Livestock Research for Rural Development 30 (6) 2018 Guide for preparation of papers LRRD Newsletter

Citation of this paper

The effect of essential oils of Tagetes minuta and Tithonia diversifolia on on-host behaviour of the brown ear tick Rhipicephalus appendiculatus

W Wanzala1,2, A Hassanali2,3, W R Mukabana2,4 and W Takken5

1 Department of Biological Sciences, School of Science and Information Sciences, Maasai Mara University, P O Box 861-20500 Narok, Kenya
osundwa1@yahoo.com
2 Behavioural and Chemical Ecology Department, International Centre of Insect Physiology and Ecology, African Insect Science for Food and Health, P O Box 30772-00100-GPO, Nairobi, Kenya
3 Chemistry Department, School of Pure and Applied Sciences, Kenyatta University, P O Box 43844-00100-GPO, Nairobi, Kenya
4 School of Biological Sciences, University of Nairobi, P O Box 30197-00100-GPO, Nairobi, Kenya
5 Laboratory of Entomology, Wageningen University and Research Centre, P O Box 8031, 6700 EH Wageningen, The Netherlands

Abstract

On–host behaviour of Rhipicephalus appendiculatus was studied in the field in Bungoma County in Kenya to evaluate the putative repellent effects of essential oils of Tagetes minuta and Tithonia diversifolia at its predilection feeding site. Oils of both plants caused a disruption of orientation, movement and attachment behaviour of ticks. More ticks dropped off in the treatments with the two essential oils than with the control.

Treating the ear pinna with the essential oil of T. minuta caused the highest percentage of ticks to drop off the host body. No tick reached the ear pinna treated with the essen­tial oil of T. minuta and up to 30% of ticks (from the forehead release site) reached the ear base. When the ear pinna was treated with the essential oil of T. diversifolia, one tick reached the ear pinna and up to 40% of ticks (from the dewlap release site) reached the ear base. The results show that T. minuta repels ticks more strongly than T. diversifolia. However, both essential oils offer possibilities for exploitation of potentially effective and environmentally acceptable tools for on–host tick control.

Key words: cattle, feeding site, Kenya, on-host orientation behavior


Introduction

Ticks and tick–borne diseases (TBDs) are a major constraint to the development of livestock industry throughout the tropics. Worldwide, an estimated 600 million cattle are exposed to anaplasmosis and babesiosis, whereas 200 million cattle are exposed to theileriosis, all being economically important TBDs. In eastern, central and southern Africa, East Coast fever (ECF), caused by Theileria parva (Theiler 1904) Bettencourt, Franca & Borges, 1907 and transmitted by the vector, Rhipicephalus appendiculatus Neumann, 1901, is considered to be the most significant tick–borne disease of cattle and a costly disease in Africa (Norval et al 1991; Taylor et al 2016). The parasite infects cattle as well as African buffalo (Cape buffalo), Syncerus caffer, Sparrman 1779, large antelopes and the vector is also found on other small animals such as hares, dogs and warthogs in 12 countries of the region (Mukhebi et al 1992; Walker et al 2005). It is estimated that about 28 million cattle in the region (eastern, central and southern Africa) are at risk of ECF and the disease kills at least 1 million cattle per year with more socio-economic losses being felt by small-scale resource-poor livestock farmers (Minjauw and McLeod 2003; Gachohi et al 2012; Mans et al 2015; Perry 2016; Nene et al 2016).

For many years, the control of ticks and the diseases they transmit has been largely through the application of acaricides as dips, dust, and pour–ons, using ear tags, sprays or systemic acaricides. The repeated application of acaricides prevents transmis­sion of the parasites and this method has been used very successfully throughout the sub–Saharan Africa, in conjunction with control of animal movement, quarantine and slaugh­ter of infested animals (Lawrence 1991). However, the development of resistance in ticks to successive acaricide compounds has been a major problem (Norval et al 1992). This problem has been compounded by the increasing costs of acaricides, unregulated cattle movement, civil unrest, poor management and inadequate maintenance of dips (abuse of acaricides and dips, under dilution of acaricides, use of counter fake acaricides), especially at the communal level. The devastating extent of recurring prolonged droughts in some parts of Africa has made many dip–tanks non–operational, due to lack of sufficient water to main­tain them. Another complication associated with the use of acaricides is that they are environmental pollutants and have been found to contaminate milk and meat (Mitchell 1996).

