CHARACTERIZATION OF CATTLE ANAL ODOUR BLEND . RESPONSmLE FOR REPELLENCY AGAINST Rhipicephalus appendiculatus, . THE VECTOR OF EAST COAST FEVER By MARGARET WANGECm KARIUKI (BSc.) Reg. No 156/15192/2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY NOVEMBER,2013 ii DECLARATION 1 hereby declare that this thesis is my original work and has not been presented forthe award of any degree in another university. Margaret Wangechi Kariuki (156/15192/2008) Signature ~: . ~b III I Q.tJ)3Date . Declaration by Supervisors This thesis has been submitted with our approval as University supervisors Dr. Margaret Mwihaki Ng'ang'a Department of Chemistry Kenyatta University Signature ~ . 33°C) experienced in some ecologically marginal areas within the range of the ticks' distribution do not allow development of theileria in the vector ticks (Young et al., 1981). Further still, in areas of extreme climatic conditions ECF may fail to establish itself due to low tick numbers for long periods (Speybroeck et al., 2003). Contrary to this general observation, Bazarusanga et al. (2007) reported a not yet fully explained phenomenon in Rwanda, of higher T parva prevalence in cattle in a region with lower tick numbers compared to two regions with higher tick numbers. 10 • Locality records Of R. appenrllculatu5 _ Reported distribution of T.parva Figure 2 1: Distribution range of T.parva and R. appendiculatus (Chaka, 2001) 2.2.3 The control of East Coast Fever East Coast Fever can be controlled using a number of measures singly or in combination. These measures may be divided into three i.e. T. parva parasite control (chemotherapy), immunization of susceptible hosts and tick vector control (Mtarnbo et al., 2006). T parva parasite control aims at managing the parasite in the mammalian host, which mostly takes the form of treatment of clinical cases. Three effective theilericidal compounds are available for this, i.e. halofuginone, parvaquone and buparvaquone (Dolan, 1999). For some years now immunisation has been and still is by an infection and treatment procedure (Radley et al., 1975; Marcotty et ai., 2001; Mbao et al., 2006). This involves injecting cattle with live sporozoite material with the concomitant application of long-acting tetracyclines. Long acting tetracyclines slow down the 11 division of schizonts and the schizont infected cells against which cellular immune responses are directed (Mtambo et al., 2007). Other vaccines tried include the 67kD circum-sporozoite antigen protein (P67) recombinant forms of which induce high antibody titres in cattle (Kaba et al., 2004; Musoke et a1., 2005) and the Polymorphic Immunodominant molecule (pIM) (Toye et al., 1996). However the p67 only offers partial protection under field conditions. Vector control activities include practices such as movement restrictions which essentially entails keeping clean cattle away from infested pastures or herds, application of acaricides which take the form of plunge dips, sprays, pour-ons or hand-dressing preparations like "tick grease" and selection of tick resistant cattle (Mtambo et a1., 2007). Choosing control options from the above in the context of integrated ECF control strategies depends on the production system, the prevalence of other tick-borne diseases and the epidemiological state ofECF for the area in question (Uilenberg, 1996; Billiouw, 2005). As already stated, the epidemiological state of an area is influenced by among other variables the vector factors. The vector factors of seasonality, abundance, vectorial competence and capacity are modulated by the environment and factors inherent to the vector itself (Mtambo et al., 2007). Factors inherent to the vector itself may be genetic, phenotypic or a combination of both. Therefore an understanding of both genetic and phenotypic variation in the vector ticks infesting livestock in an area would contribute to the understanding of the ECF epidemiology of that area and subsequently aid in the choice and design of control strategies (Mtambo et al., 2007). 12 2.3 Tick Biology Ticks are obligate haematophagous parasite of mammals' birds and reptiles throughout the world (Parola et al., 2001; Rajput et al., 2006). They belong to the phylum, arthtropoda and they are divided into two main families, Argasidae and Ixodidae (Sirnlinge, 2005). Argasids are often called soft ticks because they do not have hard chitnous plates on dorsal surface of their bodies but a flexible cuticle. On the other hand Ixodids have sclerotised dorsal plates and are commonly called hard ticks (Walker et al., 2003). Hard ticks have a number of attributes that enhance their parasitism. They feed for extended periods of time varying from several days to weeks. The length of the feeding period is influenced by factors such as life cycle host type and tick species (Sonenshine, 1991). Their bites are usually painless and may go unnoticed for a long time. Soft ticks feed briefly and usually on a single host species (Sonenshine, 1991). Many argasids have uncanny resistance to starvation and can survive for many years without starvation (Furman et al., 1984). The cuticle of the hard ticks expand but does not grow to accommodate the large volume of blood ingested, which may be from 5 to 10 times their unfed body weight (Rajput et al., 2006). 2.4 R. appendiculatus as a vector of East Coast Fever Brown ear tick is a three host tick found in most animal species. It transmits T parva, Babesia SPP and other protozoan and viral diseases including Nairobi sheep disease and looping ill. It is the principal vector of East Coast Fever (Ndung'u et al., 1995). Rhipicephalus appendiculatus is a widely spread tick in Africa occurring in Eastern, Central and Southern parts of the continent. No populations of this species have been 13 reported in West Africa (Hoogstral, 1956). Some information on distribution shows it has spread to 15 countries. The potential of the species spreading to new areas has been predicted based on their suitability (Norval et al., 1991, 1992). 