Repellency Activity of Synthetic Blends of Semio-Chemicals from Xylopia aethiopica and Dennittia tripetala Against Prostephanus truncatus (Horn)

Laboratory bioassays were conducted in Calabar, Nigeria, to evaluate repellencies of the essential oils extracted from Xylopia aethiopica and Dennittia tripetala fruits, chemical constituents of the oils and their blends as well as compare repellencies of the blends with those of the constituents using a 3-day old adult Prostephanus truncatus, a primary pest of stored maize and dried cassava chips, cultured in the laboratory. Fresh fruits of X. aethiopica and D. tripetala, 100 g each was collected, washed and dried, then pounded separately using laboratory pestle and mortar for the extraction of essential oils. The synthetic blends were prepared with 0.3 mg/ml of 1,8-Cineole, 0.4 mg/ml of b-Phellandrene and 0.3 mg/ml of b-Pinene, extracts of X. aethiopica, and the extracts of Dennittia tripetala (0.3 mg/ml of 1,3- Cyclopentadien, 0.5 mg/ml of 1,6- Cyclodecadien and 0.2 mg/ml of 2-Undecene. The essential oils extracted were tested for repellence and toxicity against P. truncatus at 10 μl per essential oil in a 4-way Olfactometer. Gas chromatography-Mass spectrometry (GC-MS) was used to isolate and identify the chemical constituents of the essential oils. Synthetic blends of the chemical constituents were prepared on the basis of their natural ratios and were also tested for bioactivity against the insect pest. Results indicated that the essential oils were significantly (P<0.05) repulsive and toxic to P. truncatus. However, the synthetic blends of the chemical constituents of the essential oils were more repellent and toxic to the insect pest than the individual component of the essential oils. The blends provided a broad spectrum of bioactivity against the insect pest and demonstrated the potential for their development in stored product protection especially at the small scale resources poor farmer’s level in Nigeria.

Keywords:Essential oils (EOs); Xylopia aethiopica; Dennittia tripetala; Prostephanus truncates; 4-way Olfactometer

In Nigeria, and indeed in most African countries, farmers usually dry and store their grains traditionally in open storage facilities that are capable of accommodating only about 1000 to 1500 kg of the total harvested grains [1]. A lot of the stored grains are often destroyed by insect pest attack culminating in varied levels of food and economic loss. Ukeh et al. (2010) reported 10 to 20 % crop loss world wide and 25 to 40 % loss in the tropics; in West Africa, stored grain insects inclusive of Prostephanus truncatus (Horn), Rhizopertha dominica, Sitophilus spp, Trogoderma granarius, Sitotroga cerealella and Tribolium castaneum have been reported to cause losses as high as 30 % [2-4]. P. truncatus is an exotic pest whose larvae and adults feed on stored grains and cassava resulting in losses in weight, quality, nutrient, market and seed value besides food poisoning arising from mycotoxins of fungal contaminants [5-7]. Over a short duration of storage, this pest caused the loss of 20-30 % of stored grains in Ghana, Togo, and Tanzania [5, 8-10]. The control of this pest in developing countries will foster their drive towards food security and self –sufficiency.

The use of synthetic insecticide is the most prevalent method of controlling stored product pests; the benefits have been overshadowed by negative impacts such as development of resistance by the insect pests, destruction of the ecosystem and mammalian toxicity [1]. There is therefore a need to adopt and integrate other methods of pest control that are less hazardous. The main objective of this research was to evaluate the potential for controlling P. truncatus using essential oils extracted from the fruits of guinea pepper (X. aethiopica) and pepper fruit (D. tripetala), constituents of the oils and their blends.

Insect culture was established in the Laboratory of Department of Crop Science, University of Calabar. with adult P. truncatus, collected from infested maize in a food shop in Obudu Local Government Area main market. The plant materials used, undamaged dry seeds of maize seeds, fresh fruits of X. aethiopica and D. tripetala, were purchased from the same market.

