Antony M. Hooper

Characterisation of Bombyx Mori Odorant-Binding Proteins Reveals that a General Odorant-Binding Protein Discriminates between Sex Pheromone Components.

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

Control of witchweed Striga hermonthica by intercropping with Desmodium spp., and the mechanism defined as allelopathic

During investigations into the control of insect damage to maize crops in subsistence farming in Kenya, which involved intercropping with repellent plants, the fodder legumes silverleaf (Desmodium uncinatum) and greenleaf (D. intortum) were also found to reduce dramatically the infestation of maize by parasitic witchweeds such as Striga hermonthica. This effect was confirmed by further field testing and shown to be significantly greater than that observed with other legumes, e.g., cowpea, as were the concomitant yield increases. The mechanism was investigated, and although soil shading and addition of nitrogen fertilizer showed some benefits against S. hermonthica infestation, a putative allelopathic mechanism for D. uncinatum was observed. In screenhouse studies, a highly significant reduction in S. hermonthica infestation was obtained when an aqueous solution, eluting from pots in which D. uncinatum plants were growing, was used to irrigate pots of maize planted in soil seeded with high levels of S. hermonthica. Growth of the parasitic weed was almost completely suppressed, whereas extensive infestation occurred with the control eluate. Laboratory investigations into the allelopathic effect of D. uncinatum, using samples of water-soluble chemical components exuded from cleaned roots, demonstrated that this involved a germination stimulant for S. hermonthica and also an inhibitor for haustorial development.

A Novel Approach for Isolation of Volatile Chemicals Released by Individual Leaves of a Plant in situ

A glass chamber designed specifically for collecting volatile chemicals from individual leaves of a plant in situ is described. The effectiveness of the chamber was demonstrated by collecting volatile chemicals from single leaves of two plant species, potato (Solanum tuberosum) and broad bean (Vicia faba), before and after mechanical damage. The glass chamber, in conjunction with thermal desorption, enables reduction of the entrainment time and thereby allows the monitoring of compounds released by leaf damage in successive 5-min periods. An intact broad bean leaf, in the middle of the day, produces small amounts of the green leaf volatiles (E)-2-hexenal and (Z)-3-hexen-1-ol. However, during the first 5 min after mechanical damage, large amounts of (Z)-3-hexenal, (E)-2-hexenal, and (Z)-3-hexen-1-ol are produced. The decline in production of (Z)-3-hexenal and (E)-2-hexenal is fast, and after 10 min, these compounds reach very low levels. (Z)-3-Hexen-1-ol shows an increase for the first 10 min and then a gradual decline. An intact potato leaf, in the middle of the day, produces very small amounts of the sesquiterpene hydrocarbons β-caryophyllene and germacrene-D. After being damaged, the profile of released volatiles is different from that of broad bean. In potato, damage is associated with release of large amounts of green leaf volatiles and sesquiterpene hydrocarbons. Compounds such as (Z)-3-hexenal, (E)-2-hexenal, and (Z)-3-hexen-1-ol are released in high amounts during the first 5 min after damage, but after 10 min, these drop to very low levels. High release associated with damage is also observed for β-caryophyllene, (E)-β-farnesene, germacrene-D, and β-bisabolene. The highest level is reached 5 min after damage and 15 min later, these compounds drop to low levels. The significance of compounds released after plant damage is discussed.

New genetic opportunities from legume intercrops for controlling Striga spp. parasitic weeds

In smallholder farming in East Africa, intercropping of maize with the cattle forage legume, Desmodium uncinatum Jacq., prevents parasitism by Striga hermonthica (Del.) Benth. (witchweed) through an allelopathic mechanism. Isoschaftoside, a di-C-glycosylflavone, isolated from the root extract and root exudate of Desmodium, interferes with in vitro radicle development of germinated Striga. The biosynthetic pathway of this class of compound is already mostly present in edible legumes and in cereals, so characterisation of the enzyme and genes that control C-glycosylflavone biosynthesis has the potential to create this protection mechanism in other agriculturally important plants.

Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana

The mechanism of ixodid tick (Acari: Ixodidae) repellency by gum haggar, a resin produced by Commiphora holtziana (Burseraceae), was investigated by evaluating activity against the cattle tick, Boophilus microplus. In an arena bioassay, a hexane extract of the resin of C. holtziana exhibited a repellent effect lasting up to 5h. The hydrocarbon fraction of the resin extract was shown to account for the repellent activity, and was analysed by coupled gas chromatography-mass spectrometry (GC-MS). Major sesquiterpene hydrocarbons were tentatively identified as germacrene-D, delta-elemene and beta-bourbonene. The identity and stereochemistry of the former compound was confirmed as the (+)-isomer by peak enhancement using enantioselective GC, whereas the latter 2 compounds, which are most likely degradation products of germacrene-type precursors, were identified through isolation by preparative gas chromatography followed by microprobe-NMR spectroscopy. GC comparison of gum haggar with another resin, C. myrrha, which was inactive in the tick bioassay, showed that the latter contained much lower levels of these hydrocarbons. To assess the suitability of the gum haggar resin as a general acarine repellent, further tests were made on a major acarine pest of European and US animal husbandry systems, the red poultry mite, Dermanyssus gallinae (Acari: Dermanyssidae). Gum haggar extract, and the isolated hydrocarbon fraction, showed strong repellent effects in an olfactometer assay, and again gum myrrh showed no effect. These findings provide a scientific basis for the observed anti-tick properties of gum haggar, and demonstrate the potential for its development as a general acarine repellent for use in animal husbandry systems.

Isoschaftoside, a C-glycosylflavonoid from Desmodium uncinatum root exudate, is an allelochemical against the development of Striga

In East African small-holder farming of maize, the cattle forage legume, Desmodium uncinatum is used as an intercrop due to its allelopathic inhibition of parasitism by Striga hermonthica, an obligate parasitic weed that can devastate the maize crop. Bioassay-guided fractionation of the root extract of D. uncinatum revealed isoschaftoside to be the main compound in the most potent fraction inhibiting growth of germinated S. hermonthica radicles. Bioassays repeated with isoschaftoside isolated from a different plant source, Passiflora incarnata, proved it to be a biologically active component. Analysis of the root exudates produced by hydroponically grown D. uncinatum showed isoschaftoside to be present in the hydroponic media at biologically active concentrations of 10-100 nM.

(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

9-Methylgermacrene-B is confirmed as the sex pheromone of the sandfly Lutzomyia longipalpis from Lapinha, Brazil, and the absolute stereochemistry defined as S

The structure of the sex pheromone produced by the male sandfly Lutzomyia longipalpis, from the Lapinha Cave (Minas Gerais State) region of Brazil, previously proposed tentatively as the novel homosesquiterpene 9-methylgermacrene-B, is confirmed and the absolute stereochemistry defined as S by comparing physical and biological properties of the synthetic enantiomers with the natural product.

High-throughput ESI-MS analysis of binding between the Bombyx mori pheromone-binding protein BmorPBP1, its pheromone components and some analogues

Chip-assisted high-throughput ESI-MS analysis of the pheromone-binding protein of the silkworm moth Bombyx mori, BmorPBP1, incubated with its pheromone components bombykol, bombykal and analogues was developed. The protein bound to bombykol ((10E,12Z)-hexadecadien-1-ol) and all 3 of its geometric isomers to a lesser extent, and showed relaxed specificity toward different chain lengths possessing unsaturation. BmorPBP1 did not bind to bombykal ((10E,12Z)-hexadecadienal), demonstrating molecular recognition of the insect pheromone components.

Companion Cropping to Manage Parasitic Plants

Parasitic plants, through a range of infestation strategies, can attack crop plants and thereby require management. Because such problems often occur in resource-poor farming systems, companion cropping to manage parasitic plants is an appropriate approach. Many examples of companion cropping for this purpose have been reported, but the use of cattle forage legumes in the genus Desmodium as intercrops has been shown to be particularly successful in controlling the parasitic witchweeds (Striga spp.) that afflict approximately one quarter of sub-Saharan African cereal production. Through the use of this example, the development of effective companion crops is described, together with developments toward widespread adoption and understanding the underlying mechanisms, both for sustainability and ensuring food security, and also for exploitation beyond the cropping systems described here.