2.4.1 General life cycle of R. appendiculatus The life cycle of R. appendiculatus consists of four stages (Figure 2.2): eggs, larvae, nymphs and adults. Larvae, nymphs and adults each go through a parasitic and free- living phase by a pattern of host seeking, feeding and off the host moulting. This developmental pattern is a typical three-host cycle where each unfed life stage (larvae, nymphs or adults) feeds on a separate host After moulting, followed by a period of hardening and in certain instances dormancy (quiescence or diapause), immatures and adults alike seek hosts, a process called questing (Sonenshine, 1993). This involves leaving their niches at the soil vegetation interface to vegetation tips to acquire hosts by direct contact. Variations in seasonal ambient conditions have an influence on the questing activity (Punyua et al., 1979, 1984; Speybroeck et al., 2003). Figure 2.2: Life cycle of three host tick R appendiculatus (Speybroeck, 2003) 14 Successful attachment on a suitable host at the predilection site is followed by feeding. Feeding periods vary depending on the host and ambient temperatures (Branagan, 1974). On cattle, feeding may take 6 days for larvae and 8 days for nymphs and adults. These periods may be longer depending on host resistance and environmental conditions like temperature. After feeding for at least 4 days adult males and females mate on the host. Complete engorgement of females follows after mating. Fed females detach and seek a suitable microenvironment (Branagan, 1974). Oviposition commences after a 3-10 day period of pre-ovipositional development. Females lay a large number of eggs of approximately 4000 eggs. Period of oviposition may last up to a month depending on ambient temperatures. Males may remain on the host for 4-6 weeks and mate with successive batches of females (Branagan, 1974). Larvae fed to repletion drop from their host and find a sheltered microenvironment and undergo moulting and nymphs will emerge. Nymphs that dropped from their host undergo metamorphosis and moult into adults (Branagan, 1974). Studies on drop-off rhythms of larvae, nymphs and females of R. appendiculatus on cattle under natural conditions of light and temperature in Zimbabwe have been carried out. Most engorged larvae dropped off between 10:00 and 14:00 hours, nymphs between 12:00 and 18:00 hours (Minshull et al., 1982). Both periods are associated with increased activity of the host (cattle) that normally would be at pasture during these periods. Majority of engorged adults dropped off between 06:00 and 08:00 hours when cattle are in their night paddocks or enclosure and therefore less active (Minshull et al., 1982). A related study in Kabete in Kenya (Mwangi et al., 1991) reported similar observations but did not observe any pattern in 15 larvae drop offs. The different stages seem to have synchronised their drop off times with host behaviour patterns so that they are deposited in optimal habitats for their development and offer the next instar greater chance to encounter a suitable host (Sonenshine, 1993). 2.4.2 Morphological description of R. appendiculatus R. appendiculatus was originally described by Neumann m 1901. Several redescriptions have since been given (Hoogstral, 1956; Walker et al., 1981). The following brief description of the instars of R. appendiculatus is derived from Walker et al. (2000). Adult R. appendiculatus have been described as moderate-sized reddish- brown ticks (Figure 2 3 and Figure 2.4) Dorsal view Figure 2.3: Male R. appendiculatus Dorsal view Figure 2.4: Female R. appendiculatus Ventral view Ventral view Male specimens (Figure 2.3) are characterized by a capitulum which is a lot longer than broad. The basis capitulum is variable, much broader than long in smaller males and only slightly broader in larger males, with short obtuse lateral angles at about the 16 anterior quarter of its length. Palps are short and broad. Coxa I has a distinctly pointed strongly-sclerotized dorsal projection called the anterior process. A dorsal shield, the conscuturn, extends from the tip of the scapular process to the distal end. The conscutum has scattered and moderate sized punctations. Cervical fields are broad and depressed with finely-reticulate surfaces. The marginal lines are distinct, extending anteriorly nearly to the eye level, delimiting one festoon posteriorly (Walker et al., 2003). The posteromedian groove is long narrow and distinct, while the posterolateral grooves are short and broad. In smaller specimens the pattem of grooves and punctations may be much reduced. In engorged specimens a slender caudal process is formed posteromedially. Eyes are marginal, almost flat and delimited dorsally by a very shallow groove. Ventrally spiracles are broadly comma shaped curving gently towards the dorsal surface. Adanal plates are large and well sclerotized tapering posterointernally to well-rounded points. Accessory adanal plates appear as small, short sclerotized points (Walker et al., 2003). In contrast, the capitulum of females (Figure 2 4) is slightly longer than broad and the basis capitulum has broad lateral angles overlapping the scapulae. The porose areas on the basis capituli are round. Palps are short broad and bluntly rounded appically. The dorsal shield now called the scutum is longer than broad but may be approximately equal in length and width in smaller specimens. The eyes are located at the widest point of the scutum, are marginal, almost flat and are delimited dorsally by a faint groove. Cervical fields are broad and depressed. Ventrally the genital aperture is shaped like the tip of the tongue (Walker et al., 2000). 17 2.4.3 Sensory perception Ticks locate their host by two mechanisms: ambushing and hunting or a combination of the two strategies, ticks climb foliage where they wait for a passing vertebrate host with their forelegs extended anterolaterally (Sonenshine, 1993). This behavior, known as questing, facilitates location of the host. Questing ticks will cling to a passing animal if direct contact is made (Sonenshine, 1993). Stimuli which induce ambush and hunting behavior include carbon dioxide, butyric and lactic acid, ammonia (from animal wastes), heat, shadows, and vibrations (Sonenshine et al., 2002). Ticks detect host cues using sensilla located on the tarsi of the front legs (Hess et al., 1986). Until recently, relatively little research has been conducted to determine how ticks detect repellents. Most repellency assays for ticks do not discriminate between repellency due to olfaction versus that from tactile chemoreception (Carroll et al., 2008). Olfactory sensilla are able to detect vaporized molecules (Sonenshine, 1993) and evidence suggests that olfaction is involved at least in part in repellency. For example, in a Y-tube bioassay (Dautel et al., 2006), showed that nymphal sheep ticks, Ixodes ricinus (L.) that approached a DEET-treated filter paper surface would come within 1-3 mm of the surface but not contact it. Carroll et al. (2008) in their bioassay wrapped repellent-treated fingers in organdy cloth to prevent direct physical contact with the repellent. 