with six well-known polydesmoid compounds (benzaldehyde, benzyl alcohol, benzoic acid, benzoyl cyanide, mandelonitrile, and mandelonitrile benzoate) and four phenols (phenol, p-cresol, 2-methoxyphenol, and 2-methoxy-4-methylphenol). Benzaldehyde and benzoyl cyanide were distributed from nymphs at stadium I, and other components started to become detectable at more advanced stadia. The largest content (35.1%) of methyl salicylate was detected in nymphs at stadium I, together with benzaldehyde and benzoyl cyanide, implying the reinforcement of defensive functions during or after egg emergence. The content (18.8%) of 1-phenyl-2-pentanone reached a maximum in hibernated females among all stadia and conditions. Its female-biased distribution at the mating season (ca. 5.7-fold more than in males) implies its sexually related functions.     

Biodegradation of phenolic compounds by sulfate-reducing bacteria from contaminated sediments

The biodegradation of phenolic compounds under sulfate-reducing conditions was studied in sediments from northern Indiana. Phenol, p-cresol and 4-chlorophenol were selected as test substrates and added to sediment suspensions from four sites at an initial concentration of 10 mg/liter. Degradative abilities of the sediment microorganisms from the four sites could be related to previous exposure to phenolic pollution. Time to onset of biodegradation of p-cresol and phenol in sediment suspensions from a nonindustrialized site was approximately 70 and 100 days, respectively, in unacclimated cultures. In sediment slurries from three sites with a history of wastewater discharges containing phenolics, time to onset of biodegradation was 50–70 days for p-cresol and 50–70 days for phenol in unacclimated cultures. In acclimated cultures from all four sites, the length of the lag phase was reduced to 14–35 days for p-cresol and 25–60 days for phenol. Length of the biodegradative phase varied from 25 to 40 days for phenol and 10 to 50 days for p-cresol and was not markedly affected by acclimation. Substrate mineralization by sulfate-reducing bacteria was confirmed with radiotracer techniques using an acclimated sediment culture from one site. Addition of molybdate, a specific inhibitor of sulfate reduction, and bacterial cell inactivation inhibited sulfate reduction and substrate utilization. None of the sites exhibited the ability to degrade 4-chlorophenol, nor were acclimated phenol and p-cresol degrading cultures from a particular site able to cometabolize 4-chlorophenol.Correspondence to: D. Dean-Ross     

Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15

The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p-cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ring-¹⁴C]toluene was oxidized to ¹⁴CO₂, and the stoichiometry of ¹⁴CO₂ production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p-cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p-Hydroxybenzoate was a transitory extracellular intermediate of phenol and p-cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p-cresol (p-hydroxybenzylalcohol and p-hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.     

Measurement of phenol and p-cresol in urine and feces using vacuum microdistillation and high-performance liquid chromatography

In this article, we describe a simple, sensitive, accurate, and repeatable method for the measurement of phenol and p-cresol (4-methylphenol) in human urine and feces. We examined a number of parameters to identify an optimal extraction protocol. Purification of sample extracts was achieved by low-temperature vacuum microdistillation. Separation was achieved in approximately 15 min by high-performance liquid chromatography (HPLC) with quantification by fluorescence at 284/310 nm. Limits of detection for phenol were 2 ng/ml for urine and 20 ng/g for feces, and those for p-cresol were 10 ng/ml for urine and 100 ng/g for feces. For comparison, approximate mean values for urine are 3 μg/ml for phenol and 30 μg/ml for p-cresol, and those for feces are 1 μg/g for phenol and 50 μg/g for p-cresol. An experienced analyst can process 60 samples each day using this method.