Contact and repellent effects of essential oils of Chromolaena odorata ( L . )

Macrotermes bellicosus, though a beneficial termite species causes serious damage to wood and wood products, fabrics and many agricultural crops and ornamental trees. This study was conducted to assess the potentials of essential oils of the leaves of Chromolaena odorata and roots of Uvaria chamae in the control of M. bellicosus. The repellency, knockdown and insecticidal effects of the oils to M. bellicosus were tested using the filter paper and cotton ball impregnation technique. Each test oil (0.05, 0.075 and 0.10 ml) was separately used in the repellency assay, for an exposure period of 30 and 60 minutes. For the knockdown and toxicity test, termites were exposed to 0.05, 0.075, 0.10, 0.125 and 0.15 ml of each oil for an exposure period of 60 minutes and 6 hours, respectively. Each test and control (untreated) group had three replicates and in each experiment twenty active workers of M. bellicosus were exposed. Repellency results were somewhat irregular and oil volume and exposure, time independent. Knockdown effect of 56.67 and 86.67 % were observed for C. odorata and U. chamae oils (0.15ml), respectively at the 60th minute. Contact toxicity test with 0.15 ml of the oils resulted in 100 % and 86.67 % mortalities for U. chamae and C. odorata, respectively. No knockdown or mortalities were observed in the controls. The difference between the repellency of both oils at 30 and 60 minutes was not significant (p>0.05; p= 0.842 and 0.212 respectively), whereas for knockdown and insecticidal effects of both oils the difference was significant Q3(p<0.05; p= 0.000 and 0.001 respectively).. Essential oils of C. odorata and U. chamae are potential agents for the control of M. bellicosus.


INTRODUCTION
Macrotermes bellicosus (Isoptera: Termitidae) is a species of termite that is commonly referred to as harvester termite or war-like termite or African termite. This species is mainly found in Africa, the Middle East and Asia (Oyedokun et al., 2011). Macrotermes bellicosus is relished by people living the traditional lifestyle especially children who snack on it when roasted or fried. It is regarded as a traditional delicacy in Southern as well as Northern Nigeria (Igwe et al., 2011). Adepoju and Omotayo (2014) reported that this M. bellicosus species is a good source of protein, calcium, phosphorus, iron, zinc, and antioxidants for its Nigerian consumers. They also reported the presence of vitamins A and E in their nutritional analysis of the termite species. Macrotermes bellicosus also plays an important role in ecosystem balance. It is a major agent of decomposition, thereby improving the fertility of the soil and also known to play an important role in nutrient and carbon fixation (Ajao et al., 2018). Besides the beneficial roles played by this species it is also known to cause great damage to wooden structures in buildings as a result of its wood diet. It has the ability to remain hidden until after extensive damage has been done (Okweche et al., 2015;Omoya and Kelly, 2019). It is also known to damage paper, cloth, carpets, and other cellulose materials. Its feeding activity is aided by symbiotic microorganisms such as bacteria and fungi which are capable of helping it to digest cellulose (Ohkuma, 2008, Ohkuma andBrume, 2011;Peterson et al., 2015). Owing to damages caused by M. bellicosus, control of this species becomes imperative. Efforts at control have been directed towards the chemical method. An example is the use of Thiamethoxam (Oyedokun et al., 2011), Solignum and used engine oil as preventive measure (Ugbomeh and Diboyesuku, 2019). The use of synthetic insecticides against termites' infestations has its drawbacks. These insecticides are often lethal to non-target and beneficial organisms. Runoffs from sites where these insecticides have been used can contaminate ground water sources. Another drawback is the development of resistance to these artificial insecticides by the target insects (Silva et al., 2012). The search for effective alternatives has been directed extensively to the diverse flora of the tropics. Oyedokun et al. (2011), reported on the efficacy of leaf extracts of Phyllanthus amarus, Acassia albida and Tithonia diversifolia against workers of M. bellicosus, in vitro. Similarly, Okweche et al., 2015 reported the comparative efficacy of some insecticidal plant materials against the dry wood termite Cryptotermes cavifrons. Okweche and Nnah (2018) also reported on the anti-termitic properties of Jatropha curcas against M. bellicosus. Omoya and Kelly (2019), assessed the efficacy of fungal entomopathogens against M. bellicosus. Their study revealed the lethal activity of the fungal species Metarhizium anisopliae on M. bellicosus. Chromolaena odorata (Asteraceae) is a perennial shrub, native to North America, but has been introduced to tropical Asia, West Africa and parts of Australia (Chakravorty et al., 2011). In Nigeria, it is known as independence leaf, because it was discovered shortly after Nigeria's independence from colonial rule in 1960 (Stanley et al., 2018). Chromolaena odorata has a life span of approximately ten years (Zahara, 2019). Vaisakh and Pandey (2011) reported the anti-diarrheal, astringent, antiplasmodic, antihypertensive and anti-inflammatory activities of C. odorata. Besides, the insecticidal properties of C. odorata have been reported against Periplanata americana, Callosobrunchus maculatus and Callosobrunchus subinnottus (Udebuani et al., 2015;Nyamador et al., 2017). Uvaria. chamae (Annonaceae), known as finger root or bush banana is an evergreen edible plant. The root of U. chamae is used for the treatment of nose-bleeding, heart diseases, blood in urine, pile, and fever (Etukudo, 2003). Also, the insecticidal properties of the powdered stem bark and ethanol extract of the root of U. chamae have been reported against Callosobruchus maculatus, Rhizopertha dominica and Sitophilus zeamais (Negbenebor et al., 2018). Copping, 2009 described bio-pesticides as pesticides derived from natural sources such as plants, fungi, nematodes, bacteria, and others. These pesticides are known to be target specific and are often regarded as important components of integrated pest management (IPM) programmes. The use of botanicals in the control of insect pests have been an age long tradition, especially in the African continent. Plants such as Gmelina arborea, Aframomum meleguueta, Moringa oleifera, Zingiber officinale, Morinda lucida and Jatropha curcas have been effectively used to control termites (Okweche et al., 2015;Okweche and Nnah, 2018). The objective of this research was to determine the major chemical components of essential oils of C. odorata and U. chamae and evaluate the efficacy of these oils in the management of M. bellicosus.

