Alarm pheromone mediates production of winged dispersal morphs in aphids

The aphid alarm pheromone ( E )- β -farnesene (EBF) is the major example of defence communication in the insect world. Released when aphids are attacked by predators such as ladybirds or lacewing larvae, aphid alarm pheromone causes behavioural reactions such as walking or dropping off the host plant. In this paper, we show that the exposure to alarm pheromone also induces aphids to give birth to winged dispersal morphs that leave their host plants. We first demonstrate that the alarm pheromone is the only volatile compound emitted from aphid colonies under predator attack and that emission is proportional to predator activity. We then show that artificial alarm pheromone induces groups of aphids but not single individuals to produce a higher proportion of winged morphs among their offspring. Furthermore, aphids react more strongly to the frequency of pheromone release than the amount of pheromone delivered. We suggest that EBF leads to a 'pseudo crowding' effect whereby alarm pheromone perception causes increased walking behaviour in aphids resulting in an increase in the number of physical contacts between individuals, similar to what happens when aphids are crowded. As many plants also produce EBF, our finding suggests that aphids could be manipulated by plants into leaving their hosts, but they also show that the context-dependence of EBF-induced wing formation may hinder such an exploitation of intraspecific signalling by plants.     

Emission of alarm pheromone by non-preyed aphid colonies

The sesquiterpene (E)-β-farnesene (Eβf) is the primary component of the alarm pheromone of most aphid species. It is released in response to physical stress including attack by natural enemies and causes aphids to cease feeding and disperse. Eβf also acts as a kairomonal cue for aphid natural enemies. In this study, we collected the headspace volatiles released by aphid colonies of different sizes. Gas chromatography-mass spectrometry analysis demonstrated the presence of Eβf in the absence of predator attack. A quadratic relationship was found between the released ( E )-β-farnesene amounts and aphid colony size. Behavioural impact of aphid alarm pheromone towards Episyrphus balteatus female oviposition behaviour was also demonstrated in this work. These results highlight the primary role of the small but continuous release of aphid alarm pheromone in mechanisms of decision-making by aphid predators during their foraging and egg-laying behaviour.     

Is the (E)‐β‐farnesene only volatile terpenoid in aphids?

Abstract: Herbivore insects use a broad range of chemical cues to locate their host to feed or to oviposit. Whether several plant volatiles are effective allelochemicals for insects, the latter also emit molecules which have infochemical role. The (E)‐β‐farnesene (EBF) is a well‐known aphid alarm pheromone commonly found in all previously tested species. Analysis of the released molecules from 23 aphid species, mainly collected on their natural host plant from May to July, was performed by gas chromatography–mass spectrometry. While EBF was identified as the main volatile substance in 16 species, alone or associated with other molecules, the alarm pheromone was only a minor component of the volatile molecule pattern of five other species. Moreover, two species, Euceraphis punctipennis and Drepanosiphum platanoides, did not release EBF at all but other terpenes were identified. This original observation raised the question on the utility and the source of the non‐EBF volatiles. Are these potential infochemical substances produced by the aphid or only absorbed from the host plant? Here we determined that terpenes released by insects were not only provided by the host plants. Indeed, Megoura viciae emitted additional molecules than the ones from several aphid species reared on the same host plant. Moreover, no systematic relation between the feeding behaviour of the aphid species and the volatile releases was observed. Aphid terpene composition and proportion would provide reliable cues to identify the emitting organism, plant or insect. The next step of this work will be to determine the infochemical role of terpenes found in the range of tested aphid samples to better understand the relations between the different tritrophic levels.     

