Jonathan J. Neal

Antioxidant defense response in a galling insect

Herbivorous insect species are constantly challenged with reactive oxygen species (ROS) generated from endogenous and exogenous sources. ROS produced within insects because of stress and prooxidant allelochemicals produced by host plants in response to herbivory require a complex mode of antioxidant defense during insect/plant interactions. Some insect herbivores have a midgut-based defense against the suite of ROS encountered. Because the Hessian fly (Mayetiola destructor) is the major insect pest of wheat worldwide, and an emerging model for all gall midges, we investigated its antioxidant responses during interaction with its host plant. Quantitative data for two phospholipid glutathione peroxidases (MdesPHGPX-1 and MdesPHGPX-2), two catalases (MdesCAT-1 and MdesCAT-2), and two superoxide dismutases (MdesSOD-1 and MdesSOD-2) revealed high levels of all of the mRNAs in the midgut of larvae on susceptible wheat (compatible interaction). During development of the Hessian fly on susceptible wheat, a differential expression pattern was observed for all six genes. Analysis of larvae on resistant wheat (incompatible interaction) compared with larvae on susceptible wheat showed increased levels of mRNAs in larvae on resistant wheat for all of the antioxidant genes except MdesSOD-1 and MdesSOD-2. We postulate that the increased mRNA levels of MdesPHGPX-1, MdesPHGPX-2, MdesCAT-1, and MdesCAT-2 reflect responses to ROS encountered by larvae while feeding on resistant wheat seedlings and/or ROS generated endogenously in larvae because of stress/starvation. These results provide an opportunity to understand the cooperative antioxidant defense responses in the Hessian fly/wheat interaction and may be applicable to other insect/plant interactions.

Tissue and life stage specificity of glutathione S-transferase expression in the Hessian fly, Mayetiola destructor: Implications for resistance to host allelochemicals

Abstract Two new Delta and Sigma glutathione S-transferases (GSTs) in the Hessian fly, Mayetiola destructor (Diptera: Cecidomyiidae), were characterized and transcription profiles described. The deduced amino acid sequences for the two M. destructor Delta GSTs (MdesGST-1 and MdesGST-3) showed high similarity with other insect Delta GSTs including the conserved catalytic serine residue. The deduced amino acid sequence for the M. destructor Sigma GST (MdesGST-2) showed high similarity with other insect Sigma GSTs including the conserved glutathione and substrate binding sites. Quantitative tissue expression analysis showed that mRNA levels for MdesGST-1 were predominant in fat body, whereas for MdesGST-2 and MdesGST-3 expression was predominant in the midgut. Temporal expression during development showed peak mRNA levels for MdesGST-1 during larval development, but in the pupal stage for MdesGST-2. MdesGST-3 showed a constitutive expression pattern throughout development. M. destructor feeds on wheat, and expression analysis after feeding indicated that mRNA levels for MdesGST-1 were significantly higher in incompatible interactions in which larvae fed on resistant wheat, while MdesGST-3 was significantly higher in compatible interactions when larvae fed on susceptible wheat. MdesGST-2 showed an equivalent expression pattern during both interactions. These results suggest that the M. destructor Delta GSTs are important in detoxifying wheat allelochemicals during feeding, while Sigma GST participates in metabolism of endogenous substrates.

Cytochrome P450 purification and immunological detection in an insecticide resistant strain of German cockroach (Blattella germanica, L.).

A German cockroach strain, Munsyana (MA) had 80-fold resistance to the pyrethroid insecticide cypermethrin, 4.5-fold greater total cytochrome P450 content and 2.5-fold greater cytochrome P450-mediated N-demethylation of 4-chloro-N-methylaniline compared to the susceptible Johnson Wax (JWax) strain. Immobilized artificial membrane high performance liquid chromatography (IAM-HPLC) of microsomal proteins from the MA strain enriched cytochrome P450 greater than 70-fold. Following purification, a single protein band of M(r) = 49,000 (P450 MA), was detected by silver-staining SDS PAGE gels. Antiserum to the purified protein from the MA strain (anti-P450 MA) was produced in mice. Anti-P450 MA inhibited cytochrome P450-mediated N-demethylation by 4-fold in both MA and JWax strains. In Western blots of microsomal proteins, anti-P450 MA differentiated single MA and JWax individuals by recognizing and M(r) 49,000 protein band in only the MA strain. In JWax cockroaches, the M(r) 49,000 band was only detectable in Western analysis following induction with pentamethylbenzene (PMB). PMB induction also increases N-demethylation 2.6 and 8.0-fold in the MA and JWax strains, respectively. These results are consistent with the hypothesis that insecticide resistance in the MA strain is due to over-expression of a cytochrome P450.

