Buenos Aires, sede de congresos y seminarios

This trial was conducted on plants of two distinct plant genotypes (N and S) and one of four endophyte statuses [in symbiosis with one of three endophyte strains (AR1, AR37 or common-toxic CT), or endophyte-free (NIL)] (‘clone-plants’; Section 2.3.1.1) as represented in Figure 3.1. In five batches of 6 tillers/batch, a total of 30 tillers per plant genotype-endophyte status (PG-E) combination was trimmed back to 4 cm shoot length and 0.3 cm root length without adventitious side roots. The trimmed tillers were weighed and one tiller from each batch sacrificed to estimate the dry matter content of the others. Each of the remaining five tillers of a batch was set into 20 to 30 mL tap water (in a glass vial of 7.6 cm length and 2.4 cm diameter) and left to recover for two weeks in the Conviron® climate chamber 2 (Section 2.2.4) set to a photoperiod of 14 h light/10 h dark and an air temperature of 17 °C. The water in the tubes was changed every 24 to 48 h. The recovered plants were individually embedded in unvented Petri dishes filled with ca.

45 to 50 g modified Bollard medium (MBM) and a moist, autoclaved cotton ball was placed on the tiller base and roots (Section 2.3.1.3). These Petri dishes were sealed with GLAD® wrap, each one wrapped into one black polyethylene sheet and a panda sheet (white side out), tied together in groups of two plants and placed in an upright position on a hygiene tray in the climate chamber, in a random order. To further standardise the conditions over the experiment, all plants were systematically rotated, moving them once a day by four positions within the trays. The plants were left in the climate chamber until the plant precondition assessment, immediately before aphid placement (≥ 30 days after the initial plant trim), returned after placement, and removed periodically for checks on the root aphids until aphid death (see below). The temperature in the wrapped root area averaged 17.5 ± 2.4 °C (mean ± standard deviation).

The genitors of the neonates that were to be placed into the Petri dishes (‘starter aphids’) had been kept for at least 3 generations in conditions similar to what was just described (climate chambers set to 14 h light/10 h dark, air temperature of 17 to 18 °C, on clone-plants embedded in MBM agar) and were, whenever possible, collected from colonies on plants of the same PG-E group as the one they were transferred to. They were maintained in the climate chamber for approx. 12 to 18 h, in 1.5 to 2 mL microcentrifuge tubes in light-proof wrapping (Section 2.3.2). The offspring viviposited in that time were transferred to a new microcentrifuge tube until placement (> 6 h after transfer, but before the end of the neonates’ third day of life). The neonate starter aphids were photographed before being moved with a fine paintbrush onto the plants’ roots, at rates of one or five neonates per Petri dish (‘solitary’ vs. ‘group’ setting; n = 5 to 13 Petri dishes per PG-E setting). Solitary starter aphids that died before maturity or were killed by handling were replaced by one new neonate. This resulted in the placement of 206 solitary starter aphids in 82 Petri dishes over the complete Biology II experiment.

Aphid handling was carried out in a dark room, under a 6-W white LED light (Austrabeam/stylelux, model L3-857391/PLU7080; Mercator Lighting Pty Ltd, Coolaroo, Australia) and a Zeiss stereo microscope (Stemi SV6; Carl Zeiss AG, Oberkochen, Germany) at 50x magnification. Photographs were taken with a digital camera (Canon PowerShot A620, Canon Inc., Tokyo, Japan) fitted to the microscope, with the flash function turned off.

(i) First two weeks: checks once a week (aphid age: 7 to 9 and 14 to 15 days, respectively). Survival and activity at the check were recorded with a site description if the aphid was feeding (see Chapter 5). A fine mist of previously autoclaved tap water at room temperature was sprayed over the aphids to remove the wax. Living aphids were triply photographed.

(ii) Subsequently: the aphids were monitored every second day up to death, recording their survival, activity (Section 5.2.1), a site description if feeding (Section 5.2.1) and the number of offspring they had produced since last checked. Three photographs were taken at the appearance of the first offspring to determine the mature adult size, and several more times thereafter if possible. All offspring were removed from the Petri dishes, counted, and triply photographed. First instar aphids were then placed together in one

microcentrifuge tube per Petri dish (= a cohort) and used for follow-up

experiments (Section 3.2.1.4). Older instars were discarded.

Approx. 0.8 g water was sprayed onto the agar to prevent water stress at each check if the agar filled ≤ ¼ of the Petri dish. Once an aphid had completed a full life span (i.e. achieved maturity and died a natural death on its plant), the plant it lived on was destructively harvested. The plant was divided into green shoots, senescing and dead shoots, and roots, weighed fresh, freeze-dried for ≥ 48 h and finally re-weighed for dry matter measurement. All green tillers were blotted for confirmation of endophyte presence before freeze-drying (Section 2.3.3).

90

[Age of plant] Start: Trim and weigh tillers, keep them in water [ 1]

24 h Recovery: Photograph tillers 24 h after trim _{[ 2]}

RECOVERY

(in water)

Embedding: Tillers mounted into agar [15-25]

RECOVERY (in agar)

BIOLOGY II

(CC2, 18 ºC, 14/10 h L/D)

[Age of plant] Precondition assessment + root aphid placement:

 Precondition: tiller + leaf count, root system ranking

 Photographs for green shoot area (GSA) + colour

[22-38] [1] Start: Trim and weigh tillers, keep them in water RECOVERY

(in water)

[16-22] Embedding: Tillers mounted into agar (1/PD)

APHID

OCCUPATION RECOVERY (in agar)

Root aphid harvest + final plant harvest:

Harvest aphids, divide them into 1st_{I/OI/Ad and count}

Tiller, green and senescent and dead leaf count

Tiller dissection + immunoblot on tiller base

 Fresh and dry matter weighing of plant material

Water

Stress [60-140] [31-258] _{APHID ESTA-}

BLISHMENT +DEVELOP-

Precondition assessment + root aphid placement:

 Precondition: tiller, green and senescent and dead leaf count, water stress

Root aphid checks + replacement (if killed/died before maturity):

Checks: 1x/week in first 2 weeks, then 1x/2-3 days

Photographs, survival,behaviour and root use records

MENT + REPRO-

Offspring harvest BIOLOGY II FOLLOW-UP

(CC1, 18 ºC, no light)

DUCTION

Water Stress

Root aphid transfer

1 cohort/ micro- centrifuge tube

[63-322, at aphid

death]

Final plant harvest:

Tiller dissection + immunoblot

Fresh and dry matter weighing of plant material

Root aphid checks

1x/2 days

Survival

Figure 3.1. Diagram of experimental set up for Population experiment, Biology II experiment and Biology II follow-up experiment. 1st_{I/OI/Ad: first} instar, older immatures, adults; The age of a plant [Age of plant] at the events is given in days; CC1, CC2: climate chamber 1, 2 (Section 2.2.4); GH9: glasshouse 9 (Section 2.2.3); L:D: light/dark; PD: Petri dish (i.e. plant sample); photographs taken.

M ot he r M ot he r M ot he r M ot he r C O L O N IE S 0, 1, or 5 neonates/PD M ot he r M ot he r (0), 1, or 5 neonates/PD M ot he r Offspring cohort