Insecticidal Activity of Some Traditionally Used Ethiopian Medicinal Plants against Sheep Ked Melophagus ovinus

Twelve medicinal plants and a commercially used drug Ivermectin were examined for insecticidal activity against Melophagus ovinus sheep ked at different time intervals using in vitro adult immersion test. The findings show that at 3.13 µL/mL, 6.25 µL/mL and 12.5 µL/mL concentration of Cymbopogon citratus, Foeniculum vulgare and Eucalyptus globulus essential oils respectively, recorded 100% mortalities against M. ovinus within 3 hour of exposure. Significantly higher insecticidal activity of essential oils was recorded (P = 0.00) when compared to 10 μg/mL Ivermectin after 3-hour exposure of M. ovinus at a concentration of ≥1.57 μL/mL, ≥3 μL/mL, and ≥12.7 μL/mL essential oils of C. citratus, F. vulgare, and E. globulus, respectively. Among essential oils, C. citratus has showed superior potency at a three-hour exposure of the parasite (P = 0.00) at a concentration of ≥0.78 μL/mL. Strong antiparasitic activity was recorded by aqueous extract of Calpurnia aurea (80% mortality) at a concentration of 200 mg/mL within 24 h among aqueous extracts of 9 medicinal plants. The results indicated all the four medicinal plants, particularly those tested essential oils, can be considered as potential candidates for biocontrol of M. ovinus sheep ked.


Introduction
In Ethiopia, a livestock sector contribute for 12% of the total Gross Domestic Product (GDP), and provides livelihood for 65% of the population and also it accounts for 37-87% out of the total house hold cash income. For many years not only the export of livestock but also the export of processed and semiprocessed skins and hide constitutes Ethiopia's second largest export commodity by accounting for 12-15% of the total export earnings next to coffee [1,2]. This has been deteriorating with an increasing number of reject grades and appearance of a skin disease called "Ekek" that is mainly due to ectoparasites infestation [3,4]. Thirty years ago, tanneries in Ethiopia used to produce 70% of processed skins with grades 1-3. About 10-20% of the skins were graded as being of poor quality. Currently, only 10-15% is in the good category while the rest are downgraded or rejected due to increasing external parasite infestations [5]. Ectoparasite infections have been responsible for major economic loss in leather industry in Ethiopia in which approximately more than half of the skin reject occurs [3,4,6].
Among ectoparasites, Melophagus ovinus sheep keds are the most economically important pests of sheep [4,[7][8][9]. Melophagus ovinus sheep ked is a voracious blood sucker causing great irritation to the sheep and forcing them to scratch and bite at themselves [10]. The parasites are reported to cause discomfort and annoyance that leads to reduction of weight gain, wool growth, and milk production due to nervousness and improper nutrition of the animals in which they spend less time feeding. Moreover, sheep ked is reported to cause inflammation, and as the host attempts to alleviate this irritation, wool loss and skin damage can be caused.
Other report, showed that heavily infested animals become weak and unthrifty and show weight loss, anemia, wool staining, reduced resistance to disease, and a condition called "cockle" which is a series of pimple-like bumps on the skin of the sheep in areas where keds have fed [3,7]. They reduce the value of the skin. If skins are used in the tanning industry, the bumps will not accept dye like the rest of the pelt and the pelt no longer demands high prices.
In addition, M. ovinus sheep ked reduces the growth rates and also causes paralysis and injuries which lead to secondary infection and expose the animal to cutaneous myiasis [11]. Further economic losses result from the effects of feeding and scratching on the skin with hard nodules (cockle) reducing the value of the hide. Wool loss from scratching and biting and the staining of the fleece caused by ked feces in the fleece are further economic effects [10]. Sheep ked also creates financial burdens of diagnostic, therapeutic or preventive programs at flock, community and national levels.
As a result control strategies are looked for to reduce disease and limit losses in the animal husbandry industry to or below acceptable economic damage thresholds [12,13]. Highly potent chemical insecticides such as organophosphate compounds are routinely and extensively used interventions in ectoparasite control of livestock [5]. However, widespread intensive use of synthetic insecticide has led to pesticide resistance in parasite populations [7,14,15]. Besides, their continuous application was coupled with environmental pollution and health concerns by causing the contamination of ground water by particle leachates and toxicity of the chemicals on animals and increasing incidence of human skin, lung, nerve disease upon exposure to these chemicals [7].
Most of the pesticides that are currently used against the various animal ectoparasites in Ethiopia are organophosphorus compounds which cause both contact and stomach poison [16]. They are acting by combining with, and neutralizing cholinesterase action, an enzyme responsible for the hydrolysis of acetylcholine which is synthesized at nerve endings and is involved in the transmission of impulses from nerve to nerve or effecter cells [16,17]. Previous investigations in Ethiopia have clearly indicated depression of cholinesterase activity associated with symptoms of intoxication among agricultural workers due to lower plasma cholinesterase and erythrocyte cholinesterase of the workers [18,19].
As the traditional approach to control M. ovinus infestations has been only partially successful due to the costs of insecticides, resistance, and environmental contamination, alternative anti-sheep ked products and or control strategies that are safe, cost-effective, and environmentally friendly are therefore necessary [20]. Ethnoveterinary medicine (EVM) specially the use of medicinal plants could be a promising area of alternative sheep ked control strategies. The value of medicinal plants as commercial sheep ked control agents has yet to be proven, but their high safety and effectiveness make them promising candidates for use as commercial sheep ked biocontrol agents [21]. (Olacaceae) were mainly used in EVM [22][23][24][25][26][27][28]. The study was therefore conducted to evaluate the in vitro bioinsecticidal efficacy of indigenous plants used in EVM practice in Ethiopia for the control of M. ovinus sheep ked.

