Effects of essential oils of lemon grass, Cymbopogon citratus and the Mexican marigold, Tagetes minuta on mortality and oviposition in adult sandflies, Phlebotomus duboscqi

The efficacy of the essential oils of Cymbopogon citratus and Tagetes minuta in causing mortality in adults of the sandfly Phlebotomus duboscqi was tested in a laboratory bioassay. The effect of the oils on oviposition by female sand flies was also evaluated. Each essential oil (EO) extract was tested at graded concentrations of 0.125; 0.250; 0.500; 0.750 and 1mg/ml in Tween 80 and a positive control of dimethyl-3methylbenzamide (DEET). The oil and control preparations were applied onto the inner surface and bottom of a sterile pot and thirty adult sandflies P. duboscqi aspirated into the pot. The mortality of insects was recorded as the number of dead flies after 24, 48 and 72 h. In addition, the numbers of eggs oviposited by female flies that were subjected to the different treatments were recorded. The results showed that both the essential oils of T. minuta and C. citratus were highly potent against adult sand flies, P. duboscqi with mortality levels of 100.00 and 82.22 % on female sandflies and 100.00 and 88.89 % on male sandflies, respectively after 72h. Comparing the mortality levels caused by the two oils, which of C. citratus was significantly higher (P < 0.05) than the oil of T. minuta on male and female sandflies. However, at the shorter periods there was no statistical difference in mortality levels of males and females subjected to each of the two oils. With regard to oviposition, female sandflies treated with the oil of C. citratus oil laid significantly lower number of eggs than those laid by sand flies treated with T. minuta oil. Furthermore, gas chromatography-mass spectrometric analysis done on samples of the two oils showed a wide range of candidate compounds, including terpenes. In conclusion, the two essential oils are promising natural insecticides due to their safety advantage over chemical insecticides. It remains to carry out further studies in the field using human subjects before their adoption for use against Phlebotomine sandflies. In addition bioassays with individual and combinations of the constituent compounds of the essential oils may determine the candidate biologically active compounds.


