Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.     

The inhibition enzymatic hydrolysis of acetylthiocholine by acetylcholinesterase using principal alkaloids isolated from celandine and macleya and their derivatives

A study was made of a possible inhibitory action on the enzymatic hydrolysis of acetylthiocholine by human erythrocyte acetylcholinesterase of principal alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa (namely sanguinarine, chelidonine, berberine), and of drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Chelidonium majus L.) and "Sanguirythrine" (a mixture of unseparated closely related to benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine, isolated from Chelidonium majus L. and other plants of Papaveraceae family). All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. On the basis of the kinetic data it has been determined that chelidonine belonged to reversible inhibitors of a competitive type. All other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction (the values of generalized inhibitory constants being 0.23, 0.23 and 0.29 microM, respectively) and cheliodonine and "Ukrain" were much weaker (2.0 and 2.5 microM, respectively). Judging from the data obtained, sanguinarine and chelerythrine exert similar inhibitory effects on the reaction of enzymatic hydrolysis of acetylthiocholine, since sanguinarine and "Sanguirythrine" have nearly equal generalized inhibitory constants.     

The impact of biochemistry vs. population membership on floral scent profiles in colour polymorphic Hesperis matronalis

Background and Aims

Studies of floral scent evolution often attribute variation in floral scent to differences in pollinator behaviour, ignoring the potential for shared biochemistry between floral scent and floral colour to dictate patterns of phenotypic variation in scent production. To determine the relative effects of shared biochemistry and/or localized population-level phenomena on floral scent phenotype, floral scent composition and emission rate were examined in five wild populations of colour polymorphic Hesperis matronalis (Brassicaceae).

Methods

Floral scent was collected by in situ dynamic headspace extraction on purple and white colour morphs in each of five wild populations. Gas chromatography–mass spectroscopy of extracts allowed determination of floral scent composition and emission rate for all individuals, which were examined by non-metric multidimensional scaling and analysis of variance (ANOVA), respectively, to determine the contributions of floral colour and population membership to scent profile variation.

Key Results

Despite the fact that colour morph means were very similar in some populations and quite different in other populations, colour morphs within populations did not differ from each other in terms of scent composition or emission rate. Populations differed significantly from one another in terms of both floral scent composition and emission rate.

Conclusions

Shared biochemistry alone cannot explain the variation in floral scent phenotype found for H. matronalis. Such a result may suggest that the biochemical association between floral scent and floral colour is complex or dependent on genetic background. Floral scent does vary significantly with population membership; several factors, including environmental conditions, founder effects and genetics, may account for this differentiation and should be considered in future studies.Key words: Hesperis matronalis, floral scent, floral colour, plant volatiles, population differentiation, scent composition, scent emission rate, terpenoids, aromatics     

Cellular and subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers.

The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.     

Extreme divergence in floral scent among woodland star species (Lithophragma spp.) pollinated by floral parasites

Backgrounds and Aims

A current challenge in coevolutionary biology is to understand how suites of traits vary as coevolving lineages diverge. Floral scent is often a complex, variable trait that attracts a suite of generalized pollinators, but may be highly specific in plants specialized on attracting coevolved pollinating floral parasites. In this study, floral scent variation was investigated in four species of woodland stars (Lithophragma spp.) that share the same major pollinator (the moth Greya politella, a floral parasite). Three specific hypotheses were tested: (1) sharing the same specific major pollinator favours conservation of floral scent among close relatives; (2) selection favours ‘private channels’ of rare compounds particularly aimed at the specialist pollinator; or (3) selection from rare, less-specialized co-pollinators mitigates the conservation of floral scent and occurrence of private channels.

Methods

Dynamic headspace sampling and solid-phase microextraction were applied to greenhouse-grown plants from a common garden as well as to field samples from natural populations in a series of experiments aiming to disentangle the genetic and environmental basis of floral scent variation.

Key Results

Striking floral scent divergence was discovered among species. Only one of 69 compounds was shared among all four species. Scent variation was largely genetically based, because it was consistent across field and greenhouse treatments, and was not affected by visits from the pollinating floral parasite.

Conclusions

The strong divergence in floral scents among Lithophragma species contrasts with the pattern of conserved floral scent composition found in other plant genera involved in mutualisms with pollinating floral parasites. Unlike some of these other obligate pollination mutualisms, Lithophragma plants in some populations are occasionally visited by generalist pollinators from other insect taxa. This additional complexity may contribute to the diversification in floral scent found among the Lithophragma species pollinated by Greya moths.     