Experimental Parasite Collection and Maintenance.
Melophagus ovinus sheep ked parasite was used in this study to conduct in vitro insecticidal assay of medicinal plants. Sheep keds were collected from naturally infested sheep around Fiche, North Shewa, Oromia Zone, Ethiopia. The identity of the parasites was confirmed on site by veterinary professionals found in the collection area. After their identification, the parasites were maintained in plastic cups into which water soaked cottons were placed to increase the humidity of the air found in the cups. The cups were covered by gauze to allow the free circulation of air into the cups. And the parasites were transported to the Fiche Animal Health Clinic Laboratory where the experimental works were conducted.

Plant Material Collection, Identification, and Extraction.
Eucalyptus globulus and Cymbopogon citratus essential oils were obtained from Wondo Genet Agricultural Research Center, Ethiopia. Essential oils of Foeniculum vulgare Mill. were extracted by hydrodistillation of the fresh leaf which was collected from Shashamane, Ethiopia. Fresh plant materials (250 g) were placed in a 5 L round-bottom distillation flask and the plant material was wetted with 3 L distilled water. The essential oils were obtained by hydrodistillation using Clevenger-type apparatus for continuous 3 h. The volatile oil was taken from the upper layer. The excess aqueous layers were further portioned using dichloromethane extract and enrich the essential oil from the water layer. The organic layer (dichloromethane extract) was filtered and dried with anhydrous sodium sulfate and concentrated using rotary evaporator to give the crude essential oil.
Three hundred grams of dry powdered leaves of Calpurnia aurea (leaves), Syzygium guineense (leaves), Bersama abyssinica (leaves), Croton macrostachyus (leaves), Nicotiana tabacum (leaves), Vernonia auriculifera (leaves), Aloe species (aerial part), and the fruits of Jatropha curcas and Ximenia caffra was macerated with 25-50 mL water. The aqueous extracts were filtered using gauze and freeze dried using lyophilizer. The volatile oils and the amorphous residues from the aqueous macerate are kept in the refrigerator and  desiccators, respectively, until used for the in vitro insecticidal susceptibility assay.  13 L/mL, 6.25 L/mL, and 12.5 L/mL, respectively. Sheep ked mortality increased as concentration and exposure time to the essential oil increased. Statistical results (one-way ANOVA (unstacked)) have showed significantly higher insecticidal activity of essential oils ( < 0.01) after a 3 h exposure of M. ovinus to concentrations of ≥1.57 L/mL, ≥3 L/mL, and ≥12.7 L/mL essential oils of C. citratus, F. vulgare, and E. globulus, respectively, as compared with 10 g/mL Ivermectin. Among the essential oils, at 3 h exposure of the parasite, significantly higher ( < 0.01) insecticidal activity was exerted by C. citratus at a concentration ≥0.78 L/mL.

