Whiteflies and plants
Whiteflies, T. vaporariorum (Westwood), originated from a lab culture at Rothamsted Research which was first collected in 1960 in Kent on French bean and had subsequently been maintained in a large laboratory population. Prior to experiments, the culture had been maintained in CRTs lab for around a year as a population of several thousand individuals on pre-flowering aubergine (Solanum melongena) of the ‘Moneymaker’ variety (Marshalls Seeds Cat. 1020-2017) at 20 °C, 16:8 light/dark.
All plants used for culture, volatile collection, and EPGs were propagated approximately 60 cm away from a Harrier HR400SH 400W lamp housing a 400W Son-T bulb. Light was on a 16-h light, 8-h dark cycle in sync with the light regime that EPGs were conducted under. Temperature was 25 °C during the light cycle and 20 °C during the dark. All plants were grown from seed in Clover Multipurpose Compost (http://www.cloverpeat.co.uk/CLOVER-RETAIL-COMPOST-1.html) in 9-cm-diameter and 8.7-cm-depth pots, one plant per pot (with the exception of watercress which was grown two to four plants per pot as plants are small), with liberal watering. Tomato (Lycopersicon esculentum Mill., ‘Elegance’, Cat. E/12/11, Batch 0113479253) was used pre-flowering with three to five fully emerged compound leafs. Cucumber, dwarf French bean and courgette were housed together (three plants of each; see Fig. 2b for general arrangement) in the first of two volatile delivery experiments. Cucumber (Cucumis sativus, ‘(F1 hybrid) La Diva’, Chiltern Seeds, Cat. 1359J) was pre-flowering with one fully emerged mature leaf at the time of placement into the volatile collection chamber. Dwarf French bean (Phaseolus vulgaris, ‘Canadian Wonder’, Chiltern Seeds, Cat 1816c) was pre-flowering with two fully emerged mature leafs at the time of placement into the volatile collection chamber. Courgette (Cucurbita pepo, ‘Nero di Milano’, Chiltern Seeds, Cat 1826) was pre-flowering with one fully emerged mature leaf at the time of placement into the volatile collection chamber. Watercress, watermelon and Savoy cabbage were housed together (three plants of each; see Fig. 2b for general arrangement) in the second of two volatile delivery experiments. Watercress (Nasturtium officinale, ‘Aqua’, Sutton’s Seeds, Cat. 161689) was pre-flowering with five to six fully emerged mature leafs at the time of placement into the volatile collection chamber. Watermelon (Citrullus lanatus, ‘Red Star F1’, Sutton’s Seeds, Cat. 171550) was pre-flowering with two to three fully emerged mature leafs at the time of placement into the volatile collection chamber. Savoy cabbage (Brasica oleracea, ‘Ormskirk(1)-Rearguard’, Sutton’s Seeds, Cat.155962) was pre-flowering with two to three fully emerged mature leafs at the time of placement into the volatile collection chamber. Plants in both volatile delivery experiments were placed in the volatile collection chamber for approximately 2 weeks with liberal watering before being replaced by fresh plants. They were all pre-flowering at the time of removal. Plant species were all chosen as hosts of T. vaporariorum based on literature (Mound and Halsey 1978; Roditakis 1990; Gorman et al. 2002; Moreau and Isman 2011). The two control treatments consisted of nine pots with damp compost and nine pots each with a single tomato plant, placed in the volatile collection chamber.