Tick control by the use of repellents is considered an alternative strategy (Mut­huswami and Nisha 2006). Some tick– and insect–repellents are available and widely used for protection by humans (Frances and Wirtz 2005). The use of topical repellents provides an effective prophylactic measure against biting arthropod vectors and arthro­pod–borne diseases at an individual level (Gupta and Rutledge 1994; Hoch et al 1995; Nentwig 2003), especially in areas where suppression of arthropod vectors is not practi­cal or feasible. Examples of such repellent materials, include, but are not limited to N,N–diethyl–3–methylbenzamide (DEET), p–menthane–3,8–diol (PMD), permethrin, allethrin, piperonyl butoxide, lemongrass oil, citronella oil, eucalyptus oil, camphor, geranium oil, ethyl hexanediol, ethyl butylacetylaminopropionate and hydroxyethyl–isobutyl–piperidine. Repellents commonly available to consumers contain the active ingredients DEET, a few repellents contain permethrin, while none or very few repellents contain botanical essential oils (New York State Department of Health (2737/04) 2004). The development of botanical essential oils as arthropod repellents has been on the increase in the recent decade, in order to replace DEET due to its reported environmental pollution and toxicity in human population using it (Waka et al 2004; Seo et al 2005; Jaenson et al 2006; Kegley et al 2007; Walschaerts et al 2007).

In the livestock industry, repellents are less commonly used than in human health, although traditional livestock owners may use a range of ethnobotanical products to protect their animals from tick bites (Martin et al 2001; Guarrera et al 2004; Van de Putte 2005; Mathias 2005; Passalacqua et al 2006; Bond 2007). Commercial repellent prod­ucts containing botanical (plant–based) oils, such those of geranium, cedar, lemon grass, soy and or citronella have been available. There is limited information, however, on the effectiveness of botanical oils individually and when combined with other ingredients. Available information indicates that, compared to the effectiveness of DEET or permethrin, botanical essential oils generally do not provide the same duration of protection (Consumers Union 1993; 2000). The efficacy of these natural products is poorly understood, whilst commercial products are rare. There is need therefore to focus research on the development of effective and safe livestock tick repellents that can be incorporated in the existing Integrated Tick Management (ITM) for livestock tick control programmes (Mount et al 1999).

The life cycle of an ixodid tick often has a total duration of 6 years and host attachment may constitute less than 2% (1½ months) of this time. Ticks spend most of their life cycle away from their hosts, hiding either in the soil and vegetation or in the nests of their hosts. However, ticks select habitats with good opportunities to encounter a host for feeding. Host–seeking and–recognition are two most important and challenging activities in the life cycle of ticks. Many ticks, in particular certainHyalomma, Amblyomma, Ornithodoros and Dermacentor species (Sonenshine 1991; Anon 2008), seek their hosts by hunting, whereas others use an ambushing strategy, e.g., the larvae of Boophilus spp and most ixodid ticks (Sonenshine 1993; Eckert et al 2005). Other tick species respond to volatile host emanations, vibrations, visual cues, radiant heat and touch with questing behaviour, e.g., R. appendiculatus (Speybroeck et al 2003). However, little is known on how different host specificities are encoded in the odours. Questing ticks climb up the stems of grass or perch on the edges of leaves on the ground in an erect posture, while the first pair of legs is waved in the direction of the stimuli of the host passing by. Subsequently, the ticks climb and grab onto the potential host body using their front–leg claws. Once on the host, gustatory and olfactory cues seem to aid the ticks in deciding whether it will remain on the host. By ‘push’ and ‘push–pull’ action modes of the host’s volatiles, ticks may be guided to particular feeding sites (Wanzala et al 2004). Some tick species prefer feeding sites where they are out of reach of attacks by the grooming behaviour of the hosts. On artificial substrates, ticks orientate towards certain host skin extracts (Akinyi 1991; Sika 1996), but the chemical nature of the directing cues is not yet known. However, gas chromatography–coupled electrophysiology recordings using different vertebrate odours has shown that lactone, methylsalicylate, carbon dioxide, sulfide, benzaldehyde, 2–hydroxybenzaldehyde, aliphatic aldehydes, 2,6–dichlorphenol, nitrophenol, pentanoic acid, 2–methylpropanoic acid, butanoic acid, ammonia, and 3–pentanone, may be in­volved in host identification by the ticks (Anon 2008). Masking these natural volatiles and diverting ticks away from their prospective hosts may be an attractive strategy to incorporate into the existing ITM for livestock tick control and management (Olwoch et al 2008).