Results
Mortality of M. ovinus sheep keds exposed to aqueous extract of C. aurea, X. caffras and J. curcas at concentration of 6.25, 12.5, 25, 50, 100, and 200 mg/mL is shown in Table 2. The maximum antiparasitic activity was exerted by aqueous extract of C. aurea. Eighty percent, 60% and 66.3% mortality of M. ovinus sheep keds were observed after 24 h of exposure to 200 mg/mL of C. aurea, X. caffra, and J. curcas, respectively. The highest insecticidal efficacy was recorded by C. aurea with (73.3%) mortality followed by X. caffra (60%) and J. curcas (56.7%) at a concentration of 200 mg/mL, within 3 hours of the parasite exposure.    3.3 ± 0.6 de 6.7 ± 0.6 d 0.0 ± 0.0 c 6.6 ± 0.6 de 6.7 ± 0.6 bc 6.7 ± 0.6 de 25 13.3 ± 0.6 cd 13.9 ± 0.6 cd 6.6 ± 0.6 c 16.7 ± 0.  Table 3 indicated the antiparasitic activity of different concentrations of aqueous extracts of S. guineense, N. tabacum, and V. auriculifera against M. ovinus sheep keds. A 43.5% mortality of sheep keds was recorded by S. guineense at a concentration of 200 mg/mL within 3 h of exposure, while a comparatively lower mortality rate (20%) was recorded by both N. tabacum and V. auriculifera within 3 h of exposure. All extracts exerted significantly lower insecticidal effect when compared to 10 g/mL Ivermectin ( < 0.05).
The effects of Aloe sp., B. abyssinica, and C. macrostachyus on M. ovinus sheep ked are also shown in Table 4. The exposure of M. ovinus sheep ked to the dose of 200 mg/mL of Aloe sp., were showed mortality of 30% within 3 h. Relatively, a higher antiparasitic potential was recorded by Aloe sp. than the other species.