Das Duftorgan der Honigbiene und die physiologische Bedeutung ihres Lockstoffes

Summary The scent organ of the honeybee was discovered by Nassonoff in 1883 (Nassonoff or Nasanov gland). Sladen (1901) was the first to recognize its true nature. He observed that bees had exposed the scent gland while they exhibited the so called joyful hum, that behaviour by which they produce a stream of air through rapid fanning of the wings, the head being bowed deeply forward and the tail raised sharply (Fig. 1). At the same time an pleasant smell can be perceived, which has been proven to be produced by the Nassonoff gland. The odour is quickly dispersed through the rapid fanning of the wings. That it is an odour with an alluring effect on other bees was demonstrated in 1923/24 by v. Frisch. Moreover, he found that the bees make use of their scent organs not only during fanning and scent dispersal (in German sterzeln) but also that they expose it at plentiful crops. In the latter case a rapid vibration of the wings does not take place (Fig. 3).In 1926 v. Frisch and Rösch tried to find out whether the odour of the scent gland is specific for each colony and attracts only the hive-companions of that bee, which makes use of the scent gland, or wether it is of a general nature and has an alluring effect on all bees, regardless from what colony they come. Based on the results of this experiment, which pointed to a colony-specific effect, and on the grounds of a surmise which was made by v. Buttel-Reepen in 1915, many beekeepers and zoologists were up till now convinced that the colony odour of the bees receives its individual note through the odour of the scent organ. Also Kaltofen (1951) and Kalmus and Ribbands (1952) found the attractant to be colony-specific, while the experiments of Wojtusiak (1934) gave contrary results.My observations and experiments brought the following results: an analysis of those situations in which the bees make use of their scent gland, makes it probable that the workers always fan and disperse scent — sterzeln —, after they have had lost for some time the customary (gewohnten) contact with their companions or with the queen. The ending of this situation, that is to say the renewal of contact, apparently causes the fanning and scent dispersal. A series of observations, which are reported, speak for the correctness of this hypothesis.The scenting of the feeding place and the fanning (with scent dispersal) of bees at the hive-entrance and at the nesting place (of a swarm) serves in all cases to attract companions. In a few cases the purpose of the scent dispersal becomes only obvious, if one takes into consideration that the bees are insects, whose entire behaviour is directed to the society.The colony odour of the bees is composed of at least 12 different components. That it receives its colony-individual note from the odour of the Nassonoff gland is unlikely, because the bees expose their scent organs only outside of the hive and never in it.Twelve Zwei-Völker-Versuche according to the methods of v. Frisch and Rösch (1926) showed on the average a colony-specific behaviour of the newcomers, but the preference for the population's own feeding place was relatively insignificant. In 5 cases one of the two test colonies flew to the feeding place of the foreign colony.This method (which also was used by Kalmus and Ribbands) has a disadvantage: since the recruits alight where the foragers with their functioning scent glands are collecting sugar water, it is not possible to separate the effect of the odour of the Nassonoff gland from the effect of the smell adhering to the bodies of the bees. On the other hand, unanimous results would be expected if the alluring odour could be obtained free of other components. This is relatively easy done in the following way: if one squeezes fairly firmly the abdomen of a bee held between the thumb and index finger, the scent gland becomes exposed. The odiferous substance can now easily be wiped away with a piece of filterpaper held with a pointed forceps (Fig. 6).Both training and spontaneous choice experiments were carried out with odiferous substance obtained in this manner. In the case of the former, 20 to 30 forager bees were trained for several hours to the odour of the scent gland obtained from recruits from their own colony and thereafter they were tested in a choice-experiment, whether they were capable to distinguish the attractant on which they were trained from that which was obtained from bees of an other colony. Also reverse experiments — training to the scent gland odour obtained of bees of another colony — were carried out.The spontaneous choice experiments are a type of Zwei-Völker-Versuche but by suitable provisions it was arranged, that the feeding places of the both groups of foragers (from A and B, which were alarming the newcomers) were seperated from those places where the newcomers sought for sugar-water. There 3 feeding dishes were set up in linear arrangement. One contained scent gland odour taken from recruits from colony A, the other the same from bees of colony B. The middle dish was free of alluring substance. Since in these experiments (as in most Zwei-Völker-Versuchen) colonies of different races were used, which differed distinctly from one another in their coloring, it could be recognized without further ado from which hive an arriving newcomer originated. Immediately after alighting they were caught and killed.Both methods yielded unanimous results: again the strongly attractting effect of the scent gland substance was demonstrated. In the spontaneous experiments of the year 1954, for example, 1260 bees flew to the dishes with the odour and only 49 (less than 4%) to those dishes free of it. The odour of the scent gland definitely is differentiated by bees from the odour of stinger-poison and from other odours; it is, however, neither colony nor race specific. It attracts without distinction all races which I investigated (Apis mellifica ligustica, A. m. nigra and A. m. carnica). If one scent gland odour is given preference, this is an expression of the quantitative differences of both alluring substances used. The effect can be reversed by variation of the quantities. Kalmus and Ribbands (1952) believed to have proven with their experiments that the type of crop influenced the quality of the odour of the scent gland or other surface-glands. The kombinierten Versuche which I made in 1954 (see p. 455) show, however, that a colony-specific behaviour of bees is to be attributed to the colony odour adhering to the surface of their bodies. With purebred Italian and Nigra colonies I carried out alternately Zwei-Völker-Versuche, in which the body odour of the forager bees could influence the decision of the newcomers as to where they would alight, and spontaneous choice experiments, in which these odiferous components were excluded. First of all I tested the untreated colonies, then the Nigra colony — and later both colonies — were scented in the hives with geranium oil; finally these odiferous substances were removed and a mixture of a half pound of honey and two tablespoons of black treacle was placed in the Italian colony in such a way that the bees could not feed on the sweet substance, nevertheless the smell of the treacle was able to exercise its effect. After additional experiments I removed the wire screen blocking the access to the mixture, so that Italian bees could now eat and store it. Then the behaviour of the bees was tested anew. Table 10 shows distinctly the different effects of body odour (in this case = colony odour) and the odour of the scent gland. If the colony odours are the same, the distribution of the newcomers to both feeding places in the Zwei-Völker-Versuche is approximately 11; if they are differrent, the colony's own feeding place is preferred. In the spontaneous choice experiments the distribution of the newcomers in every case is nonspecific, for even then, when one of the odours was preferred, it always was preferred by the newcomers of both colonies, and this surprisingly universally.The reported experiments show that if bees are able to distinguish the companions of their own colony from foreigners, this is to be attributed to the colony odour, which adheres to their bodies. The odour of the scent gland is nonspecific; it attracts without distinction all bees.