2.4.4 Behaviour of R. appelldiculatus 2.4.4.1 Host-seeking behaviour of R. appendiculatus Blood-feeding arthropods such as have, over time, developed a complex relationship with their mammalian hosts. Broad variations occur in host-specificity of ticks, 18 duration and multiplicity of contacts, and in host location behaviour (Gibson et al., 1999). From this broad range of a spectrum in host-specificity, generalists and specialists in host/prey location can be discerned (Steidle et al., 2003). This behaviour has been considered either as the result of evolutionary adaptation processes to the host-derived stimuli (Steidle et al., 2003), pathogen-induced behaviour in the vector, normal feeding habits, visual cues, host food and or its products such as feaces, urine or exuviae (Steidle et aI., 2003) or combinations of these factors. In other arthropods such as mosquitoes, the role of olfaction in host-seeking behaviour has been explored (Takken, 1991), including cues as human breath, body odours etc. (Mukabana, 2002). Previous studies indicated that various attractive host- derived stimuli (e.g., host texture, host skin humidity, host body temperature and chemical factors (kairomones/ allomones/ synomones) such as skin emanations, breath, urine and feaces, influence host-seeking behaviour in ticks (Sika, 1996). Kairomones are the main sensory cues used by haematophagous organisms to find their hosts (Mordue and Mordue, 2003). Environmental factors complement these kairomones in influencing host-seeking behaviour in ticks (Speybroeck et aI., 2003). Adult R. appendiculatus search their hosts for a blood meal when they are active early in the day. They become active under specific sets of temperature, rainfall, humidity, length of the rainy season, number of rainy and cloudy days, and day length (Pegram et al., 1989). Numbers of adult R appendiculatus on the host increase after the onset of the rains (Berkvens et aI., 1998). 19 The main factor responsible for this phenology is thought to be day length, where a long photoperiod terminates the slate of diapause and induces host-seeking behaviour in the wet months (Madder et al., 2002). Diapause in ticks is considered to be a pre-adaptive behaviour to allow the ticks to survive unfavorable conditions of a given season. Near the equator, ticks are non- diapausing and may usually feed throughout the year and their numbers vary less (Speybroeck et al., 2004). Once the weather conditions become favorable for the ticks to become active, the chemical cues elicit long-range responses while the physical cues elicit short-range responses in the host-location processes (Mordue and Mordue, 2003). The integration of physical factors and kairomones in the light of the pivotal role played by environmental factors in influencing host-seeking behaviors of ticks has not been explored in building up tick control device(s) that suit different climatic conditions (Wanzala, 2009). 2.4.4.2 Differential selection of predilection feeding sites by R. appendiculatus Once on the host animal, many tick species will not probe until they have arrived at the preferred feeding site and are not at risk of being removed from the host. This preference for feeding site may serve to avoid competition amongst the tick species feeding on the same host animal and perhaps inter-species mate confusion between closely related species, thereby enhancing their survival and reproductivity (Chilton et al., 1992). This may be as a result of evolutionary adaptations of certain tick species to the attraction of specific stimuli originating from the predilection feeding site on the host animal (Wanzala, 2009). 20 Pheromones are used to attract members of the opposite sex for reproduction, to mark food trails or location and territory, and are used as warnings or alarm systems. Each insect (or animal) has its own set of complex chemical pheromone, some of which have been identified and used in traps to monitor and sometimes control pest populations. In other cases, pheromones are used to confuse or lure insect pests away from target crops. Just as during host location, a wide range of factors ranging from physical to chemical are involved in the feeding site location on the host by ticks (Wanzala, 2009). The role of semiochemicals (different host kairomones and non-host allomones) is to help the tick identify a suitable host and reach it (Sonenshine et al., 1982), but continues while on the host to help it identify a suitable feeding site (Norval et al., 1989; Wanzala et al., 2004). The selection of the feeding site is not a random activity or chance effect but is systematically brought about by a well-coordinated stereotyped sequence of behavioral events (Figure. 5) elicited by host-derived semiochemicals (Sika, 1996). Often, ticks have to travel relatively long distances on the host to reach the targeted feeding site (McDowell et al., 1985). The settlement at 'preferred feeding sites' is also related to physical characteristics of the host (texture, body temperature, skin humidity, etc.) as well as chemical factors (skin emanations, host-derived microbial odours, breath, feaces and urine), which are likely to provide optimum conditions for the attachment of ticks (Doube et al., 1979). Observations have shown that ticks tend to avoid desiccation by choosing to occupy less exposed areas or parts on the host (Roberts, ]97]). 21 Micro-environmental conditions specific to certain body areas of the host, e.g. the hygrometric index in the ear cavity, skin temperature and humidity have also been suggested to playa role in the feeding site preference by certain tick species (Waladde et al., 1991). Chemical compounds in sweat and other skin secretions, detectable by olfactory and/or tactile receptors, are believed to facilitate the selection of suitable feeding sites (Waladde et al., 1982). Some of the compounds identified as kairomones from cattle ears, also produced by feeding females and which function as sex pheromone, include 4-methylphenol (1), 2,6-dichlorophenol (2) and 2- hydrox'ybenzaldehyde(3) amongothers (Woodet aI., 1976;Nyongesa,1998). 