Discussion
Though Ethiopia has a potential of supplying livestock and livestock products, the sector is facing multiple challenges and ectoparasites continued to be highly prevalent; even the most commonly used insecticides are becoming ineffective due to the development of resistance in addition, environment pollution and health risk of the insecticide [7]. These problems had stimulated a great deal of research to look for alternative treatment from natural products. For this purpose we evaluated activity of twelve traditionally used Ethiopian medicinal plants for the alternative management of sheep ked infestation. Application of plants essential oils or plant extracts for the control of pest has been used for hundreds of years by the community and practitioners of traditional medicine. Similarly, various studies showed the antiparasitic and insecticidal properties of the plant essential oils against phytophagous pests, ticks [31], and mites [22,23].
Our findings have confirmed that essential oils from C. citratus, E. globulus, and F. vulgare have antiparasitic activity against M. ovinus. Essential oil of C. citratus has pronounced insecticidal activity followed by F. vulgare and E. globulus. Complete immobility of the sheep ked parasite was recorded at a concentration of 3.13 L/mL, 6.25 L/mL, and 12.5 L/mL essential oils of C. citrates, F. vulgare, and E. globules, respectively. The insecticidal effect of the essential oils are depends on both dose and type of plant species as the significant effect was recorded at higher dose level. Moreover, the highest mortality effect against M. ovinus was observed by C. citratus which is considered as the best insecticidal agent among the investigated plants. Our finding that essential oils have insecticidal activity is in line with several studies where they indicated that many essential 6.7 ± 0.6 cd 6.7 ± 0.6 c 6.7 ± 0.6 d 6.7 ± 0.6 d 0.0 ± 0.0 d 0.0 ± 0.0 d 25 13.3 ± 0.6 c 13.3 ± 0.6 c 6.7 ± 0.6 d 6.7 ± 0.6 cd 0.0 ± 0.0 d 6.7 ± 0.6 cd 50 13.3 ± 0.6 c 13.3 ± 0.6 c 6.7 ± 0.6 cd 6.7 ± 0.6 bc 6.7 ± 0.6 cd 16.7 ± 0.6 bc 100 30 oils could possibly substitute synthetic chemicals [32], with higher insecticidal effect of C. citratus against mite [26], insecticidal potentials of F. vulgare fruit against mite and a significant dose dependent insecticidal effect of E. globulus against tick [33]. The mortality of the parasite may be due to the octopaminergic nervous system interference by these essential oils [34]. Similarly, the activity could be due to complex mixture of secondary metabolites like terpenoids, phenolics of mono-, di-and sesquiterpenes, specifically citral, elemicin, (+)-fenchone, and p-anisaldehyde, and 1,8-cineole in the essential oils [34]. The difference in bioactivity between the essential oils may be due to the difference in major compounds, as biological activity of essential oils depends on chemical structure of their components [35,36]. Moreover, chemical components may be toxic to various pests individually or in combination. Activity may be also influenced by the arrangement of multiple components in the essential oils as a result of synergistic effects [23]. Another notable finding of the current study was that aqueous extract of plants has insecticidal activity. Promising activity (80% mortality) was recorded by C. aurea, while 60% mortality by X. caffra, and about 57% mortality by J. curcas are still worth a credit as potential source of botanical insecticide. Our study that confirmed C. aurea has remarkable antiparasitic activity replicates a previous study [24], which reported 85% mortality of tick at 5% concentration of C. aurea. Moreover, aqueous extracts of J. curcas and X. caffra have a potential of killing or immobilizing M. ovinus sheep keds which is in line with previous study [27].
Moreover, the finding of current study showed that aqueous extracts of S. guineense, N. tabacum, and V. auriculifera have moderate toxicity against M. ovinus sheep keds. A mortality of 47% was recorded within 24 h of exposure of sheep ked parasite to 200 mg/mL of S. guineense. The antiparasitic effect observed by S. guineense was also supported by the findings of closely related species [25] on the ethanolic extract of Syzygium cumini that showed the highest antiparasitic (98.5% mortality) effect. Our findings also replicate the finding of Zaman et al. [28] whereby mortality of tick parasite was reported to be in combination with application of Azadirachta indica and N. tabacum leaves, Calotropis procera flowers, and Trachyspermum ammi seeds aqueous extracts.
Many of the plants used in this study which showed insecticidal activity are reported to have ethnoveterinary medicine practices in Ethiopia as elsewhere in the world [37]. However, our finding showed that aqueous extracts of Aloe sp., B. abyssinica, and C. macrostachyus have lower lethality activity against M. ovinus when compared to other plants and that of the commercially used antiparasite Ivermectin. This could be due to the extraction method we used; one can find higher activity by using other extractants than water. Similarly, various studies reported the antihelminthic activity of Aloe sp. [38] and the antimicrobial activity of B. abyssinica [39] and C. macrostachyus [40]. This could be an indication of the broad spectrum activity of the plant extracts.

Conclusion
The current study showed that the essential oils of C. citratus, E. globulus, and F. vulgare and the aqueous extract of C. aurea have strong insecticidal activity. The results of this study offer some scientific credence to the traditional uses of the Ethiopian medicinal plants evaluated for the management of Sheep ked parasites. There are many opportunities for the use of plant essential oils as alterative to synthetic insecticide; one is their insecticidal potency and environmental sound nature, easy accessibility by the rural pastoralist community, and superiority over synthetic antiparasite Ivermectin. Another important opportunity is their easy application as fumigant and topical treatment. Regarding C. aurea, it can be one potential alternative to synthetic insecticide to control sheep ked which is advantageous by its wide occurrence in Ethiopia and its capacity to resist drought and overgrazing. There is, however, a need for further investigation on their safety and efficacy (in vivo and under natural conditions) as well as costeffectiveness of the products that exhibited strong insecticidal activity with a view of substituting the conventional organophosphorus drugs. And also we recommend bioassay guided fractionation, isolation, and characterization of the responsible active components of the plant materials.