Some tick species exhibit specialization in selecting their feeding site on their hosts. Such specificity may serve to maximize survival and reproduction of the species (Chilton et al 1992). Adult R. appendiculatus have a marked preference for feeding mainly inside the ears of bovids, whereas their immatures show less selectivity, and are found feeding on many parts of the host body in considerable numbers (Walker 1974). A host–derived odour–based ‘push’ and ‘push–pull’ pair of stimuli has been suggested to be responsible for the orientation behaviour of adult R. appendiculatus to its preferred feed­ing site (Sika 1996; Wanzala et al 2004).

In this study, we investigated the on–host behaviour of unfed adults of the brown ear tick R. appendiculatus and assessed the effect of the application of essential oils of Tagetes minuta L. (Family: Asteraceae) and Tithonia diversifolia (Hemsl.) A. Gray (Family: Asteraceae) on this behaviour in natural field sites in Bungoma County, western Kenya.


Material and methods

The study site

The experiments were conducted under outdoor conditions at the study site in Bungoma County, western Kenya, which lies between latitude 0o 25’N and 0o53’N and longitude 34o21’E and 35o04’E (Wanzala et al 2012). The altitude ranges from 1200–3500 m above sea level. The mean annual temperature ranges from 21 to 22 oC with variations in mean maximum and minimum temperature ranging from 25 to 32 oC and 11 to 13 oC, respectively. Rainfall in the district has a bimodal pattern and varies on average from 1 000–2 000 mm annually. The major rainy season is from March to July, while the minor one starts in August and continues into October.

Experimental ticks

The tick species used (Rhipicephalus appendiculatus) was obtained from colonies reared in the insectary at the International Centre of Insect Physiology and Ecology (ICIPE), Nairobi, Kenya. Rearing conditions and handling were as described previously (Bailey 1960; Irvin and Brocklesby 1970). The newly hatched and 24 h–starved adult R. appendiculatus were transported in glass vials buried in moist sand from ICIPE, Nairobi to Bungoma (a distance of about 500 km), and used within 48 h after hatching.

Behavioural studies

Observation of on-host behaviour was done using indigenous zebu steers (body weight 150–230 kg), bought from livestock farmers near and or within the study site (Figure 1). The ani­mals had not been exposed to acaricides or other chemicals. The animals were held in a crutch facility built at Mwibale village, Bungoma County, Kenya (Wanzala et al 2012). The responses of ticks and their navigation patterns were monitored starting from six different body locations on zebu steers, representing varying distance by the observer (Wycliffe Wanzala) from preferred feeding sites and possible areas of attachment by the ticks from their questing positions on the vegetation (Browning 1976) (Figure 1). One tick at a time of mixed age and sex was placed at one of six sites on the host animal and observed for up to 14 h, between 07:00 h and 24:00 hours (Figure 1). All observations were made during dry weather with day temperatures in the range of 24–28 oC and relative humidity ranging between 60–85%. Each observation was replicated 20 times, each time using a naïve tick. The eventual pheromone trail left by each tick (Sonenshine 2006) was removed by wiping the animal with 99% alcohol and leaving it to evaporate before a new tick was placed on the animal.