Influence of 9-β-l(+)-adenosine on malate dehydrogenase activity in rice

The activity of malate dehydrogenase (MDH, EC 1.1.1.37) in rice ( Oryza sativa L. cv. California M-201) roots was increased within 25 min by a foliar application of 37 n M of 9-β- l (+)-adenosine [ l (+)-adenosine]. A similar concentration of 9-β- d (-)-adenosine [ d (-)-adenosine] did not affect MDH. Triacontanol (2.7 n M ) which elicits l (+)-adenosine in plants also increased MDH activity in roots of rice seedlings. whereas neither octacosanol nor a mixture of equimolar concentrations of triacontanol and octacosanol had any effect on MDH activity. l (+)-Adenosine increased MDH activity of rice seedlings grown at 10.20 and 40°C. The effect was measurable 24 h after application at 10 and 20°C, but not 6 h after treatment at 40°C. Ninety minutes after l (+)-adenosine was applied to the foliage of plants grown at 10, 20 and 40°C, MDH activity in the roots was 40, 30 and 9% more, respectively, than in the untreated controls. The concentration of water soluble protein was also increased by l (+)-adenosine and was positively correlated with MDH activity. When measured 90 min after application of l (+)-adenosine at different times of day, MDH activity and dry weight were increased most when l (+)-adenosine was applied 9 and 12 h after the lights came on in a 16-h photoperiod. The optimum light intensity for the response of rice to l (+)-adenosine, as measured by MDH activity, was 450 μmol m⁻² s⁻¹.     

Inhibition of Monoamine Oxidase Within Monoaminergic Neurons in the Rat Brain by (E)-β-Fluoromethylene-m-Tyrosine (MDL 72394)

The irreversible inhibition of the monoamine oxidase (MAO) activity within monoaminergic neurons in the rat brain 24 h after single or repeated administration of (E)-beta-fluoromethylene-m-tyrosine (FMMT, MDL 72394) was examined. The enzyme activity was determined by incubating synaptosome-rich homogenates of hypothalamus or striatum with low concentrations of 5-[14C]hydroxytryptamine (5-HT), [14C]noradrenaline (NA), or [14C]dopamine (DA) in the absence and presence of the selective amine uptake inhibitors citalopram (5-HT), maprotiline (NA), and GBR 12909 (DA). After a single subcutaneous injection of FMMT, the inhibition of MAO within the noradrenergic and dopaminergic neurons was significant but only slightly greater than that outside these neurons. The opposite relationship was observed for the serotonergic neurons. After 7 days' treatment of rats with carbidopa, 20 mg/kg p.o., + FMMT once daily, the preference for the inhibition of MAO within the noradrenergic and dopaminergic neurons was accentuated further. The inhibition outside the serotonergic neurons was still greater than within these neurons. The NA uptake inhibitor CPP 199 antagonized the selective inhibition of MAO within the noradrenergic neurons, which indicates that this preference is due to the accumulation of the active metabolite (E)-beta-fluoromethylene-m-tyramine by the NA transporter.     

Characterization of an endo-1,3(4)-β-d-glucanase gene from Cellvibrio mixtus

Abstract An endo-1,3(4)-β- d -glucanase gene ( cwd2 ) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb Pst I fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli , degraded both β-1,3 glucans and β-1,3–1,4 mixed-linkage glucans, was entohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40°C respectively. The M _r of specifically labelled Cwd2 was approximately 34 000. This gene was quite distinct from two other C. mixtus β-1,3 glucanases previously described.     

Alarm pheromone emission by pea aphid, Acyrthosiphon pisum, clones under predation by lacewing larvae

Genetic variation in anti-predator traits has been shown for a variety of species. Aphid alarm pheromone, ( E )-β-farnesene, is released by attacked aphids and causes a variety of behavioral defense reactions in the signal receivers. In pea aphids, Acyrthosiphon pisum Harris (Homoptera: Aphididae), ( E )-β-farnesene mediates the production of winged offspring in the presence of natural enemies. While variation in the propensity for pea aphids to produce winged offspring is well-documented, little quantitative information is available about clonal differences in ( E )-β-farnesene emission or the amount of alarm pheromone released in aphid colonies. We tested the wing induction response of four clones when attacked by a predatory lacewing larva, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), and found that three of the four clones increased the proportion of winged offspring under predator attack. We then investigated the emission of aphid alarm pheromone of these clones of pea aphid under attack. Alarm pheromone emission in aphid colonies of initially 25 adults varied from 81.2 to 10 851.0 ng per aphid colony over 24 h. There were no differences between clones in total emission or in emission per consumption event. These results show that there is substantial variability in alarm pheromone emission within clones and that the propensity to produce winged offspring in some clones is not a simple function of the propensity of alarm pheromone production in these clones.     

Proteolytic inactivation of an extracellular (1 → 3)-β-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH

Abstract The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1 → 3)- β -glucanases produced by this fungus, although the activities of the remaining two (1 → 3)- β -glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1 → 3)- β -glucanase inactivation.     