Introduction
The blood-feeding females of phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are usually considered to be the only natural vectors of protozoan Leishmania species (Euglenozoa: Trypanosomatidae), the causative agents of the neglected tropical disease leishmaniasis [1,2]. Of approximately 900 sand fly species, no more than 70 have been implicated in leishmaniasis transmission [3][4][5]. However, fewer have been associated with Phlebovirus and other arboviruses of biomedical importance [3,4,6] and only one species, Lutzomyia verrucarum sensu lato is the vector of the alphaproteobacterium Bartonella bacilliformis, which causes Carrion's disease in a limited Andean region in South America [7,8].
The World Health Organization (WHO) estimates that over 2.3 million new cases of leishmaniasis occur each year and that, at least 12 IJBR (2015) 6 (09) www.ssjournals.com million people are presently infected worldwide [9]. In Kenya, phlebotomine sandflies transmit both visceral and cutaneous leishmaniases. Visceral leishmaniasis (VL), caused by Leishmania donovani is transmitted by Phlebotomus martini (Diptera: Psychodidae) [10,11]. On the other hand, Phlebotomus duboscqi sandflies transmit L. major, one of the causative agents of cutaneous leishmaniasis (CL) [12]. The current management strategy for leishmaniasis in Kenya is mainly based on chemotherapy for treatment of infected cases and use of insecticides in vector control to reduce vectorhuman contact, hence minimize transmission of the protozoans [13,14]. Vector control using insecticides has been recommended by the WHO [15]. However, acquired resistance and environmental pollution due to the repeated application of persistent synthetic insecticides have led to increased interest in new natural chemicals [16]. In addition, usage of highly persistent and toxic synthetic insecticides has led to development of resistance in vector populations. Further, environmental pollution due to the repeated applications is a challenge. Thus, the harmful side effects of these chemicals on both animals and humans have progressively limited their usage and have led to increased interest in alternative new natural chemicals that are environmentally safe, affordable and effective in management of leishmaniases. In this context, screening of natural products has received the attention of researchers around the world. Since many diseases that are transmitted by insects such as malaria, dengue fever, yellow fever, leishmaniasis and Chaga's disease are endemic in developing countries, the search for insecticides and repellents of botanical origin has been driven by the need to find new products that are effective, but also safer and more affordable than currently available products [17].
In recent years, the use of essential oils (EOs) derived from aromatic plants as low-risk insecticides has increased considerably owing to their popularity with organic growers and environmentally conscious consumers [18]. EOs are easily produced by steam distillation of plant material and contain many volatile, low-molecular-weight terpenes and phenolics. EOs have repellent, insecticidal, and growth-reducing effects on a variety of insects [19]. They have been used effectively to control preharvest and postharvest phytophagous insects and as insect repellents for biting flies and for home and garden insects. The compounds exert their activities on insects through neurotoxic effects involving several mechanisms, notably through GABA, octopamine synapses, and the inhibition of acetylcholinesterase.
With a few exceptions, their mammalian toxicity and environmental persistence are low.
Essential oils of an appreciable number of plants have been shown to be repellent against various haematophagous arthropods [20,21]. The oils of lemongrass, Cymbopogon spp., are the most widely used natural repellents worldwide [22]. For example, essential oils from Cymbopogon martinii martinii elicited 100% repellency against Anopheles sp. mosquitoes in field tests for 12 hours [23]. Essential oil of Cymbopogon winterianus, mixed with 5% vanillin, gave 100% repulsion against Aedes aegypti, Culex quinquefasciatus and Anopheles dirus for 6 hours [24]. Lemongrass, Cymbopogon citratus essential oil is obtained from the aerial parts of the plant. The plant has been widely recognized for its enthnobotanical and medicinal usefulness [25]. Other documented effects of essential oils of plants include insecticidal [26][27][28][29][30][31], antifungal [32], antimicrobial [33,34], and the therapeutic properties [25]. However, there are relatively few studies that have been carried out to determine the efficacy of essential oils from citronella as arthropod repellents [35], in particular, against sandflies (Diptera: Psychodidae).
Furthermore, the essential oil of the Mexican marigold, Tagetes minuta L., has extensively been tested against several species of mosquitoes and shown to have both larvicidal and adulticidal effects on mosquitoes [36][37][38]. The active components were isolated from different parts of the plant. Green et. al. [36], reported mosquito larvicidal activity in the extract of Tagetes minuta flowers. Perich et al. [ 37] compared biocidal effects of the whole-plant steam distillates of three Tagetes spp. and showed that, T. minuta had the greatest biocidal effect on the larvae and adults of Ae. aegypti (L.) and Anopheles stephensi (L). Recently, Ireri et al. [39] demonstrated that, methanol and ethyl acetate crude extracts of the aerial parts of T. minuta had significant mortality against both male and female P. duboscqi, Neveu Lemaire (Diptera: Psychodidae). Further, Mong'are et al. [40] found that, similar crude extracts reduced the fecundity of P. duboscqi by 53%. No similar work has been reported for the essential oil of C. citrates. In addition, the causative candidate compounds in the essential oils of T. minuta and C. citratus have not been identified.
In the light of the foregoing, the present study sought to evaluate the insecticidal effects of the essential oils of the lemon grass, C. citratus and T. minuta against adult sandflies, P. duboscqi. The effect of the oils on oviposition by female flies was also determined. In addition, the constituent compounds in these oils were identified using coupled gas chromatograph-mass spectrometric (GC-MS) analysis.

Sand fly colony
Sandflies were obtained from a colony of P. duboscqi Neveu Lemaire that originated from Marigat Division, Baringo district, Rift Valley, and were maintained at the Centre for Biotechnology Research and Development (CBRD) insectaries in Kenya Medical Research Institute, Nairobi. The colony of P. duboscqi was established using fieldcaptured females that were held in cages and maintained according to the methods of Beach et al. [47], with some modifications. Briefly, female sandflies were fed on blood using Syrian golden hamsters that were anaesthetized with sodium pentobarbitone (Sagatal®). The hamsters' under bellies were usually shaved using an electric shaver for easy access for feeding by sandfly. The sandflies were reared at 28 ± 1°C, and an average RH of 85-95% and 12:12 h (light: dark) photoperiod in Perspex® insect rearing cages. Sandflies were fed ad libitum on slices of apple that were supplied daily as a source of carbohydrates.