Figure 1. Sites on the host animal (bovid) used for the study of on-host navigation behaviour of R. appendiculatus
toward the predilection feeding site (the ear) in the presence of essential oils. Marked site 1 =
Forehead, 2 = Dewlap, 3 = Foreleg, 4 = Rear leg, 5 = Escutcheon and 6 = Shoulder.
Dispensing of essential oil volatiles

Essential oil of T. minuta and T. diversifolia had been obtained by the hydrodistillation method using a Clevenger type distillation apparatus (Sereshti and Samadi 2007). Essential oils were diluted to 10% in odourless vaseline petroleum jelly (BP–USP 100% Grade) (Unilever, Kenya)–skin protectant. This carrier material contains no colours, fragrances or irritants. One ml of the stock 10% formulation of essential oils was placed in a 5 cm3–Eppendorf tube, which was attached by a string to a plastic ear tag on the inner side of the ear pinna. The Eppendorf tube was left open so that there was continuous release of either essential oil volatiles or petroleum jelly. The implication was that at all times a dose of 100 ml of pure essential oil in 900 ml of vaseline petroleum jelly was exposed to the ticks on the inner side of the ear of the animal. The odour from the applied essential oils was presumed to gradually contaminate the ear and its surrounding area. Both ears were simultaneously treated and ticks were released from previously marked sites (Wan­zala et al., 2004) (Figure 1). The 20 ticks were released one at a time and monitored until the final destination was determined.

Legal use of experimental animals in the field

All procedures requiring the use of experimental animals in the field were approved by Bungoma County Veterinary Officer (CVO) of Bungoma County, western Kenya. The importance, seriousness and risk–free nature of the project were further explained to the Bukusu community in western Kenya by the CVO and agricultural extension officers working within the study area. The field experiments were performed in compliance with guidelines published by Kenya Veterinary Association and Kenya Laboratory Animal Technician Association, regarding the ethical use and handling of laboratory and farm animals in the field (KVA and KLATA 1989). Informed consent was obtained from the livestock farmer volunteer from whom we rented field experimental plots.

Statistics

Data were entered in Excel database structure and then entered into a Statistical Products and Service Solutions (SPSS version 15 for Windows) database for analysis. Data for on–host tick behaviour were analysed by one–way analysis of variance (ANOVA) and univariate analysis using the general linear model (GLM) procedure for SPSS.

The mean differences were compared and separated using Student–Newman–Keuls test at α = 0.05 (Sokal and Rohlf 1995).

Differences between responses of the ticks under different treatments and at different release points (locations) on the host body were analysed using the Kruskal Wallis H–test (Kruskal and Wallis 1952). Any significant results obtained by the Kruskal Wallis H–test, between and within groups of release points and treatments, were analysed using Wilcoxon–Mann–Whitney U–test with Bonferroni correction (Wilcoxon 1945; Mann and Whitney 1947; Bergmann et al 2000).


Results

The stereotyped sequence of on–host orientation behaviours of R. appendiculatus revealed a set of sequential activities (particularly in the control experiment). These activi­ties ranged from an inactive stationary/scanning phase to the onset of erratic movements that became increasingly rapid and directional and finally to arrestment at the predilection feeding site. Although variations occurred in the duration of each set of orientation behaviours for every tick observed, the stereotyped sequence of orientation behaviours consistently occurred, irrespective of type of treatment, release point on the hosts’ body and age, sex and body size of candidate ticks. As observed previously in a controlled environ­ment (Wanzala et al 2004), these stereotyped responses comprised runs and strides of varying intensities, alternating with stops, and occasionally, walk–away and back–up movements. At the predilection feeding site, ticks behaved differently, each taking time to start the attachment process by mouth insertion into the host integument before imbibing blood much later.

The results of oriented responses and behavioural movements of R. appendicula­tus to the ear treated with the essential oils of T. minuta and T. diversifolia demonstrated a disruptive effect (Tables 1 and 2).