Kupffer cells play important roles in the metabolic degradation of a soluble anti-tumor (1 → 3)-β-d-glucan, SSG, in mice

Abstract Metabolic degradation of a soluble highly branched (1 → 3)-β- d -glucan, SSG, was examined in mice using a macrophage blocker, gadolinium chloride (GdCl₃). Intraperitoneally administered SSG distributed in the liver was slowly degraded, and after 5 weeks about 30% of the SSG became anionic. In addition, it is suggested that the metabolites would contain fewer branching points as assessed by the reactivity to limulus factor G. On the other hand, in the spleen, the molecular weight and the degree of branching of SSG were not changed for at least 5 weeks. Blockade of Kupffer cells by GdCl₃ did not significantly change the distribution ratio of SSG in the liver. However, the treatment significantly delayed the degradation of SSG. These results suggested that Kupffer cells play important roles, not in the distribution, but in the oxidative degradation of SSG in the liver. In addition, splenic macrophages did not significantly contribute to the metabolic degradation of SSG.     

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

(11Z)-hexadec-11-enal enhances the attractiveness of Diatraea saccharalis main pheromone component in wind tunnel experiments

Abstract:  GC-EAD and GC-MS analysis of pheromone gland extracts of sugarcane borer, Diatraea saccharalis , revealed two antennally active compounds, (9 Z ,11 E )-hexadeca-9,1-dienal and (11 Z )-hexadec-11-enal, in approximately 10 : 1 ratio. Various doses of identified compounds were investigated in wind tunnel experiments individually and in a 10 : 1 ratio. At all tested doses (9 Z ,11 E )-hexadeca-9,1-dienal alone elicited upwind orientation and source location only in a minority of tested males. An admixture of (11 Z )-hex-11-enal enhanced the attractiveness of (9 Z ,11 E )-hexadeca-9,11-dienal significantly. This two-component blend (100 pg) was as attractive as natural pheromone extracted from three female pheromone glands. The data suggest that (11 Z )-hexadec-11-enal is a part of the D. saccharalis sex pheromone.     

Inhibition and labeling of red beet uridine 5'diphospho-glucose: (1,3)-β-glucan (callose) synthase by chemical modification with formaldehyde and uridine 5'diphospho-pyridoxal

The effects of the lysine-reactive chemical modification reagents, uridine 5’ diphospho (UDP)-pyridoxal and formaldehyde (HCHO), on the activity of membrane-bound and solubilized UDP-Glc: (1,3)-β-D-glucan synthase (callose synthase) from red beet (Beta vulgaris L.) storage tissue were compared. Exposure to micromolar levels of UDP-pyridoxal, or millimolar levels of HCHO in the presence of NaCNBH₃, resulted in complete enzyme inactivation. Conditions for inhibition of membrane-bound enzyme activity by the two reagents were markedly similar; divalent cations were required for inactivation, and complete protection of activity was obtained with EDTA or EGTA. The substrate, UDP-Glc, protected membrane-bound callose synthase against inactivation by UDP-pyridoxal or HCHO, but protected the solubilized enzyme only against inhibition by UDP-pyridoxal, suggesting that the lysine residue modified by both these reagents is at the enzyme active site, and that the site is more open or has a certain conformational flexibility in the solubilized enzyme. Potential UDP-Glc-binding polypeptides of callose synthase were identified by a two-step labeling procedure. First, nonessential lysine residues were blocked by irreversible modification reaction with HCHO or UDP-pyridoxal in the presence of UDP-Glc to protect lysines at UDP-Glc-binding sites. In the second step, proteins were recovered, reacted with [¹⁴C]-HCHO in the absence of UDP-Glc, and polypeptide labeling patterns analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. This procedure reduced incorporation of label by 5- to 8-fold compared to a procedure omitting the preblocking step, and with enzyme partially purified by solubilization in CHAPS followed by product entrapment, labeling was limited to a small set of polypeptides. Taken together with the results of other studies, the data suggest that one or more polypeptides migrating in the 54–57 kDa region are good candidates for the UDP-Glc-binding components of callose synthase.     

