Aphid Olfaction: unpublished Electroantennogram Studies from 1993-1996
by J.H. Visser & P.G.M. Piron, DLO Research Institute for Plant Protection, Wageningen, The Netherlands
We studied the sense of smell in aphids by recording electroantennogram responses (EAGs). In the old days the grain aphids Sitobion avenae and Metopolophium dirhodum (Yan & Visser, 1982; Visser & Yan, 1995) were probed with oscilloscope and paper recorder. In 1990 the first author developed the present setup which allowed digitized recordings and efficient software calculations of electroantennogram waveforms. In this way the following aphid species have been studied: the vetch aphid Megoura viciae (Visser & Piron, 1994; Visser & Piron, 1995), the black bean aphid Aphis fabae (Hardie et al., 1994; Hardie et al., 1995), the peach-potato aphid Myzus persicae and the cabbage aphid Brevicoryne brassicae (Visser et al., 1996; Visser & Piron, 1997). Here we present the comparison between the odour response profiles of two aphid species, namely Myzus persicae and Brevicoryne brassicae both being reared on Chinese cabbage.
EAG recordings. The recording procedure of aphid electroantennograms is illustrated. For the antennal preparation the head of an aphid is removed just behind the eyes. One antenna is amputated and the very tip of the remaining antenna is cut leaving all olfactory sensilla intact. The electrodes consist of glass capillaries filled with 0.1 M KCl. The ground electrode is inserted into the open side of the head while the recording electrode is sleeved over the antennal tip. Ag-AgCl wires connect the antennal preparation to the high-impedance input of an amplifier and a transient recorder (12 bits ADC) computer combination. In order to control release rates, plant odour compounds are dissolved in paraffin oil at 1% (v/v). Fresh stimulation cartridges are prepared by applying 25 µl of each paraffin oil solution onto a piece of folded filter paper, which is subsequently placed into a Pasteur pipette. Through a glass tube clean air is blown over the antennal preparation at 40 cm/s. The tip of the Pasteur pipette is inserted into a small hole in the side of the glass tube and by blowing air through the Pasteur pipette (2s: 1ml/s) the odour compound is delivered to the antenna. Odour molecules penetrate the antennal olfactory sensilla through small pores. Inside they are transported to receptors in the dendritic membrane. On binding of the odour to the receptors, and in a cascade of biochemical reactions, the resting membrane potential drops. These electrical changes leak to the central haemolymph space in the antenna and are recorded by the instruments as electroantennogram (see for methodological details Visser & Piron, 1995).
EAG waveform analyses. In our setup EAG recordings were digitized at a rate of 25/s by a transient recorder connected to a computer. The first author developed Asyst software which allowed electroantennograms to be corrected for DC drift and noise (Visser & Piron, 1995), and the analyses of waveform characteristics (Visser & Piron, 1994). From each recording the following values were calculated: (1) µV Peak is the largest deflection in the first 2.5 seconds, (2) µV Mean response is the mean deflection in the 1.5 to 2.0 seconds period, (3) % Rise is the mean deflection in the first second relative to the Mean response, and (4) % Decay is the mean decrease of deflection in the 2.5 to 3.5 seconds period relative to the Mean response (see underneath).
- fast rise and slow decay for (E)-2-hexenal (2) & linalool (59)
- fast rise and fast decay for hexanal (24) & 3-butenyl isothiocyanate (126)
- slow rise and slow decay for 4-methoxybenzaldehyde (39) & geraniol (60)
- slow rise and fast decay for (Z)-3-hexenol-1 (5) & 2-methoxybenzaldehyde (37)
The EAGs of Megoura viciae aphids (apterous virginoparae) are shown in response to the compounds at 1% dilution in paraffin oil (v/v). The 2-second stimulation period is indicated and demonstrates the delay of the peak EAG response. It takes upto 1 second for plant odour molecules to reach an equilibrium at the receptors: at equilibrium the number of odour molecules arriving and leaving the receptor sites are equal and the EAG response levels (see for EAG shape characteristics: Visser & Piron, 1994).
EAG normalization. During the recordings the sensitivity of the antennal preparation decreases. Absolute EAG peaks (see underneath left) to the standard (E)-2-hexenal at 1% (v/v) dilution in paraffin oil, the blue bars, are normalized to 100% in order to compensate for such reduction in antennal responsiveness. The responses to tested compounds are expressed as percentage responses of adjacent standards (see underneath right). In all cases the recordings were finished before the response to the standard dropped below 100 µVolt. In general an aphid antennal preparation lasted for about 30 minutes.