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Die Durchführung dieser Arbeit wurde durch ein Stipendium der Deutschen Forschungsgemeinschaft ermöglicht; sie wurde außerdem unterstützt durch Mittel der Rockefeller Foundation, die Prof. v. Frisch zur Verfügung standen.     

Provenance, experience and plant utilisation by the polyphagous aphid, Aphis fabae

Twenty-nine clones of Aphis fabae Scopoli (Aphididae: Homoptera) were isolated into laboratory culture from the primary host plants Euonymus europaeus and Viburnum opulus and the secondary host plants Arctium lappus, Impatiens glandulifera, Tropaeolum majus and Vicia faba. All clones performed well on either T. majus or V. faba, with mean intrinsic rates of increase (r_m) in the range 0.35–0.41 aphids aphid^{– 1} day^{– 1}, but none performed well on both plant species. Each clone could, therefore, be described as affiliated to either V. faba or T. majus. All of the 13 T. majus-affiliated clones and 15 of the 16 V. faba-affiliated clones could not be maintained beyond two parthenogenetic generations on the contrary plant. These results confirm that the performance of A. fabae on T. majus and V. faba is negatively correlated. Exceptionally, one V. faba-affiliated clone (clone AED95/119) persisted indefinitely on T. majus, exhibiting a progressive improvement in performance across four parthenogenetic generations: larval mortality dropped from 80% to <20%, mean adult weight almost doubled from 0.28 to 0.53 mg, and mean number of embryos per adult aphid increased threefold from 8.6 to 24.4 embryos. The mean r_m of clone AED95/119 that had been reared on T. majus for more than 4 generations was 0.17 aphids aphid^{– 1} day^{– 1}, significantly lower than the value of 0.34 aphids aphid^{– 1} day^{– 1} on V. faba. It was also shown that the r_m on V. faba did not differ significantly between aphids of clone AED95/119 that had been derived from long-term cultures on V. faba and T. majus. It is concluded that the negative correlation between performance on V. faba and T. majus is not genetically fixed, but may diminish with exposure to T. majus. The implication is that this clone may have a capacity to utilise T. majus that is independent of its capacity to utilise V. faba, and is expressed only on sustained exposure to T. majus. It is suggested that, although the latent capacity to utilise T. majus is probably not exhibited widely under natural conditions, aphids possessing it may be at a selective advantage when more suitable host plants are temporarily unavailable.     