1 CIH0-P CI 2 HO O~ 3 HO-Q- More recent studies suggest that different tick species also playa role in guiding other ticks of the same species to the suitable feeding site. This is the case in certain Amblyomma species where an aggregation of the ticks at specific feeding sites is supported by the male-emitted attraction/aggregation attachment pheromone (ortho- nitrophenol, methylsalicitate and nonanoic acid) secreted by attached and feeding ticks, attract conspecific males, females and nymphs (Schoni et al., 1994). Once on the host animal, even very closely related species demonstrate predilection for feeding at different body sites as demonstrated for R. appendiculatus and R. evertsi (Figure 2.5 a and b) (Sika, 1996; Wanzala et al., 2004). 22 I Figure 2.5a: General orientation of R. appendiculatus that initiates movement at different release points (1-6) (Wanzala, 2009). Figure 2.5b: General orientation of R. evertsi that initiates movement from different release points (1-6) (Wanzala, 2009). The mechanisms underlying these species-specific interactions and selection of the feeding site on the host have not yet been understood. For instance, adult R. appendiculatus showed marked preference for feeding in the inner part of the bovine ear pinna while the immature one showed less selectivity by feeding on many other parts of the host in addition to the ear pinna (Walker, 1974~Wanzala, 2009). 23 In the case of R appendiculatus, olfactometric experiments using swabs impregnated with ear emanations evoked attraction of adults but repelled nymphs and larvae of the same species (Akinyi, 1991). Stimuli present in the host's anal region, which attractR evertsi, are of interest, as in addition to body surface volatiles, and may include effluvium from the gut and volatiles from dung (Wanzala et al., 2004). In addition, the pheromones emitted by male R. evertsi while in the preparasitic as well as in the parasitic phases, may play a significant role in feeding site location by host-seeking conspecifics (Goethe et al., 1985). The migratory bouts, which R. appendiculatus and a closely related species R evertsi perform during search and location of predilection feeding sites are guided by attracting and repelling host-derived semiochemicals in "push" and "push-pull" modes (Wanzala et al., 2004). With this pheromonal and allelochemical (kairomonal/allomonal) knowledge coupled with that of environmental factors, it is possible to explore on-host tick control interventions involving (1) tactical use of repellent botanicals near/around the feeding sites to confuse the ticks ( the 'push' tactic), (2) diverts ticks (the 'pull' tactic) to simple traps strategically placed on host body and contaminated with killing gent such as pathogenic fungi or acaricide botanicals, and concurrent protection of feeding sites with repellents and diversion of ticks to the traps (the "push" and "push-pull" tactics) (Wanzala et al., 2004). As much as differential selection of predilection feeding sites studies are shown to be important in control and management of ticks on hosts, and only casual attention has been given to feeding sites location behaviour of relevant arthropods (Wanzala et al., 2004). 24 2.5 Tick control practices Tick control measures worldwide have been primarily dependent on use of synthetic acaricides in sprays, dips, dusts etc. There is a lot of awareness on problems arising from widespread use of pesticides (Wharton et al., 1970; Mathewson, 1984). Use of acaricides is unsuitable in many ways. These includes the high cost of chemicals, the development of tick resistant strains, toxicity to vertebrates and effects on both the environment and non-target species such as parasitoids and birds (Stutterheim et al., 1981; Mathewson, 1984). As the development of new acaricides is becoming rather uneconomical (Norval et al., 1992), the current emphasis is on other improved formulations or release devices of existing acaricides such as ear tags and pour-ons (Gladney, 1976; Young et al., 1985). The development of arthropod resistance against repellent compounds such as pyrethroids has been reported (Pennetier et al., 2007). Ticks have developed resistance to the repellent effect of permethrin, which acts as a true repellent as well as an excito-repellentlirritant (Metcalf and Metcalf, 1982). A commonly used commercial arthropod repellent, N,N-diethyl-3-methylbenzamide (DEET), is still considered the best available product, repelling a wide variety of insects, ticks and mites (Fei and Xin, 2007). In humans, however, the repellent may cause insomnia, mood disturbances, impaired cognitive functions, seizures, toxic encephalopathy and allergic reactions (Robbins and Cherniack, 1986; Qui et al., 1998; Lewis et al., 2000). Though DEET is not expected to bioaccumulate, it has been found to cause considerable environmental pollution (Seo et al., 2005). Furthermore, the ability of DEET to dissolve some plastics, rayon, spandex, other synthetic fabrics, 25 leather and painted or varnished surfaces has led to a search for alternative repellents (Wanzala, 2009). Repellents have been in use for the control of arthropods for many years (Dethier, 1956). They have become a popular method for obtaining protection from biting arthropods. Studies have shown, however, that the risk of contracting an infection transmitted by a given vector when using an effective repellent against that vector goes down significantly (Schwantes et al., 2008). Repellents work in a unique way, different from any other methods used for vector control and management. True repellents rarely kill the target vector arthropods, instead, the vectors are just kept at bay (Metcalf and Metcalf, 1982). In exerting their effects, repellents interfere with mating and oviposition responses as well as feeding (Hocking, 1963). Other methods of tick control include mechanical method. This is done by handpicking, whereby in certain communities animals are held in a crutch facility and ticks are picked one by one and either burned or buried (Manna, 2001). This practice is also conducted during milking and cleaning of livestock sheds by women (Marina et al., 2001). Some ticks after being picked from respective host animals are given to chicken as food supplement. Handpicking was done as a communal cultural practice to reduce tick burden on heavily infested animals. However, this method is tedious, time consuming and is not sustainable for a large herd of cattle because it involves much labor (Mathias-Mundy et al., 1989). Another method is biological control which involves use of bio control agents, mainly natural enemies which include