Resistance to the pink potato aphid, Macrosiphum euphorbiae, in two accessions of Lycopersicon hirsutum f. glabratum

Accessions PI 126449 and PI 134417 of wild tomato, Lycopersicon hirsutum f. glabratum C.H. Mull, were assessed for resistance to the pink potato aphid, Macrosiphum euphorbiae (Thomas). Aphid populations under field and greenhouse conditions were significantly lower on the L. hirsutum f. glabratum plants compared to L. esculentum. Removal of trichome glands and exudates by wiping the leaf surface resulted in a dramatic decrease in aphid mortality. Compared to L. esculentum, aphid behavior on the L. hirsutum f. glabratum plants was characterized by 40 to 52% abandonment, delay in time for first probe, reduction of number and duration of probes and decrease in the total proportion of time probing. Removal of the trichomes reduced the percentage of abandonment and increased the total proportion of time probing. Density and type of glandular trichomes varied among plant lines and on adaxial and abaxial leaf surfaces within a line, particularly for the L. hirsutum f. glabratum plants. PI 134417 was, in general, more resistant to the aphids than PI 126449.

Xylem transport interruption by Anasa tristis feeding causes Cucurbita pepo to wilt

The plant tissues ingested by Anasa tristis (DeGeer) (Heteroptera: Coreidae) and the wilting of Cucurbita pepo L. (Cucurbitaceae) caused by A. tristis were investigated by feeding and light microscopy studies. Anasa tristis ingests both cell contents and xylem. Wilting of leaves and young plants by A. tristis is due to the interruption of all xylem transport to the plant apical to the feeding site. Xylem transport is interrupted by blockage of xylem vessels with salivary secretions or severing of xylem vessels by A. tristis stylets. Restricting feeding sites by caging demonstrates that squash bug does not produce a toxin that is responsible for wilting.

Leucine transport into brush border membrane vesicles from guts of Leptinotarsa decemlineata and Manduca sexta

Abstract 1. 1. Compared to crude homogenates, Manduca sexta brush border membrane vesicles (BBMV) had increased specific activity of marker enzymes leucine aminopeptidase and alkaline phosphatase while Leptinotarsa decemlineata BBMV had increased leucine aminopeptidase specific activity and decreased alkaline phosphatase specific activity. 2. 2. L. decemlineata BBMV were impermeable to a proton gradient while M. sexta BBMV sustained an alkali cation gradient. 3. 3. Both K + -stimulated and K + -independent unidirectional leucine transport pathways were identified in M. sexta BBMV while only cation-independent unidirectional leucine transport was identified in L. decemlineata BBMV.

Plant finding and acceptance behaviors of Anasa tristis (DeGeer)

Finding and acceptance behaviors of Anasa tristis were investigated using plants that are suitable (pumpkin) or unsuitable (cucumber, coleus) for development. A. tristis found plants in the absence of non-contact cues. Plant finding is enhanced by olfaction and vision. A. tristis found suitable and unsuitable plants equally well. The importance of olfaction in plant acceptance is suggested by intense antennation while on plant and change in behavior on pumpkin and coleus after removal of antennae. Insects on suitable and unsuitable plants dab the surface, contact the plant surface with the labium and insert stylets. Post ingestive effects are important for plant acceptance and rejection. Ingesting water and some nutrients from plants unsuitable for development can be important in extending the time available to move between resource patches.

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents.

Effect of Variegation on Stem Exudates of Coleus and Life History Characteristics of Citrus Mealybug (Hemiptera: Pseudococcidae)

Abstract Both defensive and nutritional hypotheses have been used to explain the improved performance of sucking insects on variegated plants. With portions of leaves unable to photosynthesize, variegated plants are likely to have less carbon assimilate to allocate to the production of defensive compounds than green plants. Alternatively, like plants grown in the shade, improved response of sucking insects on variegated plants may be caused by greater mobility of amino acid nitrogen and carbohydrates in the phloem to fuel compensatory production of leaves. We examined amino acid and sucrose content of stem exudate collected from red-variegated, yellow-variegated, and green-leafed coleus plants, Solenostemon scutellarioides (L.) Codd, and their relation to life history characteristics of citrus mealybug, Planococcus citri (Risso), to test these defensive and nutritional hypotheses. Although higher concentrations of both total amino acids and sucrose were found in green- and red-leaved plants, these quantitative measures were uncorrelated with life history parameters of mealybugs. In contrast, our analysis of qualitative measures of amino acid composition supported both defensive and nutritional hypotheses. Green-leafed plants had the greatest levels of shikimic acid precursors and lowest levels of other nonessential amino acids. Life history characteristics of citrus mealybugs were correlated negatively with increasing proportions of shikimic acid precursors and positively with increasing proportions of other nonessential amino acids. Thus, our studies of life history and stem chemistry indicate that improved suitability of variegated plants is consistent with both carbon- and nutrient-based theories of plant defense.

High affinity transport of histidine and methionine across Leptinotarsa decemlineata midgut brush border membrane

High affinity transport of histidine and methionine across the midgut brush border membrane of Leptinotarsa decemlineata was characterized using brush border membrane vesicles (BBMV). Histidine transport was not stimulated by sodium or potassium. Methionine transport was stimulated by monovalent cations. Sodium was more stimulatory than cesium or lithium. Divalent cations, Mg 2+ and Ca 2+ , were not stimulatory, and there was no effect of Cl − . Methionine transport was most strongly inhibited by 100-fold excess concentrations of cysteine, serine and leucine. L. decemlineata midgut has both ion-dependent and ion-independent transporters that differ from those in other insects and mammals. CryIIIA toxin did not inhibit uptake of methionine or histidine into BBMV.