Repellency and insecticidal properties of seed oil of Jatropha curcas L. against American cockroach, Periplaneta americana L

Background: This study evaluated the effect of Jatropha curcas seed oil against adult American cockroach, Periplaneta americana, a mechanical disease vector, using three bioassay methods to determine the repellent activity, contact and fumigant toxicity. This involved the use of J. curcas oil solution (diluted with acetone (20%)) and J. curcas pure oil. For repellency test, concentrations 0.30, 0.60 and 0.90% v/v were used for the oil solution while 1.0 and 2.0 ml concentrations were used for the pure oil. All test groups were exposed for 15 min. Contact toxicity test involved the use of 0.30, 0.60, 0.90, 1.20 and 1.50% v/v concentrations for the oil solution while 1 and 2 ml concentrations were used for the pure oil. Exposure period for all test groups was 24–120 h. For the fumigant test, 0.15% v/v and 0.5 ml concentrations were used for the oil solution and pure oil groups respectively; exposure period for the test groups was 24–120 h. All test and the control groups had ten cockroaches (P. americana) per group with four replicates. Results: Repellency was higher in test groups treated with pure J. curcas oil than in groups treated with the oil solution with repellency of 70–100% and 60–100% respectively after 15 min exposure period. For the contact test, a higher mortality rate was observed with the oil solution than the pure oil. Mortality was lower for 1 ml of pure oil with 20% at 24 h and 40% at 120 h than 2 ml of pure oil with 30% mortality at 24 h and 50% mortality at 120 h. A 100% mortality was recorded in the highest concentration (1.50% v/v) at 120 h. Fumigation test with 0.15% v/v of oil solution resulted in 20% mortality at 120 h while fumigation test with 0.5 ml of J. curcas pure oil resulted in 60% mortality at 120 h. Conclusion: J. curcas seed oil possesses repellent and insecticidal properties against P. americana . Thus, the menace caused by this mechanical disease vector could be reduced using J. curcas seed oil.


Introduction
Cockroaches are insects of the order Blattodea; they are the most abundant and obnoxious non-biting insect pests in residential buildings, hospitals, hotels and restaurants (Bala & Sule, 2012;Kumar & Tewari, 2015).
The damages caused by cockroaches are enormous and difficult to estimate ranging from inconvenience to disease transmission. Therefore, there is need to reduce their number below injury level. Control approaches involve the use of chemical insecticides such as pyrethroids, carbamates, organochlorines, organophosphates, hydramethylnon and sulfluramid. These insecticides have undesirable effects on the environment due to their hazard and are known to pose public health issues (Khan, Qamar, Mehdi, & Shahid, 2011;Tunaz, Er, & Isikber, 2009). Therefore, the need to introduce biodegradable insecticides that will help reduce the population of this group of insects is desirable.
Jatropha curcas belongs to the family Euphorbiaceae and subfamily Acalyphoideae. It is a multipurpose perennial shrub, native to Mexico and subtropical America and found in most tropical areas of the world including Nigeria (Gübitz, Mittelbach, & Trabi, 1999;Janick & Paull, 2008). Series of studies revealed that J. curcas possesses some insecticidal and larvicidal properties (Ahirrao et al., 2011;Rahuman, Gopalakrishnan, Venkatesan, & Geetha, 2008;Silva, Faroni, Sousa, & Freitas, 2012). Almost all parts of J. curcas are useful. The seeds of J. curcas consist of saponins, lectins (curcin), phytates, protease inhibitors, curcalonic acid and phorbol esters (Makhar, Francis, & Beckar, 2007). Isman (2006) reported that plant extracts and oils have wide range of activities against pests, including insect vectors. These natural products have fumigant, antifeedant and repellent effects as well as inhibitory effect on their reproduction (Omara, Al-Ghamdi, Mahmoud, & Sharawi, 2013). This study was carried out to investigate the repellency effect and insecticidal properties of seed oil of J. curcas on the American cockroach, P. americana.