The scent of fear

In this study we tried to find out if fear can be detected from human body odours. Female subjects wore under-arm axillary pads while watching a terrifying film. Saliva cortisol samples were taken before and after the film presentation as a hormonal measure for the fear response. The fear experience itself was measured by Spielberger's State-Trait Anxiety Inventory. A "neutral" film, shown one day after the "fear" film, was used as a control in a repeated measures design. In part two of the experiment, the axillary pads were presented to female subjects in a triple forced choice test. Results show that subjects were able to discriminate between fear and non-fear axillary pads, suggesting that women are indeed able to detect "the scent of fear". A direct correlation between induced fear, changes in cortisol levels and smell ratings could not be established. Thus cortisol levels are probably not the inducer of the scent of fear and a hypothetical fear pheromone could have other origins.     

The ownership signature in mouse scent marks is involatile

Male house mice advertise their territory ownership through urinary scent marks and use individual-specific patterns of major urinary proteins (MUPs) to discriminate between their own scent and that of other males. It is not clear whether recognition occurs through discrimination of the non-volatile proteins or protein-ligand complexes (direct model), or by the detection of volatile ligands that are released from MUPs (indirect model). To examine the mechanism underlying individual scent mark signatures, we compared investigatory and countermarking responses of male laboratory mice presented with male scent marks from a strain with a different MUP pattern, when they could contact the scent or when contact was prevented by a porous nitrocellulose sheet to which proteins bind. Mice investigated scent marks from other males whether these were covered or not, and biochemical analysis confirmed that the porous cover did not prevent the release of volatiles from scent marks. Having gained information through investigation, mice increased their own scent marking only if they had direct contact with another male's urine, failing to do this when contact was prevented. Individual signatures in scent marks thus appear to be carried by non-volatile proteins or by non-volatile protein-ligand complexes, rather than by volatiles emanating from the scent.     

Scent marking by otters (Lutra lutra): signaling the use of resources

Otters (Lutra lutra) deposit feces as scent marks ("spraints")throughout their range, and observations on this behavior inShetland were used to test the hypothesis that carnivores ingroup ranges use scent marking to signal priority of use ofresources to other group members. Sprainting was seasonal (highrates coinciding with low prey availability), and there wasno significant, overall difference in sprainting rates betweenotters of different sex or status. There were no concentrationsof spraints near group territorial boundaries. Sprainting wasassociated with the beginning of feeding bouts, as well as withthe utilization of other resources, such as fresh water anddens ("holts"). More than 30% of spraints were deposited inplaces that flooded within hours, and the spraints were functionalonly for a short time. It is argued that the temporal patternof use and subsequent replenishment of resources makes it advantageousfor otters to signal to other group members when they are exploitinga "patch" and for other members to avoid resources already partlydepleted by a prior arrival. With such a signaling system thereis no need for actual aggressive encounters to reinforce themessage of scent marking. Sprainting could be the mechanismfor the observed spaced-out use of resources among the inhabitantsof a group territory.     

Synthesis of Benzylglucosinolate in Tropaeolum majus L. (Isothiocyanates as Potent Enzyme Inhibitors)

Benzylglucosinolate accumulates in mature plants of Tropaeolum majus L. The biosynthetic capacity for synthesis of benzylglucosinolate and the total content of benzylglucosinolate have been investigated during plant development and in different tissues. The content increased from 5 mg of benzylglucosinolate in the fresh seed to between 200 and 400 mg in the adult plant, depending on size. The biosynthetic capacity was measured using L-[U-14C]phenylalanine as precursor. Incorporation levels of approximately 30% were obtained with green leaves, whereas the incorporation levels obtained with other tissues were in the range of 0 to 5%. Leaves were the primary site of benzylglucosinolate synthesis. The high amounts of benzylglucosinolate accumulated in other tissues (e.g. developing seeds) reflected transport of benzylglucosinolate from the leaves. The initial steps in the biosynthesis of glucosinolates and cyanogenic glycosides are thought to be similar and to be localized on microsomal membranes. However, a microsomal system prepared from T. majus was biosynthetically inactive. Inclusion of T. majus plant material during preparation of sorghum microsomes also inhibited their activity. Benzylisothiocyanate, generated by degradation of benzylglucosinolate during the homogenization procedure, strongly inhibited the sorghum enzyme system, and its presence may thus explain why the isolated T. majus microsomal system is inactive.     

