Subcellular effects and localization of binding sites of phytohemagglutinin in the potato leafhopper, Empoasca fabae (Insecta: Homoptera: Cicadellidae)

Abstract

To identify the means by which phytohemagglutinin (PHA) exerts its toxicity on the potato leafhopper, four different methods (thick and semi-thin sectioning combined with immunofluorescent staining, in vitro receptor autoradiography, and immunoelectron microscopy) were used to elucidate the PHA target tissue, binding site, and its effects on this tissue. Sixteen 1- or 2-day-old female potato leafhoppers were fed for 36 h on each of three treatments: a control, diet or a diet containing either the PHA-E subunit or the PHA-L subunit. The PHA-E subunit, but not PHA-L, had previously been shown to be lethal. The insects were then prepared for both light and confocal microscopy. Analysis of images showed that PHA bound only to the surface of midgut epithelial cells of the potato leafhopper. PHA-E caused severe disruption, disorganization, and elongation of the brush border microvilli, and swelling of the epithelial cells into the lumen of the gut, leading to complete closure of the lumen. Furthermore, PHA-E stimulated the division of midgut epithelial cell nuclei, leading to two nuclei in each cell. Nuclei later elongated and degraded. In contrast, PHA-L had little effect on the epithelial cells of the midgut. It did not strongly bind to the surface of epithelial cells and caused much less disruption of brush-border microvilli, less disorganization of the cells and less elongation of nuclei. Strong binding of PHA occurred solely on the cell membrane of the brush border microvilli of epithelial cells. In contrast, the controls (i.e., midgut tissue, blocking agent, PHA, and antibodies) showed that midgut tissue was not autofluorescent and showed no fluorescent binding signal. Analysis of both bright- and dark-field images obtained by autoradiography and immunoelectron microscopy confirmed these findings.

Effect of the lectin PHA on the feeding behavior of the grain aphid

“…The current study did not measure the effects of PHA on insect development and fecundity. Other studies, however, have demonstrated that PHA can reduce the growth and fecundity and can increase the mortality of herbivorous insects (Gatehouse et al 1984;Rahbe et al 1995;Habibi et al 1998;Machuka et al 1999;Sadeghi et al 2006;Sprawka and Leszczyński 2007). The results presented here suggest that the negative effects of PHA on herbivorous insects, and especially aphids, could be a consequence of shortening or suppressing of feeding processes.…”

“…Studies have also shown the toxic effect in vitro or in planta of lectins to different species of Hemiptera: leafhoppers, planthoppers, and aphids (Habibi et al 1993;Powell 1993Powell , 2001Powell et al 1993Powell et al , 1995Powell et al , 1998Rahbe and Febway 1993;Rahbe et al 1995;Sauvion et al 1996). Previous studies on a number of economically important pests have indicated that the kidney bean lectin (Phaseolus vulgaris agglutinin) phytohemagglutinin (PHA) had the most interesting toxic effects of all classes of lectins tested (Fitches et al 2001;Habibi et al 1998Habibi et al , 2000Machuka et al 1999;Rahbe et al 1995;Sadeghi et al 2006;Sprawka and Leszczyński 2007). PHA is tetrameric protein with a molecular weight of 120 kDa and sugar specificity for D-galactose/N-acetyl-D-galactosamine residue.…”

Uptake, flow, and digestion of casein and green fluorescent protein in the digestive system of Lygus hesperus Knight

“…Tissues were prepared at the 2-, 4-, and 8-h time points (Habibi et al, 1998(Habibi et al, , 2000. The control and treatment insects were fixed in 3% paraformaldehyde, dehydrated in a series of ethanol dilutions, infiltrated with low melting paraplast, and embedded in high melting paraplast.…”

Lectins and Their Roles in Pests Control

“…In general, the action mechanism of the lectin at cellular level of ingested lectin by insects showed that binding of the lectin to the midgut tract causing disruption of the epithelial cells including elongation of the striated border microvilli, swelling of the epithelial cells into the lumen of the gut lead to complete closure of the lumen, permeability of cell membrane to allow the harmful substances penetrations from lumen towards haemolymph and impaired nutrient assimilation by cells, allowing absorption of potentially harmful substances from lumen into circulatory system, fat bodies, ovarioles and throughout the haemolymph (Gatehouse et al, 1984;Powell et al, 1998;Habibi et al, 1998;2000;Fitches et al, 1998;2001b;Sauvion et al, 2004;Majumder et al, 2005). This information gave further support to previous suggestions that the XCL lectins disrupt midgut cells (Francis et al, 2003;Karimi et al, 2008Karimi et al, , 2009).…”