Materials and methods
Collection and identification of J. curcas J. curcas seeds (Fig. 1a)  Oil extraction from seeds of J. curcas The extraction of oil from the seeds of J. curcas was done at the laboratory of the Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo, Akwa Ibom State, Nigeria. Seeds collected were de-shelled, washed with clean water, dried under room temperature and pulverized using a crusher machine. The pulverized seeds (Fig. 1b) which weighed 697 g was poured into a glass jar and labeled.
The methods of Akbar, Siddiqui, Sagathevan, and Khan (2019) and Shivani, Khushbu, Faldu, Thakkar, and Shubramanian (2011) were adopted in the extraction process. A quantity of 2.5 l of n-Hexane was used for the extraction. The first half of n-Hexane (1.25 l) was poured into a glass jar containing the pulverized seeds. The jar was periodically shaken, and after 72 h, the mixture was filtered using Whatman No. 1 filter paper, and this was followed by the evaporation of n-Hexane from J. curcas oil using a rotary evaporator. This was done in vacuum and at a temperature of 40°C. Jatropha seed oil was stored in an air-tight container and maintained at room temperature until it was needed for the experiment.

Collection and identification of P. americana
Two methods were used for the collection of adults of P. americana which included the use of baited traps and hand collection method. The bait was prepared according to the method used by Abdel-Gahny, Zalat, Abo-Ghalia, and Semida (2008) where the bait consisted of attractants such as fresh fruits, sugar and wet bread. Also, cockroaches were collected by hand picking them using sterile hand gloves, around toilets, bathrooms and septic tanks. The collection was done at Itu road, Uyo (latitude 5°2′ 25.77″ N and Longitude 7°58′ 44.44″ E) in October, 2019. Cockroaches of the species P. americana collected were identified using standard taxonomic, morphological and pictorial keys as described by Ross (1965). P. americana is reddish brown to brown in colour with light yellow bands around the shield behind the head that is there is substantial variation in light and dark patterns on the pronotum.
Adults of P. americana collected were placed in 24 × 40 cm plastic containers and maintained at a temperature of 30 ± 2°C and photoperiod of 12:12 (L:D) hour, in the laboratory of the Department of Animal and Environmental Biology, Faculty of Science, University of Uyo, Nigeria. The four walls of the containers were smeared with a thin layer of vaseline (blue seal) as described by Thavara et al. (2007) to prevent escape of cockroaches, and they were fed with dry crumbled biscuits, bread and water. Pieces of facial tissue were provided as harborage. They were allowed 48 h to get used to the laboratory environment before commencement of the experiments.

Experimental design
A stock solution of the oil of J. curcas was prepared using 20% acetone as the diluent. From the stock solution, five different concentrations of the oil were obtained: 0.30, 0.60, 0.90, 1.20 and 1.50% v/v. Four replicates were used for each test concentration. A control experiment was also set up; this consisted of 3 ml acetone (20%). The experiments determined the repellency, contact and fumigant toxicities of J. curcas on adults of P. americana.
In this study, the repellency, contact and fumigant toxicities of pure (100%) J. curcas oil were also determined; this consisted of 1 and 2 ml of the pure (100%) oil of J. curcas. The control for this set up consisted of 3-ml distilled water.

Repellency test
The methods of Appel, Gehret, and Tanley (2001), Manzoor, Munir, Amdreen, and Naz (2012) and Sharififard, Safdari, Siahpoush, and Kassiri (2016) were adopted for this study. Whatman No. 1 filter paper was divided into two equal pieces. In each case, one half of the filter paper was treated with the test oil solution, using a micro sampler, and the other half was left untreated. Different concentrations of the test oil solution (0.30, 0.60, 0.90% v/v) and their replicates were used for the treatment. Each treated filter paper was left for 2 min to dry before commencement of the repellency test. This was followed by placing the filter paper at the bottom of rectangular assay plates (on the right side). The untreated paper was placed on the left side of the assay plate.
Ten (10) adults of both sexes of P. americana were released into the centre of each assay plate. The distribution of the test insects was observed at 0, 5, 10, and 15 min. After this exposure period, repellency was calculated using the formula shown below.
where T = number of cockroaches found in the treated area N = the total number of cockroaches used. The repellency effect of pure (100%) J. curcas oil was also determined. This consisted of 1 and 2 ml of the pure (100%) oil of J. curcas. The control for this set up consisted of 3-ml distilled water. Exposure period was also 0, 5, 10 and 15 min.

