CHAPTER 2: GENERAL MATERIALS AND METHODS
2.1. Experimental overview
This thesis reports the results of four pre-trials (Table 2.1), four experiments and one observation on mature plants, one experiment on seedlings and two follow-up experiments ex planta (Table 2.2). The overviews present the experiments in their execution order. To ease comparisons and discussions, the information collected during these experiments was grouped by topic into the three experimental chapters, however. All aspects related to aphid biology, plant impact and ecology or plant use were reported in Chapters 3, 4, and 5, and some experiments with multiple foci (e.g. Biology II experiment) are consequently mentioned in more than one of these chapters.
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Time Pre-trial Focus
(Result section) Experimental specifics (Location1, conditions) Plants1 endophyte statuses Aphids
June 2012 Calibration trial I
Body size changes by preservation (Appendix 9.2)
-
processed in laboratory - Root aphids of all ages collected outdoors on endophyte-free L. perenne plants of unknown cultivar
January
2013 Viviposition trial Reproduction and survival of adults ex planta (Appendix 7)
Climate chamber 1
17-20 ºC, 14 h light/day
REPEATED CHECKS2
In empty glass tubes
ex planta Adult root aphids collected from colonies living in the glasshouse, on endophyte-free L. perenne plants of unknown cultivar
April –
May 2016 measurements Instar
Size of various instars (Appendix 9.1)
Climate chamber 1
17 °C, 14 h light/day Clone-plants of genotype N and S
AR1, AR37, CT or NIL
Up to 10 neonates per Petri dish (i.e. plant)
Source: mothers kept on clone-plants at climate chamber conditions (17 ºC, 14 h light/day) for ≥ 6 generations June 2016 Calibration trial
II
Relation between body size and weight
(Appendix 9.3)
-
processed in laboratory - Adult root aphids collected in the glasshouse, from AR1-infected L. perenne plants (unnamed cultivar)
1 See Sections 2.2 (location) and 2.3.1 (plants) for more specifics; AR1, AR37, CT: infected with AR1, AR37 or common-toxic endophyte strain, respectively; NIL: endophyte-free (Section 2.3.3)
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Table 2.2. Overview of the experiments reported in Chapters 3, 4, 5.
(D ec 2 01 5 – Ja n 20 16
) Seedling Plant response to root aphids (4.3.1) Percival climate chamber 19 ºC, 12 h light/day, in open 50 mL centrifuge tubes 64 genotypes-AR1 75 genotypes-AR37 53 genotypes-CT 51 genotypes-NIL
0 or 3 neonates per tube (i.e. seedling) Source: mothers collected on endophyte-free
L. perenne plants in the outdoor area (Section 2.2.1)
1 See Sections 2.2 (location) and 2.3.1 (plants) for more specifics; AR1, AR37, CT: infected with AR1, AR37 or common-toxic endophyte strain, respectively; NIL: endophyte- free (Section 2.3.3);
2 REPEATED CHECKS: aphids repeatedly monitored (if not mentioned, aphids were observed/counted once only, at harvest)
Time Experiment Focus
(Result section) Experimental specifics
1
(location, conditions) Plants
1
endophyte statuses Aphids
(A ug – O ct 20 12
) Biology I Root aphid biology (3.3.1) 21 ºC, natural photoperiod, Glasshouse 9, cubicle 12 in Petri dishes, REPEATED
CHECKS
Source plants
(16 genotypes)
NIL
1 neonate per Petri dish (i.e. plant) Source: mothers from colonies kept in
glasshouse 9 on random source plants for ≥ 2 generations (S ep 2 01 3 – Ja n 2 01
4) Population Population development (5.3.4) Conviron climate chamber 1 18 ºC, 14 h light/day,
in Petri dishes
Clone-plants
(2 genotypes, N and S)
AR1, AR37, CT or NIL
0, 1 or 5 neonates per Petri dish (i.e. plant) Source: mothers from colonies kept in climate
chamber on clone-plants for ≥ 2 generations
(O ct 2 01 3 – Ju l 20 14 )
Biology II Root aphid biology (3.3.2) Root aphid behaviour (5.3.1) Root aphid root use (5.3.2)
Conviron climate chamber 2
18 ºC, 14 h light/day, in Petri dishes, REPEATED CHECKS
Clone-plants
(2 genotypes, N and S)
AR1, AR37, CT or NIL
1 or 5 neonates per Petri dish (i.e. plant) Source: mothers from Population experiment
and colonies kept in climate chamber on clone-plants for ≥ 3 generations
Biology II
follow-up Offspring survival (3.3.4) 18 ºC, no light, in microcentrifuge Conviron climate chamber 1 tubes, REPEATED CHECKS
