Bioassays to Investigate the Effects of Insect Oviposition on a Plant’s Resistance to Herbivores

Materials and Reagents

1. Reaction tubes, 0.2 ml (e.g., Carl Roth GmbH + Co. KG, catalog number: H560.1 )
   
2. Reaction tubes, 1.5 ml or 2 ml (e.g., Carl Roth GmbH + Co. KG, catalog number: CK06.1 )
   
3. Nicotiana attenuata Torr. ex. Watson (Solanaceae), 4-5 weeks old (rosette stage)
   
4. Spodoptera exigua Hübner (Noctuidae, Lepidoptera) or Manduca sexta Linnaeus (Sphingidae, Lepidoptera)
   
5. Liquid nitrogen
   Note: All materials and equipment used is rather general lab equipment or houseware and we just referred to example products we used.
   

Equipment

1. General equipment
   
   1. A soft paint brush [e.g., Rotmarder 122A, size 2 (Habico, catalog number: 50122A10 )]
      
   2. Featherweight forceps (e.g., Carl Roth GmbH + Co. KG, catalog number: AN00.1 )
      
   3. Rearing boxes (e.g., 14 x 21 x 5 cm), lid with gauze (nylon mesh 0.12 mm width)
      
   4. Precision balance (e.g., Sartorius AG, model: Sartorius MC210S )
   
   
2. Oviposition on plants
   
   1. Flight cages [e.g., kweekkooi, 60 x 60 x 90 cm (Vermandel, catalog number: 80.304 ) or flexarium, 42 x 42 x 76 cm (Rolf C. Hagen Inc., Exo Terra, model: PT2552 )]
      Note: Flight cages eventually with slots at the cage sides through which defined leaves can be exposed and that can be closed with small claw hair clips.
      
   2. Hanging labels [e.g., Hängeetiketten, HERMA FACHSHOP, model: 6901 ]] or a thread
      
   3. Headlamp (e.g., Petzl, model: PIXA^® 3 )
      
   
   
3. Larval performance, feeding damage and haemolymph sampling
   
   1. Vented clip cages [e.g., made of two dressing cups (e.g., Ø 7.03, 2.3 cm (KIV-KREIS, catalog number: 770405509 )) with polyurethane foam at the rims, the bottom of one cup is replaced by gauze (nylon mesh 0.12 mm width) and the cage can be closed with small claw hair clips]
      
   2. Folding magnifier [e.g., TRIPLET 20x, 21 mm (Light In The Box Ltd., catalog number: 01239576 )]
      
   3. Laboratory support stand with clamps
   4. Whiteboard with reference areas (e.g., a laminated sheet of white paper with black squares of 1 x 1 cm)
      
   5. Camera [e.g., Canon EOS 1200D (Canon Inc., model: Canon EOS 1200D ) with EFS 18-25 mm macro lenses (Canon Inc., model: Canon EF-S 18-55mm f/3.5-5.6 IS II)]
4. Standardised damage and harvest of leaf tissue for analyses of plant defence traits
   
   1. Vacuum pump (e.g., Vacuubrand, model: PC 510 )
   2. Teflon tube [e.g., outer Ø 1.6 mm (VWR International, catalog number: BOHLS1810-01 )]
      
   3. Glass vial with rubber septum cap [e.g., 4 ml HPLC vial (Techlab, catalog number: FLK 101.243 and FLK 101.972 )]
      
   4. Tracing/Pattern wheel (e.g., Nähmit, catalog number: 070210 )
   5. 10 µl pipette
      
   6. Scalpel (e.g., Carl Roth GmbH + Co. KG, catalog number: X004.1 )
   7. Dissecting forceps (e.g., Carl Roth GmbH + Co. KG, catalog number: 2690.1 )
      

Procedure