One hundred grams (100 g) of the dried fruits of X. aethiopica and D. tripetala were separately ground into powder using laboratory pestle and mortar and vacuum-distilled in 50 ml of redistilled diethyl ether. The container was immersed in an ultrasonic wave device for 5 mins to disperse and homogenize the contents. The vacuum distillation apparatus was then connected to a high vacuum pump (ES 50 vacuum pump, Edwards, England). The glass section of the apparatus was strongly heated with a hot air blower to remove any less volatile contaminants from its internal surface. The U-tube and the pear -shaped distillate collection vessel were submerged completely in nitrogen at a temperature of 196 °C. The residue was then distilled for 24 h at pressure of 0.05 mmHg. The distillates were then pipetted into 50 ml separation funnels to remove water. The extracts were dried, using Magnesium sulphate (MgSO4), filtered and concentrated to obtain 4 ml of each of X. aethiopica and D. tripetala essential oils [11]. Each of the vacuum distilled extract was sealed under nitrogen, labelled accordingly and placed in different ampoules, pending when needed for the laboratory bioassay experiment.

Essential oils extracted from X. aethiopica and D. tripetala were each impregnated into 10 μl/ x cm² filter paper and placed alongside with maize seed in one arm of the Olfactometer, while the other three arms were reserved as control. The 6 mm thick Perspex Olfactometer used had a four- pointed exposure chamber shaped like a star was fixed to a circular plate measuring 12 cm x 12 cm with a hole (3 mm) drilled into the walls at each point of the four cardinal glass frames. The floor plate measured 10.2 cm x 10.2 cm x 0.6 cm) and a similar sized plate with a 4 mm diameter hole at the centre served as cover. In order for the insect to work easily in the Olfactometer, a sheet of Fisher brand QL 100 filter paper (Springfield mill, Maidstone, Kent, England) was placed on the floor covering. An air stream was passed into the Olfactometer through a Teflon tubing of size 3.2 mm (can/ ab/ Ltd, U.K.) from an air entrainment chamber, to keep the insect mobile [12]. In the bioassay experiment, the parameters assessed were the time spent by a 3-day old adult P. truncatus in the different arms of the olfactometer and the number of times the insect visited the different odour zones over a 12-minute observation period. All treatments were replicated 10 times using a new insect and a fresh essential oil. It was observed that each time an insect is placed in an Olfactometer and air stream passed in from an air entrainment chamber through a Telfon tubing, the insect becomes active and mobile and begin to respond to the emissions from the spice plants by moving away from them and moving round the based of the Olfactometer, entering the different arms and coming out [11]. Soon after about eleven (11) or twelve (12) minutes of the commencement of the experiment, the insect gradually becomes tired, slugish and sufforcated from the toxic emissions coming from the splice plant extract, thereby affecting the insect’s ability to respond actively to the emissions, while the strength of the emissions also becomes weaker and weaker with time. Therefore, there is need to change the insect and the plant extract for fresh ones to ensure vigour in the response and full emission of the somio-chemicals [2].

Also assessed was toxicity and adulticidal effect of the plant (EOs) extracts and their synthetic blend on P. truncatus. Each plant synthetic blend extract (5,10 &15 ml) was impregnated separately into filter paper and placed at the bottom of a plastic container, while the same quantity of the EO was also separately applied round the inside of the same container. Twenty (20) pairs of the 3 day old P. truncatus were introduce into the containers for oviposition to take place. All treatment were replicated 4 times and arranged in complete randomized design (CRD). Mortality count was done every 24 hourly for 72 hrs after the introduction of the insect into the separate containers. On each count, after every two days, dead insects were removed by sieving, counted and discarded, while the emerging new insects were also counted and returned to the containers and then recorded until no more insects emerged.

Data obtained were analysed using analysis of variance (ANOVA) procedure, after which the data were further transformed using the formula log 10^x+1 in order to remove the aspect of zero and to ensure that they do not conflict with the analysis of variance.

