Digestive activity and organic compounds of Nezara viridula watery saliva induce defensive soybean seed responses

Abstract

The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.

Figure 1

Scanning electron microscopy of mouthparts of an adult specimen of Nezara viridula. (A) anterior view of the head of the stink bug; (B) beak and mandible tip; (C) enlarged view of the mandible tip; (D) lateral view of the head with details of: (a) central labrum, (b) stylets, (c) first labial segment; (E) detailed stylet fascicle showing: (a) last segment of the beak, (b) mandibles, (c) maxillary stylets; (F) detail of teeth in one mandibular stylet.

Figure 2

Damage produced in soybean developing seed’s structure by herbivory. SEM comparison of the damage produced in the developing fresh tissue by: (A) the puncture of a needle, mimicking Nezara’s stylet, and (B) the damage produced by the insect’s feeding activity; (C,D) offer a detailed view of the wounded area by mechanical damage and the insect, respectively.

Figure 3

Soybean tissue response to Nezara viridula feeding activity. Acid fuchsine staining of damaged pods: (A) mechanical damage, and (B) attacked by the stink bug, an inset of the pod surface is shown to point the jellified saliva sheath left on the surface. Soybean stained cotyledons showing in (A,E) mechanical damage, (D,F) damage produced after insect injecting saliva. Light photomicrographs and histological analysis of cross sections of (G) mechanical damaged seed and (H) attacked by the insect. Same sections stained with safranin showing (I) auto-fluorescence and (J) bright lignified cells in the puncture site and a siege marked with an asterisk.

Figure 4

NMR analysis of N. viridula saliva. Stink bug’s watery oral secretion from a group of 200 adults was collected under laboratory conditions and subjected to NMR analysis. (A) 600 MHz proton spectra of watery saliva. An enlarged view of signals at 4 and 1 ppm is shown. (B) Representative 2-dimensional ¹H–¹³C HSQC NMR spectrum of oral secretion. Complete ¹H and ¹³C chemical shift assignments are listed in Table 1.

Figure 5

Proteomic identification of the components in the stink bugs saliva. To identify proteins in the watery saliva, samples were digested and Mass Spectrometry (MS) analyses were performed. Functional annotation and relative abundance of enzymes identified from stinks bug saliva proteome are shown. Only high confidence peptide matches with a maximum protein and peptide false discovery rate of 1% were selected through a reverse database approach.

Figure 6

Zymogram analysis of several enzymes expressed in Nezara´s oral secretion. SDS-PAGE gel with 1% pectin co-polymerized as a substrate was used to test for pectinolytic activity, OS, corresponds to the oral secretion sample and C, to the enzyme pectinase from Aspergillus niger (Sigma) used as a positive control. For amylolytic activity the gel was co-polymerized with 0.5% starch, α-amylase from Aspergillus oryzae (Sigma) was included as positive control. For peroxidase activity a native gel was used and 10 µg of protein from soybean seeds extract was included as positive control. Protease activity was detected in a gelatin co-polymerized native gel, and bovine trypsin (Sigma) was included as positive control. A pre-stained molecular weight marker was used (MM, Kaleidoscope, Bio-Rad). One of three independent experiments is shown for each enzyme tested.