Efficacy of Allopatric Tephrosia Vogelii (Fabales: Fabaceae) against Pre-Emerginal Stages of Phlebotomus (Phlebotomus) Duboscqi (Diptera: Psychodidae)

The effi cacy of allopatric Tephrosia vogelii in killing eggs and larvae of the sand fl y Phlebotomus duboscqi was tested in the laboratory. In this study effects of both water and crude powder extracs of Tephrosia vogelii were tested against against pre-emerginal stages of P. duboscqi in the laboratory. Three groups of eggs were wetted with T. vogelii extracts from Kilifi (TVK), Nairobi (TVN) and Vihiga Counties (TVV) and observed for morphological changes, motility, transformation and death. Six groups of 30 fi rst instar larvae were fed on T. vogelii crude powder either alone or mixed with larvae food. All larvae given TVN powder become immortile, did not feed and died at the fi rst instar stage, whereas those fed on TVV and TVK did not die. Combining all the three extracts also led the death of all fi rst and second instar larvae, but gradual death and disintegration of third instar larvae was sequentially observed. The differences in activities of the allopatric plants can be attributed to allopatric introgression arising from geopgraphic separation leading to introgressive hybridization. The killing and anti-feedant activities of TVN could also mean that it is a chemotype 1 plant that contains rotenoid whereas TVK and TVV are chemotype 2 that lack rotenoids. Feeding deterrence has been shown to be caused by rutin and quercetin. Considering that only leaves were used, the toxicity of TVN can be attributed to single or combination actions of rotenone, elliptone, deguelin, rutin, rotenolone, tephrosin or quercetin that have been shown to be present in leaves. Results of this study suggest that T. vogelli has detrimental insecticidal effects on pre-emerginal stages of P. duboscqi and has potential for sand fl y control. This control method can be achieved through direct introduction of crude dried T. vogelii powder into termite mound ventilation shafts and animal burrows where sand fl ies are known to breed and rest. Research Article Effi cacy of Allopatric Tephrosia Vogelii (Fabales: Fabaceae) against Pre-Emerginal Stages of Phlebotomus (Phlebotomus) Duboscqi (Diptera: Psychodidae) Nathan M Aritho1, Paul K Ndungu1,3, Judith Inyangala4, Milkah Mwangi3, Michael M Gicheru1, Joseph JN Ngeranwa2 and Christopher O Anjili3* 1Department of Zoological Sciences, Kenya 2Department of Biochemistry and Biotechnology, Kenyatta University, PO Box 43844-00100, Nairobi, Kenya 3Centre for Biotechnology Research and Development, Kenya Medical Research Institute, PO Box 54840-00200, Nairobi, Kenya 4School of Biological Sciences, University of Eldoret, PO Box 1125-30100, Eldoret, Kenya Dates: Received: 23 February, 2017; Accepted: 28 April, 2017; Published: 29 April, 2017 *Corresponding author: Christopher O Anjili, Centre for Biotechnology Research and Development, Kenya Medical Research Institute, PO Box 54840-00200, Nairobi, Kenya, E-mail: https://www.peertechz.com


Introduction
Tephrosia vogelii has been shown to exhibit insecticidal activities. These properties have been attributed to the presence of isofl avonoid rotenoids in various parts of the plant [1]. Some of the active ingredients responsible for and entomocidal effects in T. vogelii are believed to be rotenoids, which are rotenone (C 22 H 23 O 6 ) (Nagai, 1902; LaForge and Haller, 1932 [2,3] [13] and quercetin (C 15 H 10 O 7 ) [14,15], fl avonoids found mainly in leaves of T. vogelii, which has been shown to have deterrent effects on the feeding behavior, survival, and development of the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) [16] and Rutin (quercitin 3-O-rutinoside), which has been shown to exhibit antibiotic and/or anti-feeding effect in several defoliating insects such as the sphinx moth Manduca sexta (Lepidoptera: Sphingidae) [17], the tobacco budworm Heliothis virescens (Lepideptera: Noctuidae) [18] and the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae) [19]. Rutin is well distributed in leaves of T. vogelii [14]. Its effects in slowing larval development of M. sexta is temperature dependent, being high at warm temperatures and slowed at low temperatures [20].
The rotenoids, especially rotenone has been shown to have low mammalian toxicity, and is extremely active as a contact and stomach poison against insect species in which it inhibits mitochondria electron transport [21]. In Kenya, where both cutaneous and visceral leishmaniases are andemic and are  conditions. The study was carried out using only the preemerginal stages because female adults feed on blood and both sexes suitable plant for carbohydrates [22]. It was envisaged that targeting the pre-emerginal stages can be achieved through introduction of the crude dried T. vogelii into the termite mound and animal burrow breeding and resting sites of the sand fl ies. Choice of pre-emrginal stages was because T.
vogelii has been shown shown to deter adults from feeding [23].