The chemical constituents of the polar and non-polar fractions of the essential oils were isolated and identified using Gas chromatography-Mass spectrometry (GC-MS). The GC-MS analysis was carried out using Agilent technology Model 7890A, interfaced with Mass Selective Detector (MSD) Model 5975c. An electron ionization was at a 70 ev with anion temperature of 250 °C. Helium gas was used as a carrier gas while Hp-5 ms (30 mm x 0.25 mm x 0.32 mm) were used as the stationary phase. The oven temperature was maintained at 75 °C for five minutes and ramped to 250 °C at the rate of 3.5 °C per minute for six minutes. One ml of the essential oil of X. aethiopica and D. tripetala was separately injected into the chromatographic column for analysis. Hexane was identified as the non-polar compound, while Florisil® diethyl ether fractions were identified as the polar compounds. Polar compounds isolated from the Florisil® diethyl ether fraction of X. aethiopica were 0.3 mg/ml 1,8-cineole, 0.4 mg/ml b-phellandrene, and 0.3 mg/ml b-Pinene (natural ratio 3:4:3); those for D. tripetala were 0.3 mg/ml 1,3-cyclopentadiene, 0.5 mg/ml 1,6-cyclodecadiene, and 0.2 mg/ml 2-undecene (natural ratio 3:5:2). The constituents were tested for repellency as described above.

Synthetic blends of 1,8-cineole, b-Phellandrene and b-Pinene florisil diethyl ether fractions of X. aethiopica was prepared by dissolving 0.3 mg/ml of 1,8-cineole, 0.4 mg/ml b-Phellandrene and 0.3 mg/ml b-Pinene in 10 ml flask, then 1ml of each compound was combined in a 10 ml volumetric flask and filled up with hexane. Blends of the major compounds of Florisil® diethyl ether fractions of D. tripetala were similarly prepared based on their natural ratios. The synthetic solutions were sealed in ampoules under nitrogen for storage prior to the bioassay experiments.

All data generated were subjected to analysis of variance and means were compared using Tukey’s simultaneous means separation, according to (Zar, 1999) or least significant difference (LSD) at 5 percent level of probability [13]. Data on the number of entries or visits made by the test insect to the test arm and control were analyzed using t-test at (0.05) level of probability. Minitab 15 statistical software was used for the analysis of data.

The extracted essential oils (EOs) from the two spice plants were tested individually for bioactivity against the insect pest (P. truncatus) of stored grains.

The time spent by the insect in the test arm containing maize seed plus essential oils from X. aethiopica and D. tripetala, impregnated in a filter paper was on separate occasions significantly (p<0.05) different compared to the time spent in the three control arms (Table 1). The number of visits the insect made to the test arm containing maize seed plus essential oils from X. aethiopica and D. tripetala on separate occasions, was significantly (p<0.05) different compared to the number of entries into the three control arms (Table 1).

The time spent by the insect in the test arm of the Olfactometer containing on separate occasions, maize seed plus non polar vacuum distilled hexane fractions of X. aethiopica and D. tripetala impregnated in a filter paper was not significantly (p>0.05) different, compared to the time spent in the control arms (Table 2). Similarly, the number of entries made by the insect to the test arms containing maize seed plus the non polar hexane fractions of X. aethiopica and maize seed plus the non polar hexane fraction of D. tripetala, was not significantly (p>0.05) differently compared to the number of entries or visits made to the control arms of the Olfactometer; signifying that the non polar hexane compounds of X. aethiopica and D. tripetala were not toxic and repellent to the insect pest (P. truncatus). The time spent by the insect in the test arm containing maize seed plus 1,8-cineole florisil ® diethyl ether, maize seed plus b-Phellandrene florisil ® diethyl ether and maize seed plus b-Pinene florisil ® diethyl ether extracts of X. aethiopica was significantly (p<0.05) different compared to the time spent in the control arms (Table 3). The number of visits made by the insect to the test arm containing maize seed plus 1,8-cineole florisil ® diethyl ether, maize seed plus b-Phellandrene florisil ® diethyl ether extract of X. aethiopica was significantly (p<0.05) different compared to the number of entries into the control arms of the 4-arm Olfactometer (Table 3).

