Figure 1.
Studied insect community of B. nigra.
(A) Tritrophic system consisting of the Brassicaceae-specialist Pieris brassicae and the generalist moth Mamestra brassicae lay eggs in clusters on B. nigra. The egg parasitoid Trichogramma brassicae attacks eggs of both. The larval parasitoid Cotesia glomerata attacks young caterpillar stages of P. brassicae. (B) P. brassicae clutch on B. nigra expressing a strong necrotic zone, i.e. hypersensitive response (HR) (Photo credits: D. Lucas-Barbosa), (C) M. brassicae egg clutch on B. nigra without necrosis (Photo credits: Nina E. Fatouros, www.bugsinthepicture.com), (D) C. glomerata wasp on P. brassicae eggs parasitizing a neonate that just hatched (Photo credits: N. E. Fatouros, www.bugsinthepicture.com).
Figure 2.
Proportions (±SD) of female insects choosing oviposition-induced plant volatiles (OIPVs) of B. nigra plants.
Plants were infested with eggs of P. brassicae or M. brassicae. Columns represent arcsine of the proportion of choice for OIPVs by gravid P. brassicae females tested in a flight chamber (dark grey), T. brassicae egg parasitoids tested in a Y-tube olfactometer (light grey), and C. glomerata larval parasitoids tested in a windtunnel (white). All experiments were conducted in a two-choice situation between plants infested with eggs of different ages (24 h, 72 h, 96 h), and clean plants. The dashed line indicates arcsine (0.5) = no preference. Numbers below the columns represent the percentage of female insects making a choice. *P<0.05, one-sample t-test. Each treatment combination was replicated with at least four plant pairs. Different phenotypes: Hypersensitive response (HR), HR−: no necrotic zone observed, HR+: necrotic zone.
Table 1.
Volatile emissiona by Brassica nigra HR+ (+) or HR− (−) plants in response to eggs of Pieris brassicae (PB) and Mamestra brassicae (MB) sampled at 24 or 72 h after oviposition.
Figure 3.
Projection to Latent Structures Discriminant Analysis (PLS-DA) on the volatile compounds emitted by egg-infested B. nigra.
HR+ (+) or HR− (−) plants were infested by eggs of Pieris brassicae (PB) or Mamestra brassicae (MB) sampled 24 or 72 h after oviposition. (A) Score plot visualizing the grouping pattern of the samples according to the first two PLS components with the explained variance in brackets. The ellipse defines Hotelling’s T2 confidence region (95%). (B) Loading plot of the first two principal components shows the contribution of each of the compounds to the two PLS-DA components. Markers of the 5 different treatments shown in the score plot are given. Hypersensitive response type, (−): no necrotic zone observed, (+): with necrotic zone. Compound numbers: (1) 2-Methylpropanal, (2) 2-methyl-2-Propenal, (3) Ethyl acetate, (4) 2-Methyl-1-propanol, (5) Tetrahydrofuran, (6) 2-Butenal, (7) 3-Methylbutanal, (8) 2-Methylbutanal, (9) 1-Methoxy-2-propanol, (10) 1-Pentene-3-ol, (11) 2-Pentanone, (12) 3-Pentanone, (13) Methylcyclohexane, (14) Pyrazine, (15) 4-Methyl-2-pentanone (16) 1,2-Dimethyldisulfide, (17) (E)-2-Pentenal, (18) 2,4-Pentanedione, (19) 4-Methyl-3-pentene-2-one, (20) (Z)-3-Hexen-1-ol, (21) Allyl isothiocyanate, (22) α-Pinene, (23) 6-Methyl-2-heptanone, (24) (Z)-3-Hexen-1-yl acetate, (25) 3-Carene, (26) (S)-Limonene, (27) α-Phellandrene, (28) Methyl-2-ethylhexanoate, (29) (E)-β-Ocimene, (30) o-Cresol, (31) 2,2,6-Trimethyl-4-methylene-2H-pyran, (32) p-Mentha-1,5,8-triene, (33) 2-Phenylacetonitrile, (34) Isopulegon, (35) p-Menthan-3-one, (36) Isomenthone, (37) Menthol, (38) Undecan-2-one, (39) 7-α-H-Silphiperfol-5-ene, (40) Presilphiperfol-7-ene, (41) 7-β-H-Silphiperfol-5-ene, (42) 4-(3-cyclohexen-1-yl)-3-Buten-2-one, (43) Silphiperfol-6-ene, (44) α-Funebrene, (45) Longifolen, (46) 2-Phenylethyl isothiocyanate, (47) Lilial, (48) Guaiazulene, (49) 2-Ethylhexyl salicylate, (50) Cembrene. Significantly increased terpenoids in volatile blends of P. brassicae egg-infested plants compared to uninfested plants are in bold (*P<0.05, Mann-Whitney U-test).
Figure 4.
Cryo-SEM micrographs of B. nigra leaf surfaces and adhering herbivore eggs and egg – leaf contact regions.
(A–E) abaxial site of B. nigra leaves. (A) Eggs of P. brassicae 72 hao with surrounding leaf surface of HR+ B. nigra and trichomes. (B) Eggs of M. brassicae (48–60 h old) with surrounding leaf surface of HR− B. nigra. (C) Contact region after P. brassicae egg removal (72 hao) on HR− B. nigra consisting of accessory reproductive gland (ARG) secretion functioning as cement (arrow). (D) Contact region after M. brassicae egg removal consisting of a part of ARG cement and healthy leaf cells with open stomata (arrow). (E) Necrotic zone on HR+ B. nigra leaf induced by P. brassicae eggs 72 hao, with some eggs removed. ARG-derived cement is removed together with parts of the cell layer (arrow) and open stomata at the border between dead and living cells. (F) Adaxial site of necrotic zone of HR+ B. nigra with open stomata (arrow) between healthy cells and necrotic zone on right side. Hypersensitive response type, HR−: no necrotic zone observed, HR+: with necrotic zone. Scale bars are given in lower right corners.
Figure 5.
Overview of the Y-tube olfactometer with simultaneous volatile trapping.
Wasps were released in groups and collected in insect trapping collection bulbs. Volatiles were trapped simultaneously or after a bioassay with Tenax TA tubes. Illustration credits: I. Figueroa.