The Neotropical brown stink bug, Euschistus heros (F.), feeds on stems, leaves, pods, and seeds of soybean, Glycine max (L.) Merrill. Knowledge of the damage that nymphs at different instars can cause to soybean pods and seeds, as well as efficient histological techniques for locating the salivary sheath are sparse. This study developed a new double-staining method to facilitate distinguishing the salivary sheath from plant tissues and to anatomically evaluate the damage caused by nymphs of different instars as they feed on soybean pods and seeds. Five insects from each of the analyzed instars (1st, 2nd, 3rd, 4th, and 5th) per pod at the R6 stage (full pod-filling) were kept in clip cages for 48 h of feeding. The salivary sheath was analyzed to localize the damage (pod, vascular bundle, and seed) and the depth reached by the damage (categorized tissue). Double staining with xylidine ponceau and toluidine blue provided the best differentiation between the salivary sheath and watery sheath (proteins stained red) and the plant tissues (stained blue). First instar nymphs do not feed. Second instar and older nymphs caused damage to seeds, which became more severe with later developmental stages. The damage consists of coalescence of protein bodies and degradation and breakdown of the cell wall, marked by darkened regions in the embryo tissue of seeds. The information generated will contribute to new studies on feeding habits and emphasizes the need to control E. heros in early development stages.
Keywords: Anatomy; Double staining; Glycine max (L.); Salivary sheath; Stink bug; Toluidine blue; Xylidine ponceau.
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