Ex planta Cohorts of neonates (siblings of 2-3 days)
Source: mothers of the Biology II experiment
(J un – O ct 2 01
5) Mature plant Plant reaction to root aphids (4.3.2) Root aphid reproduction ex planta (3.3.3) Insectary 10 ºC, natural photoperiod, in Petri dishes Clone-plants (2 genotypes, N and S)
AR1, AR37, CT or NIL
0 or 10 neonates per Petri dish (i.e. plant) Source: mothers from colonies kept in
insectary on clone-plants for 1 generation Mature plant
follow-up Offspring survival (3.3.4) 20 ºC, no light, in microcentrifuge Laboratory tubes, REPEATED CHECKS
Ex planta Cohorts of neonates (siblings of 1 day)
Source: mothers of the Mature plant experiment (S ep – O ct 20 15 ) Colony wax observations
Colony wax description (5.3.3) Colony description (5.3.3) Root aphid root use (5.3.3)
Conviron climate chamber 2
18 ºC, 14 h light/day, in Petri dishes (2 genotypes, N and S) Clone-plants
AR1, AR37, CT or NIL
10 neonates per Petri dish (i.e. plant)
Source: mothers from colonies kept in climate chamber on clone-plants for ≥ 2 generations
2.2. Facilities
Seven distinct locations at the Grasslands Research Centre, AgResearch Limited, Palmerston North, New Zealand, were used to carry out experiments, keep plants and maintain aphid colonies for the research reported in thesis: (i) an outdoor area within the Grasslands Research Centre’s nursery area, (ii) an insectary in the plant nursery, (iii) two glasshouses (glasshouse 18 and cubicle 12 of glasshouse 9), and (iv) three climate chambers (Conviron climate chambers 1 and 2, Percival climate chamber; Figure 2.1). The general settings and peculiarities of each location are summarised in Sections 2.2.1 to 2.2.4. Their use in various experiments is reported in Tables 2.1 and 2.2 (Section 2.1). In the unshaded centre of the plant nursery, a reference weather station (Section 2.4.1) was installed. This station was used to characterise the climatic conditions in the outdoors sites (Appendix 3). This station was also used in combination with a mobile light measuring device (Section 2.4.1) to estimate and compare the light intensities in all location (Appendix 4).
2.2.1. Outdoor area
This location was used to store a backup of source plants and clone-plants (Section 2.3.1). The plants in this location were naturally colonised by root aphids and served therefore as a source of root aphids for the Seedling experiment and for the setting up of colonies. The outdoor area consisted of a concrete pad within the plant nursery (Figure 2.1a). As it was partly shaded by a hedge, the light intensity in this location was slightly lower than the light intensity measured at the reference weather station (96% of the average reference value of 614 µmoles photons∙m-2∙s-1 [LRef]; Appendix 4). The other weather parameters were similar, however (local weather conditions, Appendix 3). To avoid water stress, the plants were watered daily from the top via a sprinkler. No pesticide was ever applied to the plants kept in this location.
Figure 2.1. Research locations. (a) Outdoor area; (b) Insectary (outdoors, covered with insect screen); (c) Glasshouse 18; (d) Glasshouse 9; (e) Conviron climate chamber 2 with top illumination; (f) Percival climate chamber with top and side illumination.
(d)
(c)
(b)
(e) (f)
2.2.2. Insectary
The insectary was used to perform the Mature plant experiment (Sections 3.2.1.3 and 4.2.2). It consisted of a walk-in pollination cage (2.8 m in length × 1.9 m in width × 1.85 m in height covered by dark insect screen with fine apertures of 1.3 × 1.1 mm; Figure 2.1b) fixed to the concrete pad of the plant nursery, close to a hedge. The plants were kept therein 79 cm above the ground, on temporary benches. The average light intensity in the insectary was approximately 21% of LRef (the solar radiation measured outside at the same time; Appendix 4). There were probably slight differences in the air temperature and humidity measured inside and outside (reference weather station; Appendix 4) of the insectary, but these differences were unlikely to have had any biological consequences for experiments. The plants were neither watered nor sprayed with any pesticide in this location.