insectivorous birds, parasitoid wasps, nematodes, 26 Bacillus thuringiensis bacteria, and deuteromycete fungi (largely Metarhizium anisopliae Sorok and Beauvaria bassiana Vullis) (Samish, 2000), which reduces the density of the target population or even eliminates it. Isolates of M anisopliae and B. bassiana are pathogenic against ixodid tropical ticks R. appendiculatus, A. variegatum Fabriscius, 1794, R (Boophilus) decoloratus Koch, 1844, R. sanguineus Latreille, 1806, R. (Boophilus) microplus Canestrini, 1888 and other hard ticks such as A. americanum Linnaeus, 1758 and A. maculatum Koch, 1844 in the laboratory (Kaaya et al., 1996; Frazzon et al., 2000; Benjamin et al., 2002; Kirkland et al., 2004a: 2004b). In field experiments carried out by Kaaya (2002) and Benjamin et al. (2002), aqueous suspensions of M anisopliae sprayed on vegetation, reduced R. appendiculatus larvae and I scapularis Say 1821 unfed adults. In literature, more than 257 tick biocontrol agents are mentioned, comprising 100 species of pathogens, seven parasitoids and 150 predators (Samish et al., 2001). During the past decades, interest in developing biological methods for tick control using birds (Couto, 1994), parasitoids (HU et al., 1998), entomo-pathogenic nematodes (Samish, 2000), entomo-pathogenic fungi, arthropods (Samish et al., 2001) and bacteria (Hassanain et al., 1997) has gained momentum worldwide because of limited impact of these organisms on the environment (Wanzala, 2009). 27 CHAPTER THREE MATERIALS AND METHODS 3.1 Ticks Stocks of R. appendiculatus ticks were obtained from colonies at International Centre of Insects Physiology and Ecology (ICIPE) Nairobi, Kenya. Rearing conditions were as described by Irvin and Brocklesby (1970). 3.2 Odour trapping The odour trapping was carried out on Friesian steers (Figure 6) at Kenyatta University cattle shed using adsorbent sachets (4 x 4 em) made of filter papers containing 0.2g of either reversed-phase CI8 -bounded silica, Porapak Q or Super Q. Six such traps were be placed in an oven bag (An oven bag is a special plastic bag used for the roasting of meat or other food in an oven) and this bag was attached to the anal region of the cattle as shown in Figures 3.1 using metallic clips. Prior to use, traps were cleaned first by putting them into a 200 rnl soxhlet extractor for 3 days, dried, and then flushed of any contaminant with a stream of dry nitrogen at 60 °C for 3 hours. The oven bags were heated in the oven at a temperature of 100 °C to remove any volatiles that may have been present. The sachets were held on the anal region for 6 hrs. The trappings were wrapped with clean aluminium foil, and in metallic tins. These tins were carried in a flask containing dry ice, Then transferred into a Pasteur pipette and eluted with distilled dichloromethane (4rnl, > 99.9%).elution was carried 28 out under dry ice. Elution from trapping cycles was pooled, concentrated and stored at -20°C until required for analysis and bioassay. For bioassay, volumes of 100 Ill, 200lli and 300lli were used. Figure3.t: Cattle at Kenyatta University crutch facility from which the odours were trapped 3.3 Odours from fresh feaces 0.5 Kg of fresh cattle feaces was collected and put in the beaker. This beaker was put in an oven bag containing sachet of adsorbents. Four sachets per adsorbents were used. The oven bag mouth was tied and left for 6 hours as shown in Figure 3.2. The volatiles were eluted the same way as the anal odours. The sachets containing the trapped odours were wrapped in clean aluminium foil, corked in metallic tins then transferred in a dewier containing dry ice to prevent desorption of the odours. Figure 3.2: Trapping of odours from the cattle dung using oven bag. 3.4 Tick climbing assay 29 A dual-choice tick climbing assay apparatus (Figure 3.3) was used for screening the repellency of the odours, synthetic chemical compounds the blend, all at doses of 0.001,0.01,0.1, and 1 mg respectively. DEET which was a positive control was also screened in a similar manner. The bioassays were done using a tick climbing assay in a laboratory at lCIPE (Wanzala et al., 2004). The assay protocol exploited the behavior of the adult ticks, R. appendiculatus, which climb up grass sterns to the stem tip to wait for any potential passing host (Browning, 1976; Chiera et al., 1985). An aluminum base of area 105 cm2 with two stands of 26 cm in height and 7.0 em apart was put in a basin of water 1.5 cm deep (to retain the ticks at the base) B. f I 40 em 82 B, 260m "../ 7I75 %) within the columns. Ten adult ticks were be placed at the centre of the aluminum base and observed for 60 minutes. The recording was done after every 15 minutes. Each assay was replicated 6 times. The number of ticks that climbed on treated and control columns were counted. Mean % repellency was calculated using the formula Percentage repellency (PR) = [Nc-Nt]/ [Nc+Nt] x 100. Where Nc = the number of ticks that climbed on the untreated glass rod tubes and Nt = the number of ticks that climbed on and or above the filter paper collar strip on the treated glass tube respectively. 3.5 Determination of the composition of the odeurs Anal and dung odours were analysed using gas-chromatography (GC) and gas chromatography-linked mass spectrometry (GC-MS) techniques (Tholl et al., 2006). One microlitre of the eluent sample was injected into the HP 6890 series gas chromatography interfaced to a 5973 Mass Selective Detector (MSD) and controlled by HP chemstation software (version b.02.05, 1989-1997). The chromatographic separation was achieved using a DB-5 MS capillary column (30.0 m x 0.25 mrn x 31 0.25 urn). The column stationary phase comprised of 5%-diphenyl- 95% dimethylpolysiloxane. The operating GC condition was an initial oven temperature of 50°C then programmed to 300°C at the rate of 10°C/minute and then kept constant at 300°C for 3 minutes. The injector and detector temperatures were set at 250 "C. The mass spectrometer was operated in the electron impact mode at 70 eV. Ion source and transfer line temperature was kept at 300°C. The mass spectra were obtained by centroid scan of the mass range from 40 to 800 amu. The constituents of the odours was identified by analysis of their mass spectra, direct comparison of their mass spectra to the Wiley NBS and NIST database of library of mass spectra, on the GC equipment. 3.6 Synthetic standards The synthetic standards and selected analogues used in the bioassays were purchased from Sigma Aldrich Company. 