Qualitative and quantitative analyses of flower scent in Silene latifolia

The quantitative and qualitative variability in floral scent of 98 specimens of the dioecious species Silene latifolia belonging to 15 European and 19 North American populations was determined. Floral scent was collected from single flowers using dynamic headspace methods, and analysed by Micro-SPE and GC-MS methods. The flowers showed a nocturnal rhythm, and scent was emitted only at night. The amount of emitted volatiles varied greatly during the season, from 400 ng/flower/2 min in June to 50 ng/flower/2 min in August and September. The qualitative variability in the floral scent was high and different chemotypes, characterised by specific scent compounds, were found. Female and male flowers emitted the same type and amount of volatiles. The differences in floral scent composition between European and North American populations were small. Typical compounds were isoprenoids like lilac aldehyde isomers, or trans-beta-ocimene, and benzenoids like benzaldehyde, phenyl acetaldehyde, or veratrole. Some of these compounds are known to attract nocturnal Lepidoptera species. The high qualitative variability is discussed in relation to the pollination biology of S. latifolia, and the results are compared with other studies investigating intraspecific variability of flower scent.     

The role of olfactory cues in mother–pup,groupmate, and sex recognition of lesser flat‐headed bats,Tylonycteris pachypus

Tylonycteris pachypus is a gregarious bat species with tens of individuals in a colony. The mechanisms by which mother bats recognize their pups and adult bats recognize each other are not clear. We hypothesized that such recognition is achieved by chemical discrimination and performed experiments to test the hypothesis. Results showed that mother bats were much more attracted to the scent from their own pups. For adult bats, females were attracted to the scent from both male and female groupmates but had a higher preference to the scent from female than from male groupmates. Male bats were much more attracted to the scent from male groupmates while showed no preference to the scent from female groupmates. Within a group, both female and male bats had no difference in preference to the scent from the same or opposite sex. These results suggest that mother–pup and groupmate recognition of T. pachypus can be achieved by olfactory cues.     

Origin, taxonomy and population structure of the allopolyploid peat mossSphagnum majus

The polyploid peat mossSphagnum majus shows considerable phenotypic plasticity along ecological gradients in mires. It is considered taxonomically heterogeneous, and two subspecies have been described. Isozyme analyses were carried out on populations ofS. majus from Central Norway and from eastern coast of North America in order to assess the origin, taxonomy and population structure of this species. High levels of fixed heterozygosity in the populations demonstrate thatS. majus is a genetic allopolyploid. At all loci screened, extant populations ofS. cuspidatum shared enzyme bands withS. majus. The other most likely progenitor based on morphology,S. annulatum, was fixed for enzyme bands not found inS. majus. The progenitor genotype ofS. annulatum may have been missed because of inadequate sampling or extinction. Alternatively, another extinct or undetected taxon may constitute the second progenitor. The observed patterns of genetic variation and linkage disequilibria were uncorrelated with the previously proposed subspecific classification ofS. majus. Lack of genetic divergence between continents suggests that the origins ofS. majus in Europe and North America were not independent. Low mutation rates and large effective population sizes may be important causing populations to diverge slowly, and may explain the observed patterns without hypothesising frequent long-distance dispersal.     

Spatial patterns of scent marking in wild moustached tamarins, Saguinus mystax: no evidence for a territorial function

I investigated whether scent marking has a territorial function in wild moustached tamarins. I examined the spatial distribution of scent marking within the home ranges of four groups of this neotropical primate and tested predictions from Gorman & Mills' (1984, Journal of Zoology,202, 535-547) model for border and 'hinterland' marking. Although home ranges were economically defensible, no evidence was found for increased marking along the territorial boundary or in areas of home range overlap, but there was also no evidence for hinterland marking. Observed distributions of scent marking in exclusively used and overlapping areas of the home range did not deviate from distributions that would be expected if scent marking occurred at random (expectation based both on size of area and on frequency of quadrat occupation), and there was a strong correlation between frequency of quadrat occupation and frequency of scent marking per quadrat. These results indicate that scent marking has no territorial function in moustached tamarins. This is in line with mainly qualitative findings from the majority of other studies on wild marmosets and tamarins. These and other findings on scent marking in moustached tamarins suggest that this behaviour functions mainly in intersexual communication. Copyright 2000 The Association for the Study of Animal Behaviour.     