Volatile collection, volatile delivery, EPG, data analysis
Integrating volatile collection and delivery with EPG
A standard eight-channel DC EPG system (Tjallingii 1978) was modified to allow the monitoring of whitefly feeding behaviour on tomato while supplying whiteflies with air containing volatiles from other host plants. Mixed-species plants or control pots were placed in a Perspex box measuring L 28 cm × W 28 cm × H 20 cm with four 2-cm mesh-covered holes on one side and four outlet holes on the other (Fig. 2b). Outlet holes fitted 4-mm-ID fluorinated ethylene propylene (FEP) tubing (low odour emission) tapering through three Y-splitters to a single length of the same tubing, which connected the air pump. Distance from the outlets to the pump was 40 cm. The volatile collection box was 50 cm away from two Osram L36W/840 bulbs on 16 L/8D in synch with plant growth lamps and the main lights in the experimental room. The air pump was a Boxer 3000 gas pump with no sliding seals or components in the fluid path of the pump, ensuring contamination free transfer of media. It delivered volatile-infused air at a rate of 2.2 L/min (Fig. 2b). Volatiles were pumped along a single length of 4-mm-ID FEP tube of length 312 cm to a bank of small FEP tubes and Y-splitters, splitting the flow into eight 4-mm-ID FEP tubes. The length of the Y-splitter bank was 21 cm. The eight tubes, each 119 cm in length, fed into the Faraday cage where they joined the volatile delivery box. This was made of Perspex and measured L 79 cm × W 9 cm × H 6 cm. Each inlet was covered with a 2-cm-diameter air deflector, with a 3-mm gap between the top of the deflector disk and the top of the box to allow inflow of air (Fig. 2c). Into the bottom of the box were cut eight 1.5-cm-diameter holes to allow the insertion of EPG probes, and into the back of the box, eight 1-cm holes were cut to allow the insertion of tomato leaves. These 1-cm holes were split along the middle, as was the whole box, so the box could be taken apart and leaves inserted without damage to the plant tissue. We began to deliver volatiles to the tomato plants approximately 1 h before initiation of EPGs.
The EPG method produces a waveform output, with different waveforms corresponding to defined feeding behaviour such as probing, xylem feeding and phloem feeding (Tjallingii 1978). We used an eight-channel DC EPG system supplied by EPG Systems (EPG-Systems, Dillenburg 12, 6703 CJ Wageningen, the Netherlands, http://www.epgsystems.eu/contact.htm; Tjallingii 1978). We used the terminal leaflet of the uppermost, fully emerged compound leaf of the tomato plant. The terminal and next two leaflets were inserted into the volatile delivery chamber still attached to the main plant (Fig. 2c) and the terminal leaflet propped perpendicular to the base of the chamber with a length of Blu-Tack (Fig. 2c). While we would ideally have wished to run EPGs with the leaf positioned naturally and the whitefly feeding from the underside of it, it was found that the insect was unstable during EPGs in this position (probably due to the weight of the gold wire), and most fell from the plant during the 15-h recording, rendering traces unusable. Four plants and four channels were used in each daily experiment (Fig. 2a), and a different set of plants were used for EPGs in each daily run. Experiments were continued until we achieved 20 or so 15-h recordings in each treatment. We generally recorded EPGs for around 20 h, and a failed recording (discarded) was defined as one in which non-probing (NP) began at some time before 15 h after the start of the recording and continued without additional waveforms until the end of the approximately 20-h period. Although uncommon, traces that showed NP across the 15-h threshold but showed additional probing waveforms between 15 and 20 h were considered successful as it was assumed that the NP that occurred within the 15-h period represented an insect still on the leaf ‘choosing’ not to probe. After placing the wired insects on the undersurface of the perpendicular leaf, when the EPG recording started, insects were closely monitored by the authors for 2 to 3h. Insects that fell from the leaf or became positioned on the leaf in a way that would not allow insertion of the mouthparts during this period were replaced on the leaf with a soft-tipped brush. After the 2–3-h period, the insects were left without intervention by the authors. The EPG electrode consisted of a 2-cm nail, with a stiff but bendable 3-cm length of copper wire soldered onto it and finally a 2-cm length of 12.5-μm-diameter gold wire. We found it necessary to anesthetise these tiny, very mobile insects with CO2 to determine their sex and attach them via the dorsal thorax to the gold wire with water-based silver glue. Only females of mixed age taken at random from the stock culture were used for EPGs, and they were allowed at least 15 min of recovery time after anesthetization before being placed on the plant. All equipment within the Faraday cage was connected to the cage via earth wires attached at several points. Experiments were carried out under fluorescent light at 16 h light/8 h dark, 20 °C, and EPGs were initiated between 6 and 9 h after the start of the light phase in the controlled environment (CE) room. Waveforms were identified using the waveform guide supplied with the Giga 4/8 EPG systems manual (http://www.epgsystems.eu/files/aphid%20waveforms.pdf) as well as two studies investigating whitefly-specific waveforms (Lei and Tjallingii 1997; Lei et al. 1999). Output from the analysis of raw waveforms was converted into behavioural parameters using the spreadsheet outlined in Sarria et al. (2009).