3.7 Composition of blends Two blends of anal odour constituents were tested. Blend 1 was made up of 4-methyl- 2-methoxyphenol, 3-pentanone, 3-methyl-2-pentanone, and 4-hydroxy-4-methyl-2- pentanone. Blend 2 comprised of 4-methyl-2-methoxyphenol, 3-methyl-2-pentanone, and 4-hydroxy-4-methyl-2-pentanone (i.e. without 3-pentanone). 32 3.8 Data analyses The repellency data obtained at different concentrations were subjected to analysis of variance (ANOVA) for a completely randomized design. Treatment means were separated using Student-Newman-Keuls (SNK) at p ~ 0.05 significance level. Dose- response relationship was determined using probit analysis and repellent concentrations at RD75 values obtained from the regression model Probitll'(dose.l j=Ba+x B1+ 'i Where Bo=coefficient of the model representing y-intercept, Bl = coefficient of the model representing dose. 1, Dose 1 =IOglO(dose.), i =error term in the data set of the predictor (regressor) variable (x) and p = repellency probability 33 CHAPTER FOUR RESULTS AND DISCUSSION 4.1 The repellence of cattle anal and dung odours against R. appendiculatus Repellency of odours from the anal region and dung was carried out. The repellent effects of the odours are summarized in Table 4.1 Table 4 1:Repellency by anal and dung odours Dose(J..Ll) 100 200 300 Odour Mean±SE Mean±SE Mean±SE RC50 RC75 Anal odours 28.89±6.39 62.14±3.34 84.02±3.77 2.72 3.90 Dung odours 39.54±4.74 54.24±2.66 77.97±1.69 2.94 4.05 Across a given row, means (±SE) with the same letter(s), are not significantly different at a = 0.05 (Student-Newman-Keuls test) The relative amounts of volatiles trapped from the cattle anal and dung sources were not quantified. However, much more volatiles appeared to have been collected from the dung, which suggests that the dung odour is much less repellent to the brown ear tick. 4.2: Gas chromatography-linked mass spectrometric analyses of the odours The chemical components present in the anal and dung odour were analysed using gas chromatography. The GC-MS profiles of the anal and dung odours are given in figure 4.1 and 4.2 respectively. The time provides the qualitative aspect of the chromatograms while the chromatographic peak heights or peak areas provide the quantitative aspect of the analyte. 80 70 60Abun~nce SO 40 S zo 34 10 6 10 9 Retention tlme(mln) Figure 4.1: GC-MS profile of the anal odour ss 20 Abundance 15 10 2 5 4 6 7 8 95 10 5 10 15 20 ReteDtioD time(miD) Figure 4.2: GC-MS profile of the dung odour Forty three compounds were identified in the GC-MS of the anal odour (Appendix 1a). These compounds included ketones, phenols, amine and alcohols. The order of occurrence of the 10 major compounds was as follows: a-xylene (4), 4-hydroxy-4- methyl-2-pentanone (5), 4-methyl-2-methoxyphenol (6), ethylbenzene (7), 2,6,6- trimethyl-] l S(la,(3,5a)]bicycloheptanes (8), 5-ethoxydihydro-2(3H)-furanone (9), 3- methylene-2-pentanone (10), 5-methyl-2-phenyl-lH-indole (11), 3-pentanone (12) and di-isobutylphthalate (13) (Table 4 2). 3S U Jlx ~OHOH b 0/ 4 5 6 r-o (y( °VO~ 7 8 9 Q--{C( H O=C 10 11 12 36 Table 4 2: Major constituents found in anal odour No Compound Molecular M+ RT Relative formula (g/mol) (min) (%) 4 3-Methylene-2-pentanone, CJI120 100.2 6.0 0.5 5 5-Methyl-2-phenyl Hl-Indole C15H13N 207.3 6.8 0.4 6 4-Hydroxy-4-methyl-2-pentanone C6H1202 116.2 7.3 4.6 7 Ethyl benzene CSHlO 106.2 7.7 2.7 8 O-Xylene CSHlO 106.2 7.9 9.2 9 2-Methoxy-4-methyl phenol CSHlOO2 138.2 8.5 4.3 10 5-Ethoxydihydro 2(3H)-furanone CJIlOO3 130.1 9.3 0.5 11 3-Pentanone C5HlOO 86.1 10.3 0.2 12 2,6,6- Trimethyl-I S- CloHlS 138.3 22.2 0.6 bicyclo[3.1.1 ]heptane 13 Diisobutylphthalate ClJI2204 278.35 22.6 41.27 Fifty nine compounds were identified in the dung odour using GC-MS (Appendix 1b). The order of occurrence of the 10 major compounds was, 4-hydroxy-4-methyl-2- pentanone (5), 2,4-dimethyl-heptane (14), 2-hexadecanone (15), para-ethyl acetophenone (16), 3-penten-2-one, 4-methyl (17), tridecanoic acid (18), 5- dodecyldihydro-2(3H)-furanone, (19), octadecanoic acid (20), a-pinene (21) and tetracosane (22) (Table 4.3). These compounds included ketones, alkanes, esters and carboxylic acids. 14 37 0y ~(CH2)13CH3 15 16 0 O~)lHO (CH2)11CH3 o (CH2)11CH3 18 19 &- CH3(CH2bCH3 22 21 17 20 Table 4.3: Major constituents found in dung odour No Compound Molecular M+ RT Relative formula (g/mol) (min) (%) 17 4-Methyl-3-penten-2-one, C6HlOO 98.1 6.1 1.7 14 2,4-Dimethyl-heptane, C9H20 128.3 6.6 5.4 5 4-Hydroxy-4-methyl-2-pentanone, C~1202 116.2 7.4 16.9 21 a.-Pinene ClOH16 136.2 9.4 1.1 16 p-Ethyl acetophenone ClOH120 148.2 15.4 2.4 18 Tridecanoic acid C13H2602 214.3 20.3 1.7 15 2-Hexadecanone C16H320 240.4 23.8 2.9 19 5-Dodecyldihydro-2(3H)-furanone, C12HIOO2 186.2 24.8 1.6 20 Octadecanoic acid ClsH3402 282.5 25.32 1.2 22 Tetracosane C2~50 338.7 27.4 1.0 38 From the GC-MS analysis of the odours, three was only one major compound found to be common This was 4-hydroxy-4-methyl-2-pentanone (9). In this study ketones (5, 13, 14, and 16) were most abundant in the anal odour, In a study carried out by -Bernier et al. (2006), on the analysis of the headspace above giraffe pelage hair, the most abundant volatiles were ketones that included 2-butanone, 4-methyl-2- pentanone, methyl isobutyl ketone, 3-methyl-2-butanone, 2-pentanone, 3-methyl-2- pentanone and 4-methyl-3-penten-2-one (Bernier et al., 2006). It was also noted that 2-pentanone was also present in the cattle anal odour while 4-methyl-2-pentanone was present in fresh dung odour, In another study, 2-methoxyphenol (guaiacol) was identified as tsetse repellent constituent of bovine odour Torr et al. (1996). In the current study, 4-methyl-2- methoxyphenol) was identified in the cattle anal odour, Saini and Hassanali (2007) examined the repellence of a number of 4-alkyl substituted analogues of guaiacol to Savannah tsetse (Glossina spp.). These analogues included 2-methoxyfuran, 2,4- dimethylphenol, 2-methoxy-4-methylphenol (4-methylguaiacol), 4-ethyl-2- methoxyphenol (4-ethylguaiacol), 4-allyl-2-methoxyphenol (4-allylguaiacol; eugenol), 3,4-methylenedioxytoluene, and 3,4-dirnethoxystyrene. The 4-methyl- substituted derivative (2-methoxy-4-methylphenol) was found to elicit stronger repellent responses from the flies compared with guaiacol. None of the other analogues showed significant repellent effects. 