Contact toxicity
The bioassay method of the WHO (1975) was used to determine the susceptibility of cockroaches to the fixed oil of J. curcas. Whatman No. 1 filter papers (whole) were separately treated with 0.30, 0.60, 0.90, 1.20 and 1.50% v/v of J. curcas oil. Each of these concentrations had four replicates. Each filter paper was first left to dry for 2 min and then was folded into a 500-ml capacity jar. The top inner surfaces of these assay jars were smeared with a thin layer of Vaseline (blue seal) to prevent the escape of cockroaches from the jars. Ten (10) adult cockroaches of both sexes were introduced into each of the jars and observed for mortality for 24, 48, 72, 96 and 120 h after treatment as described by Thavara et al. (2007) and Zibaee, Khorram, and Hamoni (2016). A control experiment was set up with four replicates also. Cockroaches in the control groups were exposed to filter papers treated with 3 ml acetone (20%).
Contact toxicity was also determined using pure (100%) J. curcas oil. Two volumes (1 and 2 ml) of the oil were used for this test. Ten cockroaches each were used in this test, with four replicates. For the control experiment, cockroaches (10) were exposed to filter paper treated with 3-ml distilled water. Exposure period was the same as for the cockroaches exposed to the oil solution.

Fumigant toxicity
Fumigant toxicity tests reported in this study were carried out as described by Appel et al. (2001), Thavara et al. (2007) and Zibaee et al. (2016). One-centimeter (1 cm) diameter cotton ball was treated with 0.15% v/v of J. curcas oil. To prevent direct contact of cockroaches with the fixed oil, it was injected to the centre of each cotton ball using a micro sampler. This was replicated four times. Each cotton ball was separately introduced into 1l capacity test jar.
The control groups which also had four replicates were exposed to cotton balls treated with 0.5 ml acetone (20%). Observation for mortality and other toxic effects of the oil on the test insects was made at 24, 48, 72, 96 and 120 h, for both the test and control experiments. A quantity of 0.5 ml of pure Jatropha oil (100%) was also used for fumigant toxicity tests where 0.5 ml of distilled water served as the control. Exposure time was also 24, 48, 72, 96 and 120 h. Each experimental set up had 10 cockroaches of both sexes.

Data analysis
One way analysis of variance (ANOVA) was used to determine the level of significance among the concentrations used in the various groups. Analysis was performed using statistical package for social sciences (SPSS) version 21.0.

Results
Repellent effect of J. curcas oil on P. americana The repellent effect of pure oil and oil solution of J. curcas is shown in Table 1. The study revealed that there was increasing repellent effect on P. americana at higher concentrations. For the oil solution, the highest repellency was observed at 0.90% v/v from 0 to 15 min. Also, it was observed that concentrations of 0.60% and 0.90% v/v both had 100% repellent effect on P. americana at the start of the experiment. The 0.30% v/v had the least repellent effect on P. americana with a percentage repellency of 60% at 15 min of exposure, although the repellent effect was as high as 90% (Table 1), at the start of the experiment. Reduction in repellency was observed for all concentrations with increase in exposure time. Analysis of variance revealed that there was no significant variation (p = 0.112) in the repellent effect of different concentrations of J. curcas oil on the test cockroaches. Although, the repellency was high for all the groups, acetone (the control) was observed to have a higher repellent effect on P. americana than the concentrations of J. curcas tested in this study and its effect did not appear to be time dependent.
For the effect of pure oil on P. americana, 1 ml and 2 ml of pure J. curcas oil performed well with no significant repellent effect between the two concentrations. One millilitre concentration had 70% repellent effect at the beginning of the study while 2 ml concentration had 80% repellent effect. There was no significant difference (p > 0.05) in the repellent effect between the two. It was observed that the pure oil had increasing repellency effect. The repellency increased with increase in exposure time (Table 1).
Contact toxicity effect of J. curcas oil on P. americana Mortality of P. americana exposed to J. curcas oil solution increased with increase in exposure time and oil concentration. A 100% mortality was observed for 1.20% v/v concentration at 96 and 120 h and for 1.50% v/v concentration at 72-, 96-and 120-h exposure period. No death was observed for 0.30% v/v concentration at 24and 48-h exposure period. For the control experiment, no mortality was observed at 24 h, whereas mortality of 80% was observed at 120 h. When P. americana was exposed to the highest concentration of 1.50% v/v of the oil solution at 48 h, a physical change in colour was For the pure oil, mortality was observed in both concentrations throughout the exposure time (24, 48, 72, 96 and 120 h). When test organisms were exposed to 1 and 2 ml of pure J. curcas oil, 20% and 30% mortality were respectively, after 24-h exposure period. At the highest exposure period (120 h), 40% and 60% mortality were observed for 1 ml and 2 ml pure oil respectively ( Table 2).
Comparison of the eight groups revealed that the oil solution at 1.50% v/v had the highest contact effect, followed by 1.20% v/v of oil solution and 2 ml of pure oil of J. curcas. These three groups were significantly effective compared to the other groups in their contact effect on P. americana.