Collection of Plant Materials
The experimental plants Chromolaena odorata and Uvaria chamae were harvested fresh from Uyo, Nigeria. The identities were confirmed by a plant taxonomist in the Department of Botany and Ecological Studies; University of Uyo, Nigeria. Voucher specimens were deposited in the herbarium of the same department for future reference.

Extraction of Volatile/Essential Oils
Essential oils from the leaves of C. odorata and the roots of U. chamae were extracted by hydrodistillation (5 h), using a Clevenger apparatus, as described in the British Pharmacopoeia (2018). Extraction process was carried out in the laboratory of the Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo, Nigeria. The oils were separately stored in labeled glass bottles for the experiments.

Chemical Analysis of the Oils
Each essential oil was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) on an Agilent System. This was carried out at Hussain Ebrahim Jamal (H. E. J) Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Pakistan. The components of each oil were identified by comparison of their mass spectra with library data of the GC-MS system and by comparison of their retention indices (RI) with relevant data provided by Adams, 2007.

Collection and Identification of Test Termite Species (M. bellicosus)
Worker termites were collected from a termitarium within the main campus of the University of Uyo, Nigeria, done by excavation of the termitarium. They were transported in a small plastic cage (14.4 x 28cm), to the Laboratory of the Department of Animal and Environmental Biology, University of Uyo. Identification of M. bellicosus was done using taxonomic keys of Constantino, 1999 andEngel et al., 2009. Dr. S. Okweche, an entomologist in the Department of Forestry and Wildlife Resources Management, University of Calabar, Nigeria, assisted in identification. Test termites were kept in aerated plastic cages and maintained under laboratory conditions of temperature (27 ± 2 o C) and relative humidity of (75 ± 5 %). They were fed with dry wooden materials and dry leaves. Distilled water was sprinkled on filter paper within the cage to provide moisture. Termites were kept away from direct sunlight as described by Alavijeh et al. (2014) and Ojianwuna et al. (2016). Test termites were allowed to adjust to laboratory conditions for 24 hours before commencement of experiments reported in this study.

Experimental Procedures
Repellency and contact toxicity tests were conducted in the laboratory of the Department of Animal and Environmental Biology, University of Uyo, Nigeria.

Repellency Test
The procedure of Kadir et al. (2014), was adopted for the test where Whatman filter paper (9.1cm diameter) was cut into two equal halves. One half of each was separately impregnated with 0.05, 0.075 and 0.10 ml of the pure essential oils of C. odorata and U. chamae, using a micropipette arranged in a completely randomized design with three replications. The second half of each filter paper was left untreated and served as the control. The treated/ impregnated half was allowed to dry. In each case, the treated and untreated halves of the filter paper were placed in a test plate with a space of 1.5cm between them. Twenty active worker termites were released into the middle with the aid of camel hair brush as described by Addisu et al. (2014) and Alavijeh et al., 2014 and covered with untreated nets with mesh sizes large enough for ventilation, yet too small to allow for escape of test termites. Observations for repellency was made at 30 and 60 minutes per treatment. This was done by counting the number of termites on the treated and untreated portions of each filter paper.

Contact Toxicity Test
Cotton ball impregnation technique was adopted for the contact toxicity tests. Five volumes (0.05, 0.075, 0.10, 0.125 and 0.15 ml) of the oils of the two experimental plants were separately tested on the termites (worker caste of M. bellicosus). These constituted the treatments which were replicated three times. Cotton balls of 1 cm diameter each were separately impregnated with the different concentrations of each oil and introduced into the test plates. Control plates which were also replicated thrice had untreated cotton balls. Both treated and untreated (control) plates contained termite nutrient made of chips of dry wooden materials. Contact toxicity was evaluated by exposing 20