Effects of soil-applied imidacloprid on Asian citrus psyllid (Hemiptera: Psyllidae) feeding behavior.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is one of the most important pests of citrus (Citrus spp.) because of its status as a vector of Candidatus Liberibacter asiaticus (Las), the bacterium associated with citrus greening disease. The use of insecticides for vector control is the primary method of managing the spread of this pathogen. Imidacloprid is an insecticide commonly applied to the root zone of young citrus trees to provide systemic protection from pests. The effects of imidacloprid on feeding behavior of D. citri have not been studied in much detail. The purpose of this study was to examine the effects of imidacloprid application on feeding behavior of D. citri and to determine whether use of this systemic insecticide could have any effect on pathogen transmission by D. citri. A direct current electrical penetration graph monitor was used to record D. citri feeding behaviors for 12-h periods on mature and young leaves of imidacloprid-treated and -untreated citrus seedlings. Overall, compared with untreated plants, the feeding behavior of D. citri was disrupted on imidacloprid-treated plants via reduction in the number of probes, as well as durations of average probes, initial stylet contact with phloem, phloem salivation, and phloem ingestion. The results of this study demonstrate that soil applications of imidacloprid can reduce the probability of citrus plants becoming inoculated with Las through a reduction in the number and duration of phloem salivation events by D. citri. Furthermore, Las acquisition from infected citrus is greatly reduced as a result of decreased phloem ingestion by D. citri on imidacloprid-treated plants.

Location of the mechanism of resistance to Amphorophora agathonica (Hemiptera: Aphididae) in red raspberry.

The aphid Amphorophora agathonica Hottes (Hemiptera: Aphididae) is an important virus vector in red (Rubus idaeus L.) and black (Rubus occidentalis L.) raspberries in North America. Raspberry resistance to A. agathonica in the form of a single dominant gene named Ag1 has been relied upon to help control aphid-transmitted plant viruses; however, the mechanism of resistance to the insect is poorly understood. Aphid feeding was monitored using an electrical penetration graph on the resistant red raspberry 'Tulameen' and compared with a susceptible control, 'Vintage'. There were no differences in pathway feeding behaviors of aphids as they moved toward the phloem. Once in the phloem, however, aphids feeding on resistant plants spent significantly more time salivating than on susceptible plants, and ingested significantly less phloem sap. This suggests that a mechanism for resistance to A. agathonica is located in the phloem. Reduced ingestion of phloem may result in inefficient acquisition of viruses and is a likely explanation for the lack of aphid-transmitted viruses in plantings of resistant cultivars.

Quantitative comparison of stylet penetration behaviors of glassy-winged sharpshooter on selected hosts.

New Zealand is threatened by invasion of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), an important vector of Xylella fastidiosa, a gram-negative bacterium that causes Pierce's disease in grape (Vitis spp.) and scorch diseases in many other horticultural crops. Therefore, an understanding of the host acceptability, feeding behavior, and potential vector efficiency of glassy-winged sharpshooter on New Zealand crops is important. We tested host plant acceptance and feeding behaviors of glassy-winged sharpshooter on three common horticultural crops grown in New Zealand (apple [Malus spp.], grape, and citrus [Citrus spp.]), and a native plant (Metrosideros excelsa [=tomentosa] Richard, pohutukawa), using the electrical penetration graph (EPG) technique. Probing (stylet penetration) behaviors varied among the host plants, primarily due to differences in waveform event durations. Apple and grape were the most accepted host plants, on which glassy-winged sharpshooter spent the majority of its time on the plant probing and readily located and accepted a xylem cell for ingestion. This resulted in long durations of sustained xylem fluid ingestion. In contrast, pohutukawa was the least accepted host. On this plant, glassy-winged sharpshooter spent less time probing and engaged in longer and more frequent testing/searching and xylem-testing activities, rejected xylem cells frequently, and spent less time with stylets resting, before accepting a xylem cell and ultimately performing the same amount of sustained ingestion. Citrus plants contaminated with sublethal insecticide residues were intermediate between these extremes, with some acceptance of xylem, but less ingestion, probably due to presumed partial paralysis of the cibarial muscles. Implications of the results in terms of host plant acceptance and the development of a stylet penetration index are discussed.

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae).

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.

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

Selected compounds were used to study physiological processes associated with digestion in the western tarnished plant bug, Lygus hesperus Knight. Durations of passage and rates of absorption, digestion, and excretion were determined for a digestible protein (casein), a non-digestible protein (green fluorescent protein, GFP), and a non-digestible carbohydrate (dextran). Dextran was used as a control to monitor the non-absorptive flow rate of ingesta through the digestive system. Fluorescent tracking of FITC-conjugates of casein and dextran, as well as immunoblotting and immunofluorescent staining of casein and GFP, were used to monitor the degradation (in vitro) and ingestion, digestion, and distribution (in vivo) of the respective compounds. Under our experimental conditions, L. hesperus took discrete meals, feeding and excreting at 2-3 h intervals. Rate of food passage was variable. FITC-dextran was found in the fecal material of most insects by 6-8 h after treatment initiation; by 12 h, 95% of ingested FITC-dextran was recovered from all insects. FITC-casein was digested extensively in in vitro homogenates of gut, hemolymph, and salivary gland. In vivo, FITC-casein was ingested and partially absorbed as a holoprotein into the hemolymph. Ingested FITC-casein was partially degraded in the gut and hemolymph within 2 h of ingestion, and no holoprotein was found after 12 h. In contrast, there was no detectable degradation of GFP in hemolymph, gut, and salivary gland homogenates after 24 h of incubation. Ingested GFP was not degraded in gut or hemolymph up to 8 h after treatment initiation, but did transfer to the hemolymph as a holoprotein. Analysis of immunohistological images confirmed that GFP bound to gut epithelial cell brush-border membranes. However, the mechanism by which GFP and casein pass as holoproteins into the hemolymph remains unknown.

Comparison of AC electronic monitoring and field data for estimating tolerance to Empoasca kraemeri (Homoptera: Cicadellidae) in common bean genotypes.

Two methods for estimating the tolerance of common bean genotypes to Empoasca kraemeri Ross & Moore were compared, using a yield trial carried out at Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, versus stylet penetration tactics measured by AC electronic feeding monitors. A stylet penetration index was devised based on principal component scores of three penetration tactics identified (pulsing laceration, cell rupturing, and lancing sap ingestion), combined with knowledge of the hopperburn symptoms caused by each tactic. Tolerant genotypes, as classified by the CIAT yield index, showed significantly more unprotected yield and lower hopperburn scores than the susceptible control. They also induced performance of less pulsing laceration (the tactic considered most damaging to the plant), and more of the other two, mitigating tactics, especially cell rupturing. When index values were calculated for each genotype, stylet penetration index values matched those of the yield index for three out of five genotypes: two EMP-coded tolerant lines ('EMP 385' and 'EMP 392') and the susceptible control 'BAT 41'. Thus, for these three genotypes, all subsequent hoppereburn symptoms are predictable by the type of feeding behavior performed on them. 'Porrillo Sintético' and 'EMP 84', considered borderline genotypes by the yield index, were overestimated and underestimated respectively, by the stylet penetration index. We postulate that, for these two genotypes, plant physiological responses to feeding (either compensatory or heightened sensitivity, respectively) synergize with type of feeding performed to generate the overall hopperburn condition. This multivariate analysis of electronic monitoring data was successfully used to devise an index of resistance. The implications of using the stylet penetration index and the advantages of using electronic monitoring in a bean-breeding program are discussed.

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

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.