The time spent by the insect in the test arm containing on different occasions, maize seed plus 1,3-cyclopentadiene florisil ® diethyl ether, maize seed plus 1,6-cyclodecadiene florisil ® diethyl ether and maize seed plus 2-undecene florisil ® diethyl ether extracts of D. tripetala was significantly (p<0.05) different compared to the time spent in the different control arms (Table 4). Similarly, entries by the insect to the Olfactometer arms containing maize seed plus 1,3-cyclopentadiene florisil ® diethyl ether, maize seed plus 1,6-cyclodecadiene florisil ® diethyl ether and maize seed plus 2-undecene florisil ® diethyl ether extracts from D. tripetala was significantly (p<0.05) different compared to number of entries to the control arms of the Olfactometer (Table 4). The result above showed that the time spent by P. truncatus in the test arm of the Olfactometer containing on separate occasions, maize seed plus synthetic blends from X. aethiopica and D. tripetala was significantly (p=0.01) higher compared to time spent in the different control arms (Table 5). Similarly, the number of entries or visits made by the insect to the test arm containing on separate occasions maize seed plus synthetic blends of X. aethiopica and D. tripetala was highly significant (p=0.01) compared to the number of entries or visits to the control arms [14]. The result in Table 5 showed a high significance (p<0.05) in the repellence and toxicity of the synthetic blend of X. aethiopica against P. truncatus compared to the single compounds in terms of the mean time spent by the insect in the different arms of the Olfactometer. The time spent by the insect in the test arm containing the synthetic blend of the spice plant was by far shorter than the time spent in the control and the other arms containing the individual compounds. Similarly, the number of entries or visits by the insect to the test arm containing the synthetic blend is significantly (p<0.05) less than the number of times or visits the insect made to the control arms and the other arms containing the single compounds (Table 6). The result in Table 7 also showed a significant difference in the main time spent by the insect pest in the test arm containing the synthetic blend compared to the time spent in the control arms and the single compounds. It was obvious that the insect made significantly shorter time spent in the test with synthetic blend compared to the time spent in the test arms containing the single compounds and the control arms. Also the number of visits to the test arm with the synthetic blend was highly significantly (p=0.01) different compared to the number of visits to the control arms and the single compounds (Table 7). There was no significant (p>0.05) difference in the mortality count of adult P. truncatus at 24, 28 and 72 hour at 5 ml, compared to the control treatment in the blends of the essential oil (EOs) of the two spice plants. However, there was a significant (P<0.05) difference within the plants synthetic blends applied, and between 5 ml and 10-15 ml and the control experiment. Synthetic blend of X. aethopica at 10 ml and 15 ml exhibited the highest mortality count of the insect (P. truncatus) compared to the untreated (control) and at 5 ml of the extract. Synthetic blend of D. tripetala was equally toxic to the insect (P. truncatus) at 10-15 ml, but only next to the toxicity of X. aethiopica (Table 8). On the whole, the synthetic blends were more toxic to the insect than the single compounds.

One of the most important sources of repellants is the essential oil extracts from aromatic plants commonly used domestically to flavor foods and in the industries to manufactured perfumes [14]. These aromatic plants’ essential oils consists of complex mixtures of monoterpenes, diterpenes, triterpenes and sesquiterpenes hydrocarbons, aliphatic as well as aromatic compounds, with a few major constituents [15]. In an olfactometer experiment, vacuum distilled essential oils from X. aethiopica and D. tripetala were repellant to adult P. truncatus when tested in combination with maize seed. It was therefore observed that the repellency of the essential oils (EOs) was facilitated by the polar florisil ® diethyl ether fractions from the spice plants rather than from the non-polar hexane fractions, as the repellency test was not significant (p>0.05) at 5 % level of probability (Table 2). The toxicity of the essentials oils (EOs) and their synthetic blends to the Insects pest was probably as a result of the interactions of the polar florisil ® diethyl ether with the insect nervous system, either by inhibiting the release of the enzyme acetyl cholinesterase or by antagonizing the functions of octopamine receptors [15]. Octopamine is a biogenic amine that acts as neurotransmitter, neurohormone and neuromodulator in invertebrates [16]. Effects of essentials oils (EOs) of X. aethiopica and D. tripatala on the insect, may suggest their mode of actions such as repellency and feeding deterrence. Application of essential oil of Citroneller in stored –product protection, mosquito repellency and domestic pest (cockroaches, ants and flea) control, and the application of cinnamon oil in mites and urban pests control have been reported [17,18].