Collection of plant materials
Fresh leaves of the lemon grass, Cymbopogon citratus were collected from the equatorial rainforest in Kakamega, Kenya. The plant identity was confirmed by a taxonomist and a voucher specimen was deposited at KEMRI's Center for Biotechnology Research and Development (CBRD) for future reference. The leaves were screened and dry and/or damaged ones were discarded. The remaining good leaves were used for extraction while still fresh. On the other hand, floral and foliar parts of T. minuta plants were collected from Marigat District of Baringo County, Rift Valley region, Kenya. The plant parts were packed in a cold box and transported to the International Centre for Insect Physiology and Ecology (icipe), Kasarani, Nairobi, Kenya where extraction of the essential oils was done. The plant identity was also confirmed by a taxonomist and a voucher specimen was deposited at KEMRI's CBRD for future reference.

Extraction of essential oils of Tagetes minuta and Cymbopogon citratus
Extraction of the essential oil of the lemon grass c. citratus was done as described by [41]. The fresh leaves were immersed in distilled water after which they were subjected to steam distillation. The mixture of steam and the volatile oil generated was passed through a condenser and collected in a flask. Then, a separating funnel was used to separate the oil from water. The recovered oil was dried using anhydrous sodium sulphate and kept in a refrigerator at 4 o C for subsequent use [41].
For the extraction of the essential oil from T. minuta, fresh plant material was sliced and hydrodistilled by using a Clevenger-type apparatus [42], with slight modifications [43]. Heat was provided by a heating-mantle equipped with a thermostat and the temperature maintained at 90 °C. The plant material was immersed in distilled water then placed into a 2 litre round-bottomed flask and hydro-distilled for 2 hours. The distillate was collected as the essential oil band above the water [44].

Bioassays on sand flies with essential oils
Each oil concentration (1.0 ml), and the controls, DEET and Tween 80 were applied to the inner surface and bottom of each pot using a pipette. Thirty adult P. duboscqi flies (15 males and 15 females) were released inside of the pots after the application of the oils, and the concentrations that were used were from 0.125; 0.250; 0.500; 0.750 and 1mg/ml of C. citratus and T. minuta essential oils. In this experiments, the parameters observed were insect mortality after 24, 48 and 72 h, mortality rate differences between female and male insects and the number of eggs obtained from females subjected to the oils; The percentage mortality was calculated by using the formula below; Percent mortality = Number of dead adults X 100 Number of adults introduced The corrections for mortality when necessary were done using Abbot's (1925) formula Corrected percentage mortality = % Kill in treated -% kill in control X 100 100 -% Kill in control

Ethical considerations
Approval for the study was sought from Kenya Medical Research Institute's ethical review committee (IREC) and the Board of Postgraduate Studies of University of Eldoret. The experiments were done in compliance with KEMRI's Animal Care and Use Committee (ACUC) and in conformity with Good Laboratory Practices (GLP).

GC-MS analysis of essential oil of Tagetes minuta and Cymbopogon citratus
The analysis of the essential oils was carried out in the Behavioural and Chemical Ecology Dept. laboratory at the International Centre of Insect Physiology and Ecology (icipe), Nairobi. Samples of essential oil of each of the two plants were diluted in high purity (99.9%, Sigma, Aldrich) dichloromethane were analyzed on a coupled GC-MS using a Hewlett Parckard (HP) 7890 Series A gas chromatograph (Agilent technologies, Wilmington, DE, USA) coupled to a 5975 C Series mass spectrometer fitted IJBR (2015) 6 (09) www.ssjournals.com with an 7683 B Series autosampler (Agilent technologies, Wilmington, DE, USA) and a triple axis detector [45]. The GC was equipped with a nonpolar capillary column (HP5 MS 5% with phenylmethyl silicone; 30 m long × 0.25 µm (i.d.) and 0.25 µm (film thickness)) for the separation of the chromatographic peaks. The GC was also coupled to a HP monitor (L1710) for displaying chromatographic data which will be acquired and studied using the 3365 MSD ChemStation software (G1701Ea E.20.00.493). Samples were injected in the split mode at a ratio of 1:10 -1: 100. The injector was kept at 250°C and the transfer line at 280°C. The column was maintained at 50°C for 2 min and then programmed to 260°C at 5°C/min and held for 10 min at 260°C. The MS was operated in the EI mode at 70 eV, in m/z range 42-350. Identification of the compounds was performed by comparing their retention indices and mass spectra with those found in literature [46] and supplemented by Wiley and QuadLib 1607 GC-MS libraries. The relative proportions of the essential oil constituents were expressed as percentages obtained by peak area normalization, all relative response factors being taken as one [32].