Behavioural response of winged aphids to visual contrasts in the field

To test the behavioural response of winged aphid spring migrants to visual contrasts, we conducted a field trial in which water traps (painted in seven different shades of green and yellow) were set up on uncovered soil and on coloured boards (also painted in seven different colours including black, brown and various shades of green). In total, 56 trap–background combinations were tested. Out of the 4904 aphid individuals caught, 64.5% belonged to Aphis ssp. Using spectral measurements of both traps and backgrounds, as well as information on insect spectral sensitivity, an empirical colour choice model was built based on photoreceptor adaptation to the background, and colour opponency of the green and blue photoreceptor. Specifically, the visual input variable C* represents the difference between green–blue colour opponency values of the trap and the background. When C* > 0, the number of aphids linearly increased with C*. The model explained 64% of the behavioural response of the aphids. Applied to intercropping scenarios of sugar beet, the behavioural model showed a higher visual attractivity of a monocrop sugar beet than intercropped sugar beet. Implications for the use of mulches and for increasing plant diversity in cropping systems are discussed.     

Flight of Heliothis virescens males in the field in response to sex pheromone

Abstract. The behaviour of Heliothis virescens males flying upwind in the field in a sex pheromone plume was videorecorded and analysed. Males flew faster and straighter, with less counterturning, and heading more directly into the wind when they were 9-11m away from the odour source than when they were 1–3 m away. Regardless of their distance from the source or the windspeed, they maintained an average groundspeed of c. 200 cm s^_1, except when they arrived within 1 m of the source, when their groundspeed slowed significantly. Two or more males flying in the plume at the same instant often exhibited either extremely straight and directly upwind tracks or else zigzagging tracks with significant counterturning (as did males flying through the field of view of the cameras at slighdy different times). The males' position, either in the centre of the plume's axis or along one side, might explain these differences in track straightness, which previous studies with H.virescens have shown to be caused by higher frequencies of contact with plume filaments. When a significant shift in wind direction occurred, males tended to make an initial movement in the direction of the shift, perhaps due to latencies of response in both the olfactory and visual systems associated with flying into clean air. The males' behaviour in the field overall was similar to that observed in the wind tunnel, except that their airspeeds and groundspeeds were significantly higher than those observed in the laboratory. The fact that they flew faster in the field can be explained both by the significandy higher windspeeds that males need to compensate for in the field to attain a preferred velocity of image motion, as well as by a higher height of flight over the ground in die field causing a slower apparent motion of images at a given groundspeed compared with the laboratory.     

Alate production in an aphid in relation to ant tending and alarm pheromone

1. Winged dispersal is vital for aphids as predation pressure and host plant conditions fluctuate. 2. Ant‐tended aphids also need to disperse, but this may represent a cost for the ants, resulting in an evolutionary conflict of interest over aphid dispersal. 3. The combined effects of aphid alarm pheromone, indicating predation risk, and ant attendance on the production of winged aphids were examined in an experiment with Aphis fabae (Homoptera: Aphididae) (Scopoli 1763) aphids and Lasius niger (Formicidae: Formicinae) (Linné, 1758) ants. 4. This study is the first to investigate the joint effects of alarm pheromone and ant attendance, and also the first to detect an influence of alarm pheromone on the production of winged morphs in A. fabae. 5. After a period of 2 weeks, it was found that aphid colonies exposed to intermittent doses of alarm pheromone produced more winged individuals, whereas ant tending had the opposite effect. The effects were additive on a log scale, and ant attendance had a greater proportional influence than exposure to alarm pheromone. A tentative conclusion is that ants have gained the upper hand in an evolutionary conflict about aphid dispersal.     

Removal of the vomeronasal organ blocks the stress-induced hyperthermia response to alarm pheromone in male rats

Previously, we reported that male Wistar rats release alarm pheromone from their perianal region, which aggravates stress-induced hyperthermia (SIH) in pheromone-recipient rats. The subsequent discovery that this pheromone could be trapped in water enabled us to expose recipients to the pheromone in their home cages. Despite its apparent influence on autonomic and behavioral functions, we still had no clear evidence as to whether the alarm pheromone was perceived by the main olfactory system (MOS) or by the vomeronasal system. In this study, we investigated this question by exposing 3 types of recipients to alarm pheromone in their home cages: intact males (Intact), vomeronasal organ-excised males (VNX), and sham-operated males (Sham). The Intact and Sham recipients showed aggravated SIH in response to alarm pheromone, whereas the VNX recipients did not. In addition, the results of the habituation/dishabituation test and soybean agglutinin binding to the accessory olfactory bulb verified the complete ablation of the vomeronasal organ (VNO) with a functional MOS in the pheromone recipients. These results strongly suggest that male rats perceive alarm pheromone with the VNO.