Fumigant toxicity effect of J. curcas oil on P. americana
The fumigant toxicity effect of J. curcas oil on P. americana is presented in Table 3. At 24 h, 5.0% mortality was observed for J. curcas oil solution while 30.0% mortality was recorded for groups treated with J. curcas pure oil.
At 120 h, 20% mortality was observed for the groups treated with J. curcas oil solution, whereas 60% mortality was observed for groups treated with J. curcas pure oil. For the control experiment where acetone was used, no mortality was observed at 24-and 48-h exposure period while 20% mortality was observed at 120 h. The control experiment with water gave no mortality throughout the exposure period of 120 h. Analysis of variance revealed that there was significant difference (p < 0.05) in the fumigant toxicity effect between the test oil concentrations and the controls.

Discussion
This study on evaluation of the efficacy of J. curcas oil against P. americana is a preliminary investigation. Results obtained revealed the repellent and insecticidal potentials of oils from the seeds of J. curcas and corroborate the reports of other researchers. The potency of the oil of J. curcas against the maize weevil (Sitophilus zeamais), pest of cowpea (Callosobruchus maculatus) and the pest of okro (Podagrica sp) has been documented by Adebowale and Adedire (2006),  Ohazurike , Omuh, and Emeribe (2003) and Phowichit, Buatippawan, and Bullangpoti (2008), respectively. Addisu, Mohamed, and Waktole (2014) and Yohannes (2006) also tested the effect of J. curcas on termites of the species Microcerotermes beesoni and Macrotermes sp respectively. They reported that the oil had insecticidal effect on these species of termites. Studies on the efficacy of some plant essential oils and powders against P. americana have been documented by Rejitha et al. (2014) and Thavara et al. (2007) respectively. In this study, the pure oil of J. curcas performed better than the oil solution in terms of repellency and fumigant toxicity. It was also observed that mortality was higher in the contact toxicity test than in the fumigant test. This may be due to the weak fragrance of the oil. A quantity of 3 ml of 20% acetone which was used in the control experiment demonstrated appreciable repellent effect on the test organisms. However, when acetone (20% acetone) was used in the formulation of the oil solution, it did not appear to improve the performance/potency of the oil. This is an indication that J. curcas seed oil with or without diluents holds promising potential as repellent and insecticide against P. americana.
The potency of oil from the seeds of J. curcas is attributed to its chemical constituents. Adebowale and Adedire (2006), in their studies, detected the presence of sterols and triterpene alcohols in the oil obtained from the seeds of J. curcas and commented that these chemical substances have insecticidal properties. Bashir and El Shafie (2013) reported the presence of saponins, lectins (curcin), phytates, protease inhibitors, curcalonic acid and phorbol esters in oil extracted from J. curcas seeds. They attributed the insecticidal effect of this oil on the desert locust, Schistocerca gregaria to these constituents. Thus, the repellency (60-100%) and mortality observed for the varying oil concentrations tested in this study is also attributable to the chemical compounds present in the oil. Acda (2009) and Bekele (2002) reported that an active component in the oil of J. curcas called jatrophine, a diterpene which is a dominant compound, interfered with the normal message transfer system in the mid gut cell leading to physiological discomfort and death of insects.
The chemical constituents of the seed oil of J. curcas may have exerted their effect singly or in synergy. Further investigation is needed to substantiate this. It is thus needful to isolate and characterize the active principle(s) in this test oil.

Conclusion
Oil from the seeds of J. curcas possesses repellent and insecticidal properties against P. americana. The pure oil proved more effective in repelling the cockroaches than the oil solution. In other words, J. curcas seed oil showed a considerable repellent effect on P. americana. Appreciable mortality of test cockroaches was observed in both the contact and fumigant toxicity tests. The oil of J. curcas holds potential as repellent and insecticide against the American cockroach, P. americana.

Recommendations
Due to the negative impact of the use of chemical insecticides on the environment, it is recommended that ecofriendly insecticides should be used. The observation from this study revealed that the oil of J. curcas was effective as repellent and insecticide against adult American cockroach, P. americana. J. curcas seed oil should be considered for the control of P. americana due to its high repellency and insecticidal effects. Better formulations and application methods should be developed to ensure desired results of the oil.