Data analysis
All experiments were done in replicates. Data on adult mortality was recorded using the Microsoft Excel programme. Control groups in the experimental bioassays with >20% mortality were repeated. Where mortality in the control groups fell between 5 and 20%, the observed mortality was corrected using Abbott's formula [48]. The dose mortality data was analysed by log-probit method of Finney [49] and lethal concentrations for 50% (LD 50 ) and 90% (LD 90 ) determined. Statistical significance of the recorded mortality of the various test concentrations and the controls were analyzed using one-way analysis of variance (ANOVA) at P < 0.05.

Bioassays with essential oils
Insecticidal effects of the essential oils of C. citratus and T. minuta on adults of the sandfly, P. duboscqi 24, 48 and 72 h after treatment are shown in Tables 3-5. Also, the number of eggs laid by female flies during the same period are included. Among the two oils, that of C. citratus was significantly (P < 0.05) more potent and caused higher mortality than that of T. minuta on against both male and female sand flies. The results show that, after 24 h, treatment with the oil of C. citratus at a concentration of 1 mg/ml caused a mortality of 91.11 and 88.89 % against female and male sandflies, respectively. However, the essential oil of T. minuta at the same concentration, recorded a relatively lower mortality of 71.11% 66.67 % in female and male sand flies, respectively. The results of this study demonstrate that, the effects of the oils were dose-dependent and increased with the concentration of the oil. The low concentrations tested inflicted low levels of mortality. This is clearly evident for all the concentrations tested with the lowest one (0.125 mg/ml) of C. citratus and T. minuta oils causing 51.11 and 28.89% mortality, respectively. Further, the mortality levels recorded also increased with time. Thus, the highest mortality levels were observed at 72 h after treatment for all the concentrations tested. In fact, after 72 h after treatment, the essential oils of C. citratus and T. minuta at a concentration of 1 mg/ml recorded a mortality of 100.00 and 82.22 % respectively, on female sandflies. At the same concentration, C. citratus and T. minuta oils caused mortalities of 100.00 and 88.89% respectively, in male sandflies. There was no statistical difference in mortality rates between males and females subjected each of the two oils C. citratus and T. minuta at 24 h, 48 h and 72 h (P >0.05). However, there was a significant difference between the mortality rates of C. citratus and T. minuta (P< 0.05) observed for both male and females after 24 h (P=0.00014), 48 h (P=0.0000238) and 72 h (0.00084). The LD 50 values for C. citratus and T. minuta oils were 0.07mg/ml and 0.2 mg/ml respectively.   With regard to the number of eggs that were laid by female sandflies that were treated with the essential oils, those treated with the oil of C. citratus oil were significantly lower than those laid by sand flies that were treated with that of T. minuta oil (P< 0.05; P= 0.00084). In comparison with the controls, flies subjected to Tween 80 which was a negative control laid significantly higher (P> 0.05) number of eggs than those treated with the essential oils of C. citratus and T. minuta.

Chemical composition of Cymbopogon citratus
The volatile Lemon grass essential oil obtained from hydro distillation had the usual light yellow color, a lemony scent, and an extraction yield of 0.6% (v/w) when distilled from the fresh aerial parts of the plant, as was done in the present study. Thirty compounds which constituted 98.28% of the total oil were identified. The constituents identified by GC-MS analysis, their retention times and area percentages are summarized in Table 4. The oil was dominated by monoterpene hydrocarbons. This monoterpene fraction was characterized by a high percentage of Geranial (20.45%), Myrcene (14.24%), Neral (11.57%), and Verbenene (9.26%) among others.