4.3 Repellent effects of synthetic compounds against R. appendiculatus The data in Table 4.4 summarizes the repellent effects of the synthetic compounds. At the smaller doses (0.001 and 0.01 mg), 3-methyl-2-pentanone had a negative 39 repellency, i.e. it was attractant to the tick. However, at higher dose, it was significantly repellent against R. appendiculatus However; this is not strange to insect behavior as demonstrated by locust which had been found to prefer to be within phenylacetonitrile (PAN) permeated air column at low relative doses of the pheromone, but away from PAN at high relative doses in a choice assay (Rono et al., 2008). Of the 5 compounds tested, 3-pentanone was least repellent (RD75=622.7) as compared to 3-methylene-2-pentanone (RD75=1.34) and 4-hydroxy-4-methyl-2- pentanone (RD7s=4.93). 4-Methylguaiacol (10) had the highest repellency (RD75=O.56)against R. appendiculatus. This compound was absent in the fresh dung odour, which may have contributed to the lower repellence in the dung. Interestingly, previous structure-activity studies involving the repellency of a series of guaiacol analogues against tsetse, showed 4-methylguaiacol to be most repellent (Saini and Hassanali, 2007). Diisobutylphthalate, which was the most dominant constituent, was first considered a contaminant. However, this compound was not detected in dung odour collection. 40 Table 4.4: The mean (±SE) percentage of repellency by synthetic compounds Dose mg 0.001 0.01 0.1 1 Compound Mean(±SE) Mean(±SE) Mean(±SE) Mean(±SE) RD7S 4-Methylguaiacol 33.9±3.9Db 41.4±8.4Cbc 53.3±4.8Bb 89.7±6.65Aa 0.56b Diiso butyl phthalate 39.6±7.0Cb 46.6±7.6Bb 50.9±4.2Bb 71.1± 10.4Ac 96.7e 3-Pentanone 18.3±2.4ccd 31.0±5.2Bcc 31.1 ±4.3BCcd 50.2±6.4Ad 622.7f 3-Methyl-2- -22.2±9.2Ce -13.65±5.2Ce 37.5±13.3Bc 76.6±11.4Abc 1.34c pentanone 4-Hydoxy-4- 14.1±3.5ccd 28.9±6.4BCcd 36.8±7.9BCc 81.5±8.5Ab 4.93d methyl-z-pentanone DEET 76.7 ±4.8Ba 83.1 ±5.8Aa 87.8 ±5.1 Aa 97.0±3.3Aa 0.0014a Mean (±SE) with the same lowercase letter in each column and uppercase letters in each row are not significantly different at 0.=0.05 (Student-Newman-Keuls test), respectively. 41 The repellency of the compounds against R. appendiculatus was compared with those obtained with DEET. The repellence of DEET (RD75=O.0014)proved to be better compared to that of the compounds identified in the anal region. 4.4 Repellent of selected 4-metbylguaicol analogues against R. appendicuiatus The compounds tested were guaiacol (23), 4-methylguaiacol (4-methyl-2- methoxyphenol), (24) eugenol (4-allyl-2-methoxyphenol), (25) 3,4- methylenedioxytoluene (26) and 2,4-dimethylphenol (27). Their repellent effects against R. appendiculatus are as shown in Table 4.5. HOp-b 23 24 25 26 27 42 Table 4.5: Repellency by 4-methylguaicol analogues Dose me 0.001 0.01 O.l 1 Compound Mean(±SE) Mean(±SE) Mean(±SE) Mean(±SE) RD75 Guaiacol -38.9±3.5Dd 38.7±5.5cb 46.1±11.9cb 95.2±4.8Aa 0.66e 4-Methylguaiacol 33.9±3.9Db 41.4±8.4Cb 53.3±4.8Bb 89.6±6.6Aa 0.56d Eugenol 13.7±3.3cc 16.9±4.4cb 56.1±2.9Bb 100.0±.OAa 0.21c 3,4-Methvlenedioxv toluene -2.6±10.1 Cc -11.1±8.7cc 6.6±3.0ABc 29.2±7.l Ac 10>f 2,4-Dimethvlphenol 61.7±7.9Da 76.7±1O.5Ca 87.8±7.8Ba 100.0±.OAa 0.0089b DEET 76.7 ±4.8Ba 83.1 ±5.8Aa 87.8 ±5.1 Aa 97.0±3.3Aa 0.00143 Mean±SE with the same lower case letter in each column and upper case letters in each row are not significantly different a=0.05) 43 At the lower concentration (0.001 and 0.01 mg), guaiacol and 3,4-methylenedioxy toluene had a negative repellency, i.e. They were attractant to the tick. This compares with the behaviour of locusts which had been found to prefer to be within PAN- permeated air column at low relative doses of the pheromone, but away from PAN at high relative doses in a choice assay (Rono et al., 2008). The repellent dose of 2,4-dimethylphenol at RD75 (0.0089) (Table 6) are the lowest. This implies that 2,4-dimethylphenol was more effective as a repellent compared to the other analogues. 3,4-methylenedioxytoluene had an extremely low activity and therefore would not be considered as an effective repellent. In this study, 2,4- dimethylphenol elicited the best response of all the analogues tested. 4.5 Repellenteffects of syntheticblendsagainstR appendiculatus Blend 1 comprised of 4-methylguaicol, 3-pentanone, 4-hydroxy-4-methyl-2- pentanone and 3-methyl-2-pentanone, while blend 2 comprised of 4-methylguaiacol, 4-hydroxy-4-methl-2-pentanone, and 3-methyl-2-pentanone. The repellency data are given in Table 4.6. There was no overall significant difference between the repellency of blend 1 and blend 2 as reflected in the RD75 values. The RD75 (0.0185) of blend 2 was lower than that of blend 1. Blend 2 had higher activity than blend 1. 44 Table 4.6: Repellency of selected blends Dosetme) 0.001 0.01 O.l 1 SAMPLE Mean±SE Mean±SE Mean±SE Mean±SE p-value RD75 Blend 1 56.9±5.6Ca 67.1±1.7SCab 83.0±7.7ABa 95.2±4.8Aa 0.001 0.0322b Blend 2 64.1±4.6Ca 64.2±3.9Cabc 81.2±6.1 Sa 100.0±.OAa 6.03 xlO-6 0.0196a P value 0.343 0.509 0.859 0.341 Mean±SE with the same upper case letters in each row and lower case letter in each column are not significantly different at 0,=0.05 4S When the unsaturated branched ketone, 6-methyl-5-hepten-2-one, was applied to cattle, it reduced the attraction to biting flies. Saturated ketones, particularly in the C7-C12 range have also been found to inhibit mosquitoes (Birkett, 2004). However, as larger saturated ketones within the series, like pentanone (C5) and hexanone (C6), are blended with L-Iactic acid, the attraction drops and then results in inhibition of attraction for blend (Bernier et al., 2006). High levels of lactic acid, butanone, 2-pentanone, 3-pentanone, and 6-methyl-5- hepten-2-one have been seen to be attractive to Aedes aegypti (Bernier et al., 2006). The current study showed 3-pentanone as a mild repellent against R appendiculatus. The repellency of 2-pentanone was not tested against R. appendiculatus since its occurrence was in very small amounts. 46 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Anal odour showed greater repellency than the fresh dung odour against R. appendiculatus. Although no direct comparison in the repellency of the two odours could be made for the amounts collected were relative and could not be quantified Forty three compounds were identified in the GC-MS of the anal odour, and fifty nine compounds in the dung odour. Three compounds (4-hydroxy-4-methyl-2-pentanone, N-(3-methyl-l-oxobutyl)-alanine, methyl ester and 2,4-dimethyl-benzo[h]quinoline) were common in the two odours. Of the individual compounds of the anal odour tested, 4-methyl-2-methoxyphenol was found to be most repellent and 3-pentanone least active. Of the two blends tested, blend 2 (without3-pentanone), was more repellent than blend 1. Of the analogues tested, 2,4-dimethylphenol was most repellent (~5 =0.0089), while 3,4-methylenedioxytoluene was inactive. 