Only female adults taken at random from the stock culture (mixed age) were monitored. The first experiment was a control in which nine pots filled with damp compost were placed in the volatile collection box (hereafter ‘air control’). In the second control experiment, volatiles were delivered from nine pre-flowering tomato plants (hereafter ‘tomato control’). This experiment controls for the ‘quantity’ of volatiles and is the control with which mixed host treatments (below) are compared. A difference between this control and mixed host treatments indicates that effects are due to quality and not the additional quantity of mixed host volatiles per se supplied to tomato leaves. In the third experiment, volatiles were delivered from three pre-flowering host plants of T. vaporariorum (Mound and Halsey 1978; Roditakis 1990; Gorman et al. 2002; Moreau and Isman 2011): cucumber, dwarf French bean and courgette (hereafter ‘volatile mixture 1’). In the fourth experiment, volatiles were delivered from another three pre-flowering host plants of T. vaporariorum (Mound and Halsey 1978; Roditakis 1990; Gorman et al. 2002; Moreau and Isman 2011): watercress, watermelon and Savoy cabbage (hereafter ‘volatile mixture 2’). The EPG recording lasted 15 h.
Feeding parameters from EPGs were generally not normal, so were plotted as medians with confidence intervals for the median (Zar 1998) (n ≈ 20). Within these data, proportional statistics were analysed using the chi-square test; otherwise, the Mann-Whitney U test was used. Planned statistical comparisons undertaken were as follows: mixed host volatile treatments were compared with the tomato control and each other, and the tomato control was also compared to the air control.
Settling behaviour, long-term honeydew deposition, performance, data analysis
To study settling behaviour of unconstrained individuals, these four sets of experiments were essentially repeated, but EPG probes were removed, the delivery box was sealed to prevent escape of whiteflies, and 150 whiteflies of both sexes taken at random from stock culture (mixed age) were inserted into the delivery box with tomato leaves (n = 8) still attached to plants and observed every 30 min over the next 6 h.
The volatile delivery chamber was set up as for EPGs except that eight tomato leafs were inserted into the chamber, and the chamber was made whitefly-tight using soft foam to plug petiole holes and fine gauze to cover other possible outlets. One hundred and fifty whiteflies of both sexes were taken at random from culture (hence mixed age) and split evenly between three 5-cm-diameter Petri dishes. Whiteflies in each of these dishes were anesthetised, and while still anesthetised, dishes were placed evenly spaced in the volatile delivery chamber, Petri dish lids were removed, and the chamber was sealed. Observations of the number of whiteflies on leafs were then undertaken every 30 min for 6 h. A note of the number individuals on each leaf was taken after 24 and 54 h, and the number of eggs on each leaf counted after 54 h. These procedures were repeated four times: once for the volatile mix 1 treatment (cucumber, dwarf French bean and courgette), once for the volatile mix 2 treatment (watercress, watermelon and Savoy cabbage) and once each for the controls. In each experiment, observations were begun 5–7 h after initiation of the light phase in the CE room. During settling experiments, we additionally placed aluminium foils of dimension 5 × 7 cm under each leaf to collect honeydew. Foils were weighed before use in experiments and 54 h after initiation of the settling experiments.
The data on numbers of whiteflies per leaf vs. time were analysed by fitting a cubic model (whiteflies per leaf = a + (b × time) + (c × time^2) + (d × time^3) and significant differences between settling parameters inferred from non-overlapping confidence intervals for model parameters. The 104 points (Fig. 3a) in each treatment were fitted using the Levenberg-Marquart method with an SS convergence point of 1E−8. Settling was also observed at 24 and 54 h and treatments compared using a small number of planned t-tests. The same planned comparisons as in EPG experiments above were undertaken. The number of eggs laid on each leaf after 54 h in settling experiments was analysed (n = 8), and again, the same planned comparisons as above undertaken using t-tests. The increase in weight of each foil (n = 8) was analysed using the same set of planned comparisons and t-tests.