Rapid,gregarious settlement of the larvae of the marine echiuran Urechis caupo Fisher & MacGinitie 1928

Larvae of Urechis caupo Fisher & MacGinitie, reared in the laboratory, were exposed to potential settlement stimuli, including natural sediment from adult burrows, and “scent” obtained from the skin of adult animals. Competent larvae settled rapidly and specifically in response to adult burrow sediment when compared with their responses to other natural and abiotic sediments. Larvae also responded specifically to chemical “scent” from adult animals when the “scent” of another echiuran worm, Listriolobus pelodes Fisher served as a control. Larval responses to chemical “scent” were as great as their responses to natural burrow sediment. Hence, it is likely that larvae settle gregariously in nature in response to “scent” on sediment grains of adult burrows. The chemical “scent” had a molecular weight between ≈3500 and 14000 daltons, as determined by dialysis. It quickly lost its effectiveness in promoting settlement after it was heated to 80 °C, but was relatively stable at ambient ocean temperatures, retaining its effectiveness for several days. It was soluble in sea water. However, larvae did not respond to the chemical “scent”, unless it was adsorbed onto a surface. Purely tactile stimuli, such as the shape, texture, and size-distribution of particles, were not important settlement cues during these experiments.     

Meadow Voles (Microtus pennsylvanicus, Arvicolidae) Over-mark and Adjacent-mark the Scent Marks of Same-sex Conspecifics

Scent over-marking occurs when an animal deposits its scent mark on top of the scent mark of a conspecific; adjacent-marking occurs when an animal deposits its scent mark next to the scent mark of a conspecific. Given that male rodents usually scent mark more than females and that animals spend more time investigating the odor of the top-scent donor of an over-mark, I tested the following three hypotheses. First, male meadow voles deposit more scent marks than female meadow voles. Second, male meadow voles will deposit more over-marks and adjacent-marks in response to the scent marks of a same-sex conspecific than females would. Third, meadow voles spend more time investigating the odor of the second vole placed in the arena than that of the first vole placed in the arena. To test these hypotheses, two age-matched, like-sex conspecifics (first vole and second vole) were placed successively into an arena in which they were allowed to freely explore and scent mark for 15 min. The first hypothesis was not supported. The first and second vole, independently of sex, deposited a similar number of scent marks. The second hypothesis was also not supported by the data: more conspecific scent marks were over-marked by the second female than by the second male. The third hypothesis was supported by the data. After investigating a scented arena, males and females spent more time investigating the odor of the second vole than that of the first vole. Sex differences in scent-marking behaviors of meadow voles are unlike those reported for other species of rodents.     

Honeybees Learn Odour Mixtures via a Selection of Key Odorants

Background

The honeybee has to detect, process and learn numerous complex odours from her natural environment on a daily basis. Most of these odours are floral scents, which are mixtures of dozens of different odorants. To date, it is still unclear how the bee brain unravels the complex information contained in scent mixtures.

Methodology/Principal Findings

This study investigates learning of complex odour mixtures in honeybees using a simple olfactory conditioning procedure, the Proboscis-Extension-Reflex (PER) paradigm. Restrained honeybees were trained to three scent mixtures composed of 14 floral odorants each, and then tested with the individual odorants of each mixture. Bees did not respond to all odorants of a mixture equally: They responded well to a selection of key odorants, which were unique for each of the three scent mixtures. Bees showed less or very little response to the other odorants of the mixtures. The bees'' response to mixtures composed of only the key odorants was as good as to the original mixtures of 14 odorants. A mixture composed of the other, non-key-odorants elicited a significantly lower response. Neither an odorant''s volatility or molecular structure, nor learning efficiencies for individual odorants affected whether an odorant became a key odorant for a particular mixture. Odorant concentration had a positive effect, with odorants at high concentration likely to become key odorants.

Conclusions/Significance

Our study suggests that the brain processes complex scent mixtures by predominantly learning information from selected key odorants. Our observations on key odorant learning lend significant support to previous work on olfactory learning and mixture processing in honeybees.     

Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.