Gas chromatography-Mass spectrometry (GC-MS) conducted to isolate the chemical components of the essentials oils (EOs) from the two spice plants, identified the presence of monoterpenes, sesquiterpenes, oxygenated monoterpenes and alcohols as the components found in the essential oils (EOs). The major volatile constituents of the Diethyl ether fractions of X. aethiopica include; 1,8-cineole, b-Phellandrene and b-Pinene, while those of D .tripetala were 1,3-cyclopentadiene,2-undecene and 1,6-cyclodecadiene. This result was in line with the report of Ajaiyeoba and Ekundayo (1999) who indentified and isolated four aryldecanones and eight minor compounds from n-hexane and methanoic acid extracts of A. melegueta obtained from South Western Nigeria [19].

The synthetic blends of the isolated florisil ® diethyl ether compounds from X. aethiopica and D. tripetala were very repulsive to P. truncatus, therefore both the number of entries and the time spent in the test arm of the Olfactometer containing maize seeds and the organic compounds was highly significant (p=0.01) compared to the time spent and the number of entries into the control arms. Time spent and the number of entries into the test arm containing maize seed plus synthetic blend of X. aethiopica was shorter than the time spent and the number of entries into the test arm containing maize seed plus synthetic blend of D. tripatala, meaning that the synthetic blend of X. aethiopica was more toxic and highly repellant to P. truncatus than the synthetic blend of D. tripetala. Each time the insect was entering the test arm having been attracted by volatiles from maize seed, it was in turn confronted by volatiles from the synthetic blends which were repellant and toxic to the insect, therefore it will run back to the control arms where there was no such toxic emission. This phenomenon is described as the “Pull Push” strategy (Ukeh et al. 2010), where the insect is pulled in, by volatiles from maize seed, it is pushed back by the toxic volatiles from the spice plants [2]. The repellence of the produce of the spice plants have implies that if the synthetic blends are used in stored product protection, such as maize or dried cassava chips in storage, will provide total protection of the grains. The result here showed that the blends of the compounds in their natural ratios were more toxic and repellant to the insect pest and provides a broad spectrum of bioactivity against the insect pest than the individual compound (Table 6 and Table 7). Also in terms of mortality count of the insect (P. truncatus) there was a high mortality level in the applied, compared to the control and also between the application of 5 ml and 10-15 ml of the synthetic blends from the two spice plants. The high toxicity level of the insect of 10 to 15 ml of the synthetic blends from both X. aethiopia and D. tripelela, showed that the toxicity increases as the concentration of the blends of the spice plants increase (Table 8). The blends of the essential oils (EOs) produced a percentage repellency (PR), equivalent to a class iv repellant with between 60-80 percent repellency of the spices. This is in accordance with Juliana and Su (1983) percentage repellency class of 0 to v; class 0(PR=0.15), class I(PR=0.15-20 %), class II(PR=20.1-40 %).class III(PR=40.1-60 %), class Iv (PR=60.1-80 %) and class V(PR=80.1-100 %) [20].

Both the individual components of the extracts (essential oils) from the spice plants and the synthetic blends of the extract have broad spectrum of bioactivity (repellent effect) against P. truncatus, However, the synthetic blends were more toxic to the insect than the single or individual compounds.