EAG peaks are normalized in order to compensate for the decrease in responsiveness. Blue bars represent EAGs to the standard.
EAG comparison between aphid species. The odour response profiles of the polyphagous (food generalist) peach-potato aphid, Myzus persicae, and the oligophagous (food specialist) cabbage aphid, Brevicoryne brassicae, are shown underneath. The electroantennogram responses represent means of normalized peak responses (n ≥ 10). Aphids were reared on Chinese cabbage, Brassica chinensis cultivar Granaat, under long-day conditions (L16:D8) at 22 oC, and reproduced parthenogenetically. For the EAG recordings we used the alatae (winged forms) of both Myzus persicae M3 clone and Brevicoryne brassicae Spanish clone. The compounds tested were all from reliable sources and very pure (mean purity is 97.3%). All compounds were diluted in paraffin oil at 1% (v/v) and 25 µl were applied to a filter paper in the Pasteur pipette. Stimulations and recordings were as described above.
EAG response profiles of Myzus persicae and Brevicoryne brassicae to green leaf volatiles (fatty acid metabolites), benzene derivatives, terpenes and derivatives, and Nitrogen- and Sulfur-containing compounds. Mean EAG response calculated from at least 10 individuals. • indicates significant differences between aphid species at P ≤ 0.05 (2-tailed).
All plant odour components were dissolved in paraffin oil in order to create slow-release formulations that allowed cartridges to be used for repeated stimulations, upto 15 times, without a detectable change in their stimulus intensity. In the evaluation of response profiles the different polarities of compounds should be kept in mind as, thereby, paraffin oil formulations release different odour concentrations stimulating the antenna. Despite these limitations, EAG responses can be compared in chemical groups with similar polarities. The comparison between the EAG response profiles of two aphid species does not suffer from these constraints. In the response profiles significant differences between Myzus persicae and Brevicoryne brassicae are indicated by bullets. It is striking that Brevicoryne shows higher EAG responses to (Z)-3-hexenyl acetate, 3-octanone and both carvones than Myzus. (Z)-3-hexenyl acetate is a prominent component in the odour of cabbage, so it makes sense that the oligophagous Brevicoryne exploits an increased sensitivity for this compound. However, the “classical cabbage” isothiocyanates are not so clear-cut. The nitriles elicit large responses in both aphid species, Myzus perceives hexanonitrile more intense than Brevicoryne. The 3– and 4-methoxybenzaldehydes elicit higher EAG responses in Myzus than in Brevicoryne, but methyl salicylate is the opposite.
Variation. Aphids employ different “lifestyles”: (A) in summer they reproduce parthenogenetically, without sex females deliver genetically identical daughters (clones), (B) when host plants become overcrowded wingless females (apterae) produce winged offspring (alatae), they fly away in search for new food resources, and (C) in autumn, when days become short, sexual forms are born and new aphid hybrids hatch from eggs in springtime. Besides, aphid species show a variety of feeding habits, from specialist aphids occurring on just one (monophagous) or a few plant species (oligophagous), to generalists present on a variety of plant species (polyphagous). Aphid “lifestyles” further differentiates in aphid species alternating between summer and winter host-plant species. So aphids are perfect models to study the impact of biological variation coming from the surroundings (phenotype) or just the genes (genotype). Some of such variation in relation to aphid olfaction of plant odours is presented underneath.
Comparisons were made between: (Species) Myzus persicae and Brevicoryne brassicae (present study), (Clones) Myzus persicae M1 and M3 clones, (Forms) Myzus persicae M1 alatae and apterae (winged versus wingless), and (Diets) Myzus persicae M1 diet-reared and cabbage-reared (Visser & Piron, 1997). In these comparisons absolute EAG responses to the standard (E)-2-hexenal differed solely between Myzus persicae M1 alatae (mean EAG: 407 µV) and apterae (mean EAG: 256 µV) at P ≤ 0.05 (2-tailed). Myzus persicae alatae possess a larger number of olfactory sensilla, namely the secondary rhinaria, than the apterous summer forms. The variation in aphid response profiles is largest between species, followed by clones, forms and diets.
Left: the electrophysiological setup for recording aphid EAGs. Right: Hans Visser inserting the odour cartridge, Pasteur pipette, in the air flow over the antennal preparation (photographs from 1993).