2.2.3. Glasshouses
Glasshouse 18 (ca. 18 m in length × 11 m in width with 6.4 m height at its summit; with twin skin polythene cladding; Figure 2.1c) was used to raise source plants from March 2012 to August 2012 and to maintain aphid-free clone-plants from March 2012 to January 2015 (Section 2.3.1). The plants were kept 0.9 m above ground, on a bench with a capillary mat (Transportect Ltd, Auckland, New Zealand) covered by a black woven weed control mat (Cosio Industries Ltd, Auckland, New Zealand). Light deflection by the cladding and shadows cast by surrounding structures resulted in the plants being exposed to a natural photoperiod length (Appendix 3) at reduced light intensity in this location (56% of the outdoors measured LRef intensity, Appendix 4). Water was provided by an automatic watering system embedded in the capillary mat and additional watering from the top with a hose as necessary. The climate within the glasshouse was regulated by a general control system in response to air temperature and humidity conditions monitored within the glasshouse. At plant level, the mean relative humidity was 76 ± 17.2% and mean temperature of 23 ± 6.4 °C from 16/01/2013 to 25/02/2013. From the 22/06/2012 onwards, all plants in this glasshouse were subject to a biweekly pest control spraying programme of either Orthene® (Arysta Life Science Ltd., Cary, NC, U.S.A.; 0.8 g/L), Nuvos® (Orion AgriScience Ltd, Christchurch, New Zealand; 0.6 mL/L) or Mavrik® aquaflo (ADAMA New Zealand Ltd, Nelson, New Zealand; 0.4 mL/L). These products
were mixed with DC-Tron® mineral oil (Caltex Australia Petroleum Pty Ltd, Sydney, Australia; 10 mL/L) to enhance the products’ adherence to plant surfaces. Occasionally, other compounds and preparations were also used (Appendix 5). Treated plants were not used to set up any experiments or colonies for at least double of each pesticide’s recommended withholding period after an application.
Glasshouse 9 was a small glasshouse clad by twin skin polythene and subdivided into small cubicles of 2.5 m in width × 3.96 m in length × 2.16 m (outer side) to 3.65 m (inner side) in height, equipped with 90 cm high benches (Figure 2.1d). Its cubicle 12 was used to grow source and clone-plants (Section 2.3.1), keep aphid colonies and perform the Biology I experiment (Section 3.2.1.1). This cubicle was oriented eastward and provided the plants with a natural photoperiod. The average light intensity in it was about 28% of the LRef, however. A general climate control system was set to maintain average air temperatures of 10 to 25 °C and an average relative humidity over 60%, but the measured temperature and relative humidity on the benches at plant level varied between 4.0 and 39.2 °C and 17.5 to 100%, respectively. The plants grown in potting mix (Appendix 5) in this cubicle were also kept on a water holding mat covered with a black woven weed mat (see above) and were watered from the top every other day with a hand- held hose. Mavrik® aquaflo (0.4 mL/L)/DC-Tron® (10 mL/L), Orthene® (0.8 g/L)/DC- Tron® (10 mL/L) or Nuvos® (0.6 mL/L)/DC-Tron® (10 mL/L) mixture were sprayed for pest control as required without compromising experiments (treated plants were not used in experiments or as colony plants for at least double the recommended withholding period). In two instances, Diazinon® 20G (Nufarm Ltd., Auckland, New Zealand) granules were also sprinkled over the potted plants to control porina caterpillars, at rates of approximately 10 to 15 granules/pot.
2.2.4. Climate chambers
Three different climate chambers of two distinct types (Conviron and Percival) were used. The two Conviron growth cabinets (Conviron® CMP 3023, Winnipeg, Canada; e.g. Figure 2.1e) were used to maintain aphid colonies and to conduct several aphid biology experiments (e.g. population and Biology II experiments, Sections 5.2.3 and 3.2.1.2). Illumination was provided by eight Philips cool daylight triphosphor tubes 36W/865 and four Philips Softone 100 W incandescent lamps installed in the ceiling of
each chamber, which provided a light of approx. 230 µmoles photons∙m-2∙s-1, i.e. about 38% of the average intensity measured outdoors during daytime hours (Appendix 4). Both chambers were set to maintain reference conditions of 17°C [the optimal growth temperature for perennial ryegrass roots according to Robin (2011)] and 14 h of light per day [14:10 h light/dark, a photoperiod expected to maximize shoot growth under artificial light; personal communication by Alison Popay]. The relative air humidity in the chambers was approximately 60%.
The Percival climate chamber (Model I-35LLVL, Boone, U.S.A.; Figure 2.1f) was used for the Seedling experiment only (Section 4.2.1). Illumination was provided by two short cool daylight tubes (Philips TLD 18W/865) fixed horizontally to the chamber’s ceiling, and four cool daylight tubes (Philips TLD 36W/865) arranged laterally (from the ceiling to the floor of the chamber), which provided a total light intensity of 70 µmoles photons∙m-2∙s-1 on the top shelf (11% of LRef, the average intensity measured outdoors). Following recommendations for seedling germination tests (Ellis et al., 1985), this chamber was set to maintain a photoperiod of 12 h light per day and temperatures of 19 to 20 °C. A relative humidity of 88.0 ± 4.6% was measured in it during the Seedling experiment.
As the plants kept in the climate chambers were rooting in agar, they were not watered unless the agar was close to drying out (see Biology II and Population experiments, Sections 3.2.1.2 and 5.2.3 for specifics in this case). No pesticide was ever applied to any plant in the climate chambers.