2,4- dimethylphenol was more repellent compared to all compounds and blends tested. However DEET (RD75=0.0014) still proved to be the most repellent. These results lay down some groundwork for characterizing compounds and/or blends with potent repellence against the Brown Ear Tick on cattle. 5.2 Recommendations The following are the recommendations 1. Further work needs to be done to ascertain whether the position of the carbonyl group has any effect on repellence. 47 n. Further analyses of the oven bag similarly treated need to be done to ascertain if diisobutylphthalate is an authentic constituent of the anal odour. 111. A study to compare the effects of on-host (cattle) performance of controlled- release formulations, at different doses of (i) most potent anal repellent blend, and (ii) 2,4-dimethylphenol on infestations of R. appendiculatus. IV. Screening of other individual constituents of the anal odours and their blends against R. appendiculatus to find out if their contribution is significant. v. Developing control-release devices and study the behaviour of the ticks on the host in the cattle shed and in the field. VI. 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Parasitological, 96:403-432. 59 APPENDICES Appendix la: GC-MS constituent compounds identified in the cattle anal odeurs No Compound Molecular M+ RT Relative Formula (g/mol) (min) (%) 1 3-methylene-2-Pentanone C6H120 100.16 6.04 0.51 2 5-Methyl-2-phenyl-lH-Indole C1sH13N 207.27 6.78 0.4433 3 2,3 -Dihydro- I, 4-dioxin, C4ffi;02 86.08 7.14 0.01 4 4-hydroxy-4-methyl- 2- CJI1202 116.16 7.30 4.60 Pentanone 5 Ethylbenzene CsHlO 106.17 7.72 2.70 6 O-Xylene CSHIO 106.17 7.92 9.2 7 4-methyl-2-methoxy-phenol CsHlOO2 138.16 8.51 4.30 8 5-Ethoxydihydro-2(3H)- C6HlOO3 130.14 9.30 0.52 furanone 9 N-[2-(2- CllH1SN03 209.24 9.74 0.17 methoxyphenoxy)ethyl]- acetamide 10 2-Pentanone CSHIOO 86.13 9.85 0.09 11 3-Pentanone tSHlOO 86.13 10.30 0.15 12 2-(3 Methylguanidino )ethano I CJfllN30 117.15 11.04 0.17 13 N(cyanomethyl)-Formarnide C3~N20 84.07 11.42 0.19 14 3-Pentanone CSHIOO 86.13 11.76 0.09 15 2-(3-Methylguanidino )ethanol CMllN30 117.15 12.00 0.05 16 I-Nitroso-azetidine C3ffi;N2O 86.11 12.11 0.13 17 Taurolidine C7H16N404S 284.36 12.40 0.07 60 Appendix 1a Cont: 18 Spermine ClOH26N4 202.34 12.60 0.13 19 Spermine ClOH26N4 202.34 13.12 0.06 20 N-(3 -Butyrylamino-propyl)- CllH22N202 214.31 13.41 0.02 butyramide 21 3-Ethoxy-l-propanamine CsH13NO 103.16 13.52 0.05 22 l-Nitroso-azetidine C3~N20 86.11 13.73 0.01 23 l-Hexadecanamine C16H3SN 241.4606 l3.8152 0.04 24 N-(3-Butyrylamino-propyl)- CIIH22N202 214.31 14.02 0.01 butyramide 24 2-(3-Methylguanidino )ethanol C~llN30 117.15 14.85 0.02 25 Thymol ClOH14O 150.22 15.20 0.15 26 D-Norleucine C6H13NO 131.17 16.21 0.07 27 Oxirane- 2-carboxylic acid, CSHg03 116.12 16.90 0.14 ethyl ester 28 2,2-Dimethyl- , OXIDle CSHllNO 101.15 17.85 0.02 Prop anal 29 2-Phenoxy-ethanamine CgHllNO 137.18 18.83 0.18 30 3-AuTnnopyrrolidine C~lON2 86.14 20.83 0.03 31 N-Methyl-N-(l-methylethyl)- C~lsN 101.19 21.19 0.12 l-pentanamine 32 l-Octadecene C1sH36 252.48 21.77 0.12 33 2,6,6- Trimethyl,l S- ClOHls 138.25 22.17 0.60 (la,2B,5a)- bicyclo[3.1.1 ]heptanes 34 N-Acetyl-DL-alanine, CSH9N03 l31.13 22.42 0.13 35 Diisobutylphthalate Cl~2204 278.35 22.57 41.27 36 N-acetyl-S( -)-Cathinone C~llN03 256.43 25.01 0.02 37 3-Propoxyamphetamine Cl~23N03 253.33 25.26 0.01 61 Appendix la Cont: 38 2-Cyanoacetamide C3RtN2O 84.08 26.09 0.003 39 I-Alanine, N-(3-methyl-l- C.JI17N03 187.24 26.63 0.10oxobutyl)-, methyl ester 40 4-(2- Amino-l-hydroxypropyl)- C9H13N03 183.2 26.90 0.15 1,2-benzenediol 41 Ethyl N'-isopropylureidoacetate CsH16N203 188.22 27.10 0.09 42 l-Alanine, N-(3-methyl-l- C.JI17N03 187.24 27.30 0.18 oxobutyl)-, methyl ester 43 3-Propoxyamphetamine C12H19NO 193.29 27.57 0.06 44 DL-Cystine C6H12N204S2 240.3 27.72 0.05 45 4-methyl-2-Pentanamine C6HlSN 101.19 27.79 0.07 46 2-nonyl- C2J1it40 336.60 27.97 0.03 Cyclopropaneundecanal 47 I-Alanine, N-(3-methyl-l- C.JI17N03 187.24 28.08 0.16 oxobutyl)-, methyl ester 48 2-p-Nitrophenyl-oxadiazol- CsHsN304 207.03 29.20 0.15 1,3,4-one-5 49 2,4-dimethyl- Benzo[h ]quinoline ClsH13N 207.27 29.29 0.09 62 Appendix Ib: GC-MS constituent compounds identified in the cattle dung odours No Compound Molecular M+ RT Relative formula (glmol) (min) (%) 1 4-Methyl-3-penten-2-one CJIlOO 98.14 6.11 l.73 2 2,4-Dimethyl-heptane, C9H20 128.25 6.65 5.41 3 4-Hydroxy-4-methyl-2-pentanone C6H1202 116.16 7.36 16.94 4 Alpha-pinene ClOH16 136.23 9.43 l.11 5 Camphene C1oHl6 136.23 9.76 0.45 6 Sabinene CloHl6 136.23 10.32 0.49 7 Beta -pinene ClOHl6 136.23 10.70 0.35 8 Delta-3-carene ClOH16 136.23 1l.04 0.62 9 1,8-Cineole ClOHlSO 154.25 1l.44 0.58 10 Z-beta ocimene ClOH16 136.23 1l.60 0.18 11 (E)-beta-ocimene ClOHl6 136.23 1l.78 0.29 12 Gamma-terpinene ClOHl6 136.24 1l.96 0.11 13 Camphor C1oH160 152.23 13.46 0.42 14 Para-ethyl acetophenone ClOH120 148.20 15.41 2.35 15 Daucene C1sH24 204.35 16.46 0.32 16 Decanoic acid ClOH2002 172.26 16.75 0.79 17 Alpha-copaene C1sH24 204.35 16.84 0.47 18 Beta-elemene< > C1sH24 204.35 17.04 0.69 19 Spirolepechinene C1sH24 204.35 17.47 0.23 21 Alpha-humulene C1sH24 204.35 17.89 0.74 22 2-(1,I-Dimethylethyl)-6-methyl-phenol C1sH24O 220.35 18.18 0.77 23 D-germacrene C1sH24 204.35 18.25 0.66 24 2-Tridecanone C13H260 198.34 18.32 0.71 25 Bicyclogermacrene ClsH24 204.35 18.43 0.77 26 Gamma-cadinene ClsH24 204.35 18.63 0.27 27 Delta-amorphene< > CIsH24 204.35 18.72 0.32 63 Appendix Ib; continued' No Compound Molecular M+ RT Relative formula (g/mol) (min) (%) 28 Z-nerolidol C15H26O 222.37 19.20 0.06 29 Decyl-oxirane, C12H24O 184.31 19.71 0.50 30 Alpha-cadinene C1sH24 204.35 20.20 0.44 31 Tridecanoic acid C1Ji2602 214.34 20.31 1.68 32 Tridecane-13-oxabicyclo C12H220 182.3 20.85 0.34 33 Cis-carvone oxide, C lOH1402 166.22 21.16 0.90 34 Dodecanoic acid ClOH2002 172.26 21.41 0.30 35 2-Hexadecanone Cl6ll320 240.42 21.74 0.04 36 2-(3-Methylbuta-1,3- Cl~220 206.32 22.06 0.43 dienyl)cyclohexanone 37 (2,2,6- Trimethyl-bicyclo[4.1.0]hept-1- CUH180 182.26 22.28 0.25 yl)-methanol 38 Pentadecanoic acid C1sH3002 242.39 22.44 0.74 39 2-Heptadecanone C17H34O 254.45 22.80 0.74 40 I-Nonadecene C1