Materials and Methods
Allopatric Tephrosia vogelii were collected from Kilifi County in Coast, Vihiga County in Western, and Karura in Nairobi provinces. Leaves were harvested and transported in paper bags to the laboratory where they were dried in a shade to a brittle state. The brittle leaves were ground to a fi ne powder using an electric mill and stored in glass bottles. The powder from the Nairobi plant was designated TVN, the Kilifi TVK and Vihiga TVV respectively.

Preparation of chloral hydrate mountant gum
Chloral hydrate gum which is used to clear sand fl ies was prepared according to the method of [24]. Briefl y, 8 grams of gum arabic, 70 grams of chloral hydrate crystals were weighed.
These were dissolved in a mixture of 10 ml distilled water, 5 ml glycerine and 3 ml glacial acetic acid. This mixture was stirred using an electric magnetic stirrer for uniformity and then used to mount both eggs and various larval stages of the sand fl y.

Preparation of sand fl y larvae food
Larvae food is usually prepared by mixing rabbit droppings with rabbit chow, which actually dried corn meal from Unga Feeds. Equal proportions of the two are ground together, spread on a plastic tray, sprinkled with water and covered for ten days for a fungus to grow. The fungus is then harvested, and then dried in an oven [25].

Toxicity assays
Equal proportions (200 grams) of TVK, TVN and TVV powders and larvae food were separately mixed and used as food for three groups of the thirty (30) larvae. Another group of larvae was fed on a mixture of all the three T. vogelli (TVK, TVN and TVV) powders mixed with larva food. Another set of 30 larvae was fed on normal food at the same time to serve controls. Each day after the larvae were given the mixtures, they were examined for ingestion, mortility, and death using a dissecting microscope. An additional 3 larvae per group were suffocated using CO 2 , mounted on slides using chloral hydrate mountant, left for 24 hours to clear and then examined using a light microscope for any changes internal changes. Live larvae were examined on a daily basis for any transformation to second, third, fourth in stars, pupae and adult emergence.
Comparison was made between the time taken by the treated and the controls for the emergence of adult fl ies.

Result and Discussion
When fed on larvae food mixed with T. vogelii, they were able to digest it without any detrimental effect. A summary of the activities of the allopatric T. vogelii on the pre-emerginal species of the sand fl y are given in the table 1.
The fi rst instar larvae with two caudal bristtles the third instar larvae with traces of T. vogelii in the fore gut, and with four caudal bristles are shown in Plate 1.
All larvae fed on extracts of Tephrosia vogelii from Nairobi County (TVN) exhibited 100% mortality. Results of this study showed that T. vogelii extracts from Kilifi (TVK) and Vihiga (TVV) are less potent as compared to the Nairobi plant. This could suggest that the Nairobi plant is a chemotype 1 that contains rotenoids whereas TVV and TVK are Chemotype 2 species that lack rotenoids [25]. The differences in activities of TVV, TVN and TVK could be as a result of allopatric introgression which is also known as has been reported in some plant species such as Juniperus virginiana (Pinales: Cupressaceae) [26]. Geographically separated plants of the same species usually exhibit introgressive hybridization, the movement of a gene (gene fl ow) from one species into the gene pool of another by repeated backcrossing of an interspecifi c hybrid with one of its parental species which changes chemical composition within the plant [26,27] (Plates 2,3).
Even though rotenone distribution varies in amount in most plants, leaves which were used usually contain the highest amount than in other plant parts as has been demonstrated by thin-layer densitometric analysis [27]. It has also been proven that the concentration of rotenone is high in leaves formed during the growing season and remains constant in a given leaf throughout its life on the plant [28]. Only leaves were used  in the study, the killing and anti-feedant effects displayed by the sand fl y can be attributed to single or combination actions of rotenone, elliptone, deguelin, rutin, tephrosin, rotenolone or quercetin that have been isolated from leaves [14,29].
The possibility of combination enhancement of toxicity can be because separately, the modifi ed D and E rings deguelin and toxicarol are non-toxic whereas sumatrol and elliptone are less insecticidal compared to rotenone [30]. The antifeeding effects caused T. vogelii extract which was observed in this study has also been reported for the spotted cereal stem borer, Chilo partellus (Lepidoptera: Crambidae) [31]. Feeding inhibition and lack of movement as was observed in 1 st instar larvae fed on TVN can be useful in suppressing the spread of Leishmania major by P. duboscqi if T. vogelii is used. Quercetin and rutin have been shown to display properties of inhibiting the development, and increasing mortality of larvae of the moths Lymanria dispar (Lepidoptera: Erebidae) and Spodoptera litura (Lepidoptera: Noctuidae) [32,33]. It is possible that these compounds did the same to larvae of P. duboscqi when TVN was used.
The ability of P. duboscqi larvae to feed on T. vogelii from Western and Coast and without dying can be taken to suggest these are C2 plants lacking rotenoids, and that there is need to screen allopatric species from different regions before use.
The development of natural resistance or innate immunity to rotenoid pesticides have rarely been reported despite their widespread and continued use since 1948 in Central America [34]. This is thought to be because insects are not known to detoxify rotenoids [35]. Being easy to grow, coupled with being biodegradable, T. vogelli can be used to control the leishmaniases in Kenya.