Discussion
The bioassay results of this study demonstrate that both T. minuta and C. citratus are highly potent against P. duboscqi sandflies. Between the two oils tested, that of C. citratus was significantly more potent (P < 0.05) and caused higher mortality than that of T. minuta on both against male and female sandflies. The results further demonstrate that after 24 h, treatment with the oil of C. citratus at a concentration of 1 mg/ml caused mortality of 91.11 and 88.89 % against female and male sandflies, respectively while T. minuta oil at the same concentration, recorded a relatively lower mortality of 71.11% 66.67 % in female and male sand flies, respectively. The results of this study demonstrate that, the effects of the oils were dosedependent and increased with the concentration of the oil. The low concentrations tested inflicted low levels IJBR (2015) 6 (09) www.ssjournals.com of mortality. The highest mortality levels were observed at 72 h after treatment for all the concentrations tested. In fact, after 72 h after treatment, the essential oils of C. citratus and T. minuta at a concentration of 1 mg/ml recorded a mortality of 100.00 and 82.22 % respectively, on female sandflies. At the same concentration, C. citratus and T. minuta oils caused mortalities of 100.00 and 88.89% respectively, in male sandflies. The findings of this study concur with previous studies which demonstrated that C. citratus and T. minuta essential oils are effective against arthropods. Hanifah et al [50] was able to demonstrate that the mortalities from lemongrass extract were higher than neem for both topical and contact activities against the house dust mites Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus). At 50 % concentration, both 24 hrs topical and contact exposures to lemon grass resulted in more than 91% mortalities for both species of mites. At the same concentration and exposure time, neem resulted in topical mortalities of 40.3% and 15.7% against D. pteronyssinus and D. farinae respectively; contact mortalities were 8.0% and 8.9% against the 2 mites, respectively [50].
Previous studies have demonstrated various biocidal activities of plant natural oils and products against sandfly adults. Lutzomyia longipalpis Lutz & Neiva adults were killed by water extracts of the leaves of Antonia ovata Pohl (LD 50 =233mg/mL) and water extracts of the roots of Derris amazonica Killip (LD 50 =212mg/ mL) [51]. Also, Eucalyptus spp. essential oils exhibit toxic effects in contact with L. longipalpis adults. Thus, adulticidal effects were observed for lemon ironbark (E. staigeriana F. Muell) essential oil whose major components were limonene, Z-citral, α -citral (EC50 = 0.59mg/ml), and lemon eucalyptus (E. citriodora Hook) with the major chemical constituent being β-citronellal (ED50= 5.04mg/ml). Finally, E. globulus Labill with essential oil major component being 1,8-cin-eole with an effective concentration of 7.78mg/ml. The superior toxicity of lemon ironbark is evident from these and other data and is due presumably due to the activity of the major components of its essential oil, which were not individually evaluated for biological activity [52].
With regard to the observed reduction in the number of eggs oviposited by the treated female flies, in addition to there being possible adverse physiological effects on female sand flies, the mortality of the flies before ovipositing may have been a major factor.
The qualitative and quantitative analyses of the essential oil extract obtained from T. minuta in this study showed that there are six major components in the extract. The six compounds The major components of the essential oil were Dihydro-Tagetone (21.15%), (E)-Tagetone (16.21%), (Z)-Tagetone (14.99%), (Z)-beta-Ocimene (9.84%), Limonene (7.40%), and allo-Ocimene (6.69%) represent more than 70% of the essential oil The results of this study are consistent with those found by Moghaddam et. al. [64] and Garcia et al. [65]. The T. minuta essential oil used in this study was rich in terpenes, as determined by GC and GC-MS analyses.
Hanifah et al [50] (2011) reported that mortalities from lemongrass extract were higher than neem for both topical and contact activities. At 50 % concentration, both 24 hrs topical and contact exposures to lemongrass resulted in more than 91% mortalities for both species of mites. At the same concentration and exposure time, neem resulted in topical mortalities of 40.3% and 15.7% against D. pteronyssinus and D. farinae respectively; contact mortalities were 8.0% and 8.9% against the 2 mites, respectively. There was no difference in topical mortalities of D. pteronyssinus from exposure to concentrations of lemongrass and neem up to 12.50%; lemongrass was more effective than neem at the higher concentrations [50].
In conclusion, the essential oils of the two plants, C. citratus and T. minuta are promising IJBR (2015) 6 (09) www.ssjournals.com natural repellents due to their safety advantage over chemical repellents. It remains to carry out clinical studies on human subjects prior to their possible adoption for use against phlebotomine sandflies. In addition, there is need to carry out bioassays with individual and combinations of the identified compounds to elucidate the candidate biologically active components.