Both release points and treatments (the essential oils of T. minuta and T. diversifolia and petroleum jelly) had a significant effect on the responses of ticks on the host while searching for a predilection feeding site. For all the release points, the number of ticks reaching the ear base [H (2) = 6.086, p = 0.048], ear pinna [H (2) = 14.500, p = 0.001] and those that dropped off [H (2) = 8.97, p = 0.011], were significantly affected by treatment. No tick reached the ear pinna treated with the essential oil of T. minuta, while only one tick reached the ear pinna treated with the essential oil of T. diversifolia. More ticks dropped off in the treatment with the two essential oils than in the control, with the ear pinna treated with the essential oil of T. minuta showing the highest number of ticks dropping off from various release points (Table 1). More ticks from the various release points reached the base of the ear when the essential oil of T. diversifolia was ap­plied on the ear pinna than in the case of the treatment with the essential oil of T. minuta (U = 4.5, r = –0.630, p = 0.029). The number of ticks from various release points reach­ing the ear base in the control treatment was significantly different from the treatment with T. minuta (U = 5.50, r = –0.585, p = 0.043) but not from the treatment with T. diversifolia (U = 17.5, r = –0.024, p = 0.934). The number of ticks reaching the ear pinna was significantly higher in the controls than in the treatment with either T. minuta (U = 0.00, r = –0.890, p = 0.002) or T. diversifolia (U = 0.500, r = –0.842, p = 0.004). More ticks were affected by the treatment with T. minuta and T. diversifolia than the treatment with the petroleum jelly (Table 1). However, whereas the treatment with T. minuta and T. diversifolia equally prevented ticks from getting attached at the ear pinna (U = 15.00, r = –0.289, p = 0.317), the treatment with the essential oil of T. diversifolia did not prevent the ticks from getting attached at the ear base like its counterpart.

The number of ticks that was attached in the neighbourhood of release points (NRP) [H (2) = 2.589, p = 0.274] and that representing the non–respondent ticks once placed at the release points [H (2) = 0.834, p = 0.659], were not affected by the treatment with petroleum jelly and the essential oils of T. minuta and T. diversifolia.

The site of release of the ticks on the host did affect their directional behaviour in searching for a predilection feeding site (p<0.05). The number of ticks reaching the ear base from release point 1 was the highest and significantly different from all other release points (p<0.05). However, the number of ticks reaching the predilection feeding site from release point 1 did not significantly differ from that arriving from release point 2 (p>0.05). The site of release of ticks on the host animal significantly affected the non-responding ticks [H (5) = 11.533, p = 0.042]. For example, the location of release sites 3 and 4 may not have allowed the ticks placed at these sites to receive sufficient amounts of host–derived attractants or repellents, or applied essential oils of the two plants. The number of ticks not responding from release site 4 was the highest and significantly different from all other release sites (p<0.05) (Mann–Whitney U–test) (Table 1).

The site of release of the ticks, the essential oil treatment and the interaction of these two factors had a significant effect on the behavioural responses of tick vectors on the host (p<0.05) (Table 2). Response times of ticks released at forehead, escutcheon, shoulder and dewlap were short and not significantly different (P<0.05) for all the treat­ments except for dewlap in the control. Ticks took significantly longer to respond when released at fore– and rear legs (p<0.05). For a specific release point, the three treatments had the same significant effect on response time except for the foreleg and dewlap release points (Table 2). At the foreleg, ticks responded in a significantly shorter time to the treatment with T. diversifolia than in the controls. At the dewlap, ticks took a signifi­cantly shorter time to respond to the treatment with T. minuta and T. diversifolia than in the controls; with the response time to the treatment with T. minuta being the shortest.

The initial walking speed of ticks at forehead, fore– and rear legs was not signifi­cantly different for all the treatments. At the dewlap, escutcheon and shoulder, the initial speed was highest in the treatment with the essential oil of T. minuta and lowest in the control except at the escutcheon release point. These differences are statistically signifi­cant except for the treatment with T. diversifolia and T. minuta at the shoulder release site (Table 2).

The time between release of ticks at the various locations and their arrival at the predilection feeding site was not significantly different for all the treatments except for rear leg and escutcheon release points. For both the rear leg and escutcheon release points, ticks took significantly much longer time to reach the predilection feeding site in the control treatment than in the treatment with the two essential oils (Table 2).

Generally, it was observed that the time it took ticks to reach the ear pinna and base and become attached was much longer than the time to drop off or attach in the vi­cinity of the release points. The effect of this was observed to be greater in the treatments of the two essential oils than in the control. However, these differences were not statisti­cally significant (Table 2).

Table 1. On–host behaviour of Rhipicephalus appendiculatus released at various locations on zebu steers resulting from treatment of the predilection feeding sites with either petroleum jelly (Control) or the essen­tial oil of either T. minuta (Tm) or T. diversifolia (Td) (n = 20)

Site of
tick release

Treatment

No. of ticks
attached near and
or at the ear base

No. of ticks
attached at
the ear pinna

No. of ticks
attached in
the NRP1

No. of
ticks that
dropped off

Percentage of
non-responding
ticks2

1. Forehead

Control

10

7

2

1

0

Tm

6

0

4

10

0

Td

6

1

1

11

5

2. Dewlap

Control

6

7

2

0

25

Tm

2

0

6

12

0

Td

8

0

6

6

0

3. Foreleg

Control

4

3

9

3

5

Tm

1

0

7

5

35

Td

5

0

4

5

45

4. Rear leg

Control

2

1

6

0

55

Tm

0

0

4

4

60

Td

4

0

4

0

60

5. Escutcheon

Control

5

9

4

0

10

Tm

2

0

11

7

0

Td

5

0

12

3

0

6. Shoulder

Control

5

10

3

1

5

Tm

1

0

8

11

0

Td

4

0

4

12

0

1 NRP–Neighborhood of Release Points (body locations).
2
The term ‘responding’ refers to the percentage of the total number of ticks at different release points that initiated any movements within and or away



Table 2. On–host walking behaviour of Rhipicephalus appendiculatus to the predilection feeding site; ticks were released at six different locations on zebu cattle (n = 20)

Site of
tick release

Mean response1
time (h) (± SE)

Mean initial
walking speed2
(cm/min) (± SE)

Mean time taken to
reach the ear pinna3
(h) (± SE)

Number of ticks
reaching the
ear pinna

(a) Animals treated with petroleum jelly (control experiment)

1. Forehead

0.01 ± 0.01e

2.29 ± 0.22ab

1.76 ± 0.44c

7

2. Dewlap

0.53 ± 0.28ab

1.12 ± 0.06cd

3.06 ± 0.34bc

7

3. Foreleg

0.90 ± 0.29a

0.73 ± 0.04d

3.40 ± 0.67b

3

4. Rear leg

0.92 ± 0.44a

0.70 ± 0.05d

8.60

1

5. Escutcheon

0.05 ± 0.03d

2.10 ± 0.21b

5.38 ± 0.21a

9

6. Shoulder

0.09 ± 0.03d

1.55 ± 0.21bc

3.53 ± 0.42b

10

(b) Animals treated with essential oil of Tagetes minuta

1. Forehead

0.02 ± 0.01e

2.36 ± 0.34ab

-

0

2. Dewlap

0.13 ± 0.03cd

2.53 ± 0.31a

-

0

3. Foreleg

0.71 ± 0.12ab

0.95 ± 0.17cd

-

0

4. Rear leg

0.66 ± 0.05ab

0.66 ± 0.08d

-

0

5. Escutcheon

0.06 ± 0.01d

2.98 ± 0.31a

-

0

6. Shoulder

0.02 ± 0.01e

2.71 ± 0.31a

-

0

(c) Animals treated with essential oil of Tithonia diversifolia

1. Forehead

0.04 ± 0.01d

2.01 ± 0.17b

12.84

1

2. Dewlap

0.37 ± 0.06c

1.62 ± 0.19bc

0

3. Foreleg

0.51±0.19ab

0.69±0.10d

-

0

4. Rear leg

0.98±0.28a

0.84±0.06d

-

0

5. Escutcheon

0.08±0.03d

1.93±0.11bc

-

0

6. Shoulder

0.08±0.02d

2.16±0.17ab

-

0

For a given column, means followed by the same letter are not significantly different from one another at p = 0.05 (Student-Newman-Keuls test).
1
Time taken by ticks from placement on the host animal to initial movement within and or away from the release points.
2
The walking speed of the ticks from time they initiated their first movement within and or away from the release points until they stopped.
3
Time taken by ticks from placement on a given release point to the arrival at the predilection feeding site on the host animal.
- Indicates that no tick from the corresponding site of release on the host body reached the predilection feeding site, the ear pinna
.


Discussion

In this study, we evaluated the on-host effects of essential oils of T. minuta and T. diversifolia plants from Bungoma District, western Kenya against newly hatched and 24 h–starved adult R. appendiculatus under field conditions. This is one of the few studies in which the essential oils of putative repellent plants are evaluated in the presence of host–derived attractive and repellent stimuli (Sika 1996; Dautel et al 1999; Wanzala et al 2004).

The formulated essential oil of T. minuta changed its colour gradually from yel­low to light–yellow and then to pale white–yellowish, a colour that was close to the car­rier material. This change in colour of the formulation may reflect the escape of some of the volatile compounds that constituted the essential oil composition or a biochemical change of oil components. The gradual change of the colour supported the fact that the many compounds previously identified by capillary Gas Chromatography (GC) and Gas Chromatography–Mass Spectrometry (GC–MS) (Singh et al 2003; Singh et al 2006; Anon 2006; Moghaddam et al 2007; Adekunle et al 2007), have different volatility properties and may behave differently under the same conditions. This may undoubtedly affect the repellent activity of the essential oil, particularly its efficacy and residual activity, thus causing it to take longer to cause significant effects (Cloyd and Chiasson 2007). In our studies, compounds that first escaped from the formulation could probably be the ones causing the ticks to drop off at a high rate and thereafter, the low repellency of the remnant compounds allowed ticks to reach the predilection feeding site but after a much longer time. The effect of this was greater in the treatments than in the control thus demonstrating a behavioural effect of both the essential oils. This poses great challenges to the essential oil application in the field, but improvements in efficacy and residual activity may be realized with appropriate stable formulations (Thavara et al 2007). The essential oil of T. diversifolia could be behaving in a similar manner only that a colour change was not seen, as the formulation is colourless.

On-host observations of the ticks in our field site showed the typical sequence of behavioural patterns reported previously (Sika 1996; Wanzala et al 2004). In fact, the tick response pattern in the control experiment almost followed the pattern of on-host responses of ticks observed in Wanzala et al (2004). Although the responses of ticks at all release points was the same as that obtained in Wanzala et al (2004), the number of ticks successfully reach­ing the predilection feeding site was relatively low, implying that the essential oils ap­plied had a significant repellent effect. This effect was further manifested in the high number of ticks losing their way and dropping off in the essential oil–treatments, while another sizeable percentage of ticks did not respond at all. Few ticks in the control dropped off. Although most ticks responded at all release points, the orientation and appropriate navigation toward locating and attaching at the predilection feeding site was undermined by repellent effects of the essential oils. This also meant that the previously suggested mediation of specific attractive and repellent host–derived stimuli in the proc­ess of orientation and navigation toward predilection feeding sites (Wanzala et al 2004) was masked by the volatiles of the essential oils, as initially hypothesized.

With the disruptive effect of the two essential oils, the stereotyped sequence of orientation behaviours of R. appendiculatus on the host animal observed in the control experiment and described in Wanzala et al (2004), was not evident. This implied that the percentage of ticks reaching the predilection feeding site and exhibiting the unique complex set of behaviours, did so against the repelling forces of essential oils of T. minuta and T. diversifolia. Most notably missing in these stereotyped sequences of orientation behaviours were the second and third phases, involving random and directional movements, respectively. In the two essential oil treatments, ticks were observed randomly navigating on the host body and seeming to have accidentally arrived at the predilection feeding site, as they behaved in a manner that suggested that they were not affected by a feeding–site–specific cue.

The critical key variables of this study, the reaction time, initial speed and time between release and arrival at the predilection feeding site, were evaluated. However, other variables, such as the percentage of ticks at different sites that initiated movement (respondents), the percentage of ticks reaching feeding sites and the percentage of ticks that attached in the neighbourhood of release points, the percentage of ticks that dropped off after losing the way, and that which did not respond at all, were also evaluated. By comparing the percentage of ticks responding and those reaching the predilection feeding site for all six release points, the results showed relatively low rates of successful orientation, location and attachment of the ticks to their predilection feeding site the ear pinna, with the highest numbers coming from the controls followed by the animals treated with T. diversifolia essential oil and then T. minuta essential oil, in that order. This suggests the masking effect of natural tick attractant stimuli that guide ticks to a predilection feed­ing site by the two essential oils, which again differ in the way they mask. This supports our hypothesis that intercepting the tick movement toward a predilection feeding site with repellent essential oils of T. minuta and T. diversifolia may provide a prophylactic mechanism to protect animals from tick bites and must be considered as a management strategy. This control strategy works at individual level and reduces tick–host contact and, subsequently, reduces the chances for the transmission of Theileria parva parasite that causes the East Coast fever (ECF) in the hosts (Muthuswami and Nisha 2006). It is possible that in the presence of repellent essential oils, even attached ticks may not sus­tain the attachment for the period of time (between 24 and 72 h) that is sufficient to allow an effective transmission of the tick–borne pathogen (T. parva) to the host (Ochanda et al 1988; Konnai et al 2007).

That the essential oil of T. minuta has a higher repellent effect than the essential oil T. diversifolia, is manifested in the fact that treatment by the former causes the highest percentage of ticks to drop off the host from various body locations and that none of the ticks got attached to the ear pinna, the predilection feeding site. A considerable number of ticks in the control became attached to the ear pinna and just a few dropped off, whereas only one tick got attached to the ear pinna of the animals treated with the essential oil of T. diversifolia, where a considerable percentage of ticks dropped off the host body. Although both essential oils showed significant effects on R. appendiculatus locating their preferred feeding site, the essential oil of T. diversifolia did not prevent the ticks from becoming attached to the ear base, like its counterpart.

The data further support results obtained previously in Wanzala et al (2004) regarding the re­sponses of ticks deposited on body locations further away (escutcheon, upper rear legs and forelegs) and close to (forehead and shoulder) predilection feeding sites (Wanzala et al 2004). For instance, although release site 5 (escutcheon) was relatively furthest removed from the predilection feeding site, the ticks oriented responses and behavioural navigations from this point were similar to those of ticks navigating from body locations closer to the ear pinna. For these two regions on the host animals, there was no significant difference in the mean reaction time, initial speed and time taken to reach the predilection feeding site. These observations imply the previously-noted operation of both avoidance (closer to none predilection feeding sites) and attraction (closer to the predilection feeding sites) responses of the ticks (Sika 1996; Wanzala et al 2004). However, the influence of the essential oil can not be ruled out. Also the fact that more ticks responded in the treatments than in the controls, may have been due to the repellent effects of the essential oils. For instance, from release points of the host animals treated with the essential oils of T. minuta, the ticks’ mean reaction time was lower and initial speed higher than either those from the control animals or the animals treated with the essential oil of T. diversifolia. Therefore, it is possible that these essential oils can be used to confuse the ticks while on the host, and adversely affect their feeding habits through which they transmit the etiologic agents of tick–borne diseases (Wanzala et al 2017).


Conclusions


Acknowledgements

This research was jointly supported by the International Foundation for Sciences, Stockholm, Sweden and the Organization for the Prohibition of Chemical weapons, The Hague, The Netherlands through a grant AB/12782–2. The first author wishes to acknowledge the financial and material support received from the International Centre of Insect Physiology and Ecology (ICIPE) under the African Regional for Postgraduate Programmes in Insect Science (ARPPIS) and the Wageningen University and Research Cen­tre, the Laboratory of Entomology under PhD Sandwich Fellowship. The authors wish to thank Mr. Newton Komeri Mwanga for illustrations and ICIPE staff for their technical support.


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Received 2 May 2018; Accepted 13 May 2018; Published 1 June 2018

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