Prospects of genetic engineering for robust insect resistance

Secondary plant metabolites are potentially of great value for providing robust resistance in plants against insect pests. Such metabolites often comprise small lipophilic molecules (SLMs), and can be similar also in terms of activity to currently used insecticides, for example, the pyrethroids, neonicotinoids and butenolides, which provide more effective pest management than the resistance traits exploited by breeding. Crop plants mostly lack the SLMs that provide their wild ancestors with resistance to pests. However, resistance traits based on the biosynthesis of SLMs present promising new opportunities for crop resistance to pests. Advances in genetic engineering of secondary metabolite pathways that produce insecticidal compounds and, more recently, SLMs involved in plant colonisation and development, for example, insect pheromones, offer specific new approaches but which are more demanding than the genetic engineering approaches adopted so far. In addition, nature also offers various opportunities for exploiting induction or priming for resistance metabolite generation. Thus, use of non-constitutively expressed resistance traits delivered via the seed is a more sustainable approach than previously achieved, and could underpin development of perennial arable crops protected by sentinel plant technologies

Towards objective and reproducible study of patient-doctor interaction : automatic text analysis based VR-CoDES annotation of consultation transcripts

While increasingly appreciated for its importance,the interaction between health care professionals (HCP) and patients is notoriously difficult to study, with both methodological and practical challenges. The former has been addressed by the so-called Verona coding definitions of emotional sequences (VRCoDES)– a system for identifying and coding patient emotions and the corresponding HCP responses – shown to be reliable and informative in a number of independent studies in different health care delivery contexts. In the present work we focus on the practical challenge of the scalability of this coding system,namely on making it easily usable more widely and on applying it on larger patient cohorts. In particular, VR-CoDES is inherently complex and training is required to ensure consistent annotation of audio recordings or textual transcripts of consultations.Following up on our previous pilot investigation, in the present paper we describe the first automatic, computer based algorithm capable of providing coarse level coding of textual transcripts. We investigate different representations of patient utterances and classification methodologies, and label each utterance as either containing an explicit expression of emotional distress (a ‘concern’), an implicit one (a ‘cue’),or neither. Using a data corpus comprising 200 consultations between radiotherapists and adult female breast cancer patients we demonstrate excellent labelling performance.Postprin

Novel aspects of benzyne chemistry

Chapter One describes attempts at utilising ortho-quinodimethanes in the synthesis of polycyclic ring systems, where construction of the the 1,4-elimination precursor could not be achieved. Additionally, several attempts were made to utilise the diradical behaviour which ortho-quinodimethanes are thought to possess, in similar annulative reactions. Unsuccessful attempts at generating furanyl- ,benzofuranyl- and benzenoid- derived diradical species, however, brought these particular studies to an end. The remainder of this thesis is devoted to studies that were made into developing new uses for benzynes in organic synthesis. Their discovery, general history and subsequent application is divided for convenience into three chapters; Chapter Two covers the discovery of these classic species, and approaches to these species which have been improved from the highly impractical methods which were originally developed. Chapters Three and Four give an overall review of the past and recently reported applications of benzynes in organic synthesis; Chapter Three covers the use of benzynes in nucleophilic couplings, whilst Chapter Four covers their application in cycloaddition reactions. Chapter Five describes preliminary studies that were made into developing new routes to polycyclic ring systems via the intramolecular Diels-Alder trapping of benzynes. After unsuccessful attempts at utilising the anthranilic acid route to benzynes, the remainder of the studies were concentrated on utilising the 1-aminobenzotriazole route to benzynes, a mild, efficient, yet relatively underexploited route. An improved synthesis of 7-methyl-1-aminobenzotriazole is described, as is the functionalisation of the methyl substitutent in the BOC-derivative via the formation of the dianion. However, 1,3-diene incorporation via this route was unsuccessful, due to difficulties in preparing suitable l,3-dienes. Chapter Six describes attempts at applying the metallation chemistry of BOC-protected 7-methyl-l-aminobenzotriazole to the synthesis of 2-substituted dihydrobenzofurans (n = 0) and chromans (n = 1) via the intramolecular trapping of benzynes by flanking hydroxyl functions, which were incorporated via the condensation of the dianion with aldehydes and epoxides respectively. Using H-bromosuccinimide as the reagent for benzyne generation, benzo-fused heterocycles were successfully generated in moderate yields, with bromine incorporation also being achieved (X =Br). Using lead (1V) acetate, simple dihydrobenzofurans (X=H) were obtaine4 in better yields. Excellent yields, in some cases virtually quantitative, were obtained using N-iodosuccinimide as the reagent, with the iodine substituent (X = I) being incorporated. The additional bonus of having the iodine substituent was highlighted by utilising iododihydrobenzofurans and chromans in subsequent coupling reactions. Overall, this thesis describes the advances made in increasing the role of benzynes in synthetic organic chemistry, providing novel metalation chemistry on the aminobenzotriazole ring system, and a novel annulative approach to valuable synthetic and potentially active species such as dihydrobenzofurans and chromans. Above all, this work illustrates that under certain suitable conditions, benzynes can serve as extremely efficient reactive intermediates in heterocyclic annulations

Antennal Electrophysiological Responses of Three Parasitic Wasps to Caterpillar-Induced Volatiles from Maize ( Zea mays mays ), Cotton ( Gossypium herbaceum ), and Cowpea ( Vigna unguiculata )

Many parasitic wasps are attracted to volatiles that are released by plants when attacked by potential hosts. The attractiveness of these semiochemicals from damaged plants has been demonstrated in many tritrophic systems, but the physiological mechanisms underlying the insect responses are poorly understood. We recorded the antennal perception by three parasitoids (Cotesia marginiventris, Microplitis rufiventris, and Campoletis sonorensis) to volatiles emitted by maize, cowpea, and cotton plants after attack by the common caterpillar pest Spodoptera littoralis. Gas chromatography-electroantennography (GC-EAG) recordings showed that wasps responded to many, but not all, of the compounds present at the physiologically relevant levels tested. Interestingly, some minor compounds, still unidentified, elicited strong responses from the wasps. These results indicate that wasps are able to detect many odorant compounds released by the plants. It remains to be determined how this information is processed and leads to the specific behavior of the parasitoid

Vertebrate pheromones and other semiochemicals: the potential for accommodating complexity in signalling by volatile compounds for vertebrate management

The interaction between volatile and non-volatile, e.g. proteinaceous, components of pheromone and other semiochemical-based signalling systems presents a daunting set of problems for exploitation in the management of vertebrates, good or bad. Aggravating this is the complexity of the mixtures involved with pheromones, not only by definition associated with each species, but also with individual members of that species and their positions within their immediate communities. Nonetheless, already in some contexts, particularly where signals are perceived at other trophic levels from those of the vertebrates, e.g. by arthropods, reductionist approaches can be applied whereby the integrity of complex volatile mixtures is maintained, but perturbed by augmentation with individual components. In the present article, this is illustrated for cattle husbandry, fish farming and human health. So far, crude formulations have been used to imitate volatile semiochemical interactions with non-volatile components, but new approaches must be developed to accommodate more sophisticated interactions and not least the activities of the non-volatile, particularly proteinaceous components, currently being deduced

Novel use of stir bar sorptive extraction (SBSE) as a tool for isolation of oviposition site attractants for gravid Culex quinquefasciatus

Mosquitoes such as Culex quinquefasciatus Say (Diptera: Culicidae) are important vectors of organisms that cause disease in humans. Research into the development of effective standardized odour baits for blood-fed females (oviposition attractants), to enable entomological monitoring of vector populations, is hampered by complex protocols for extraction of physiologically active volatile chemicals from natural breeding site water samples, which have produced inconsistent results. Air entrainment and solvent extraction are technically demanding methods and are impractical for use in resource poor environments where mosquito-borne disease is most prevalent. This study reports the first use of a simple, robust extraction technique, stir bar sorptive extraction (SBSE), to extract behaviourally active small lipophilic molecules (SLMs) present in water samples collected from Cx. quinquefasciatus breeding sites in Tanzania. Extracts from a pit latrine and from a cess pool breeding site attracted more gravid Cx. quinquefasciatus in pair choice bioassays than control extracts, and coupled gas chromatography-electroantennography (GC-EAG) allowed tentative identification of 15 electrophysiologically active chemicals, including the known oviposition attractant, skatole (3-methylindole). Here, we have demonstrated, using simple pair choice bioassays in controlled laboratory conditions, that SBSE is effective for the extraction of behaviourally and electrophysiologically active semiochemicals from mosquito breeding site waters. Further research is required to confirm that SBSE is an appropriate technique for use in field surveys in the search for oviposition cues for Cx. quinquefasciatus

Attractiveness of host plant volatile extracts to the Asian citrus psyllid, diaphorina citri, is reduced by terpenoids from the non-host cashew

Diaphorina citri is a vector of the bacterial causative agent of Huanglongbing (HLB = Citrus greening), a severe disease affecting citrus crops. As there is no known control for HLB, manipulating insect behaviour through deployment of semiochemicals offers a promising opportunity for protecting citrus crops. The behavioural responses of D. citri to plant volatiles, and the identity of these plant volatiles were investigated. Volatiles were collected from host plants Murraya paniculata, Citrus sinensis, C. reshni, C. limettioides, Poncirus trifoliata, and from non-host plants Psidium guajava, Mangifera indica, Anacardium occidentale. In behavioural assays, female D. citri spent more time in the arms containing volatiles from either M. paniculata or C. sinensis compared to the control arms. When D. citri was exposed to volatiles collected from A. occidentale, they preferred the control arm. Volatiles emitted from the other studied plants did not influence the foraging behaviour of D. citri. Chemical analyses of volatile extracts from C. sinensis, M. paniculata, and A. occidentale revealed the presence of the terpenoids (E)-4,8-dimethylnona-1,3,7–triene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) in higher amounts in A. occidentale. In further behavioural bioassays, female D. citri spent less time in arms containing a synthetic blend of DMNT and TMTT compared to the control arms. Female D. citri also spent less time in arms containing the synthetic blend in combination with volatile extracts from either M. paniculata or C. sinensis compared to the control arms. Results suggest that higher release of the two terpenoids by A. occidentale make this species unattractive to D. citri, and that the terpenoids could be used in reducing colonisation of citrus plants and therefore HLB infection

Effects of nicotine and tobacco-related products on the feeding behavior of the German cockroach (Blattodea: Blattellidae)

Animals use olfaction to detect developmentally significant volatile organic compounds (VOCs) in their local environment. As part of a wider study aiming to demonstrate that the olfactory responses of animals to VOCs can be modified through the creation of a drug-addicted status and association with a selected VOC, we investigated nicotine and tobacco smoke particulate (TSP) extract as possible addictive compounds for male German cockroaches, Blattella germanica (Linnaeus). In feeding experiments using an artificial food stimulus, food treated with TSP extract was preferred over untreated food. Surprisingly, nicotine, which was expected to be the most important addictive tobacco component, did not induce noticeable effects on cockroach behavior. Both TSP extract and nicotine were shown to be phagostimulants. Olfactometry assays that measured odor-mediated insect behavior demonstrated that male B. germanica did not choose TSP-extract-treated food even when attempts were made specifically to train them via this modality. These results support a hypothesis that B. germanica needs to consume TSP-containing food to show a clear preference for this stimulus and that gustatory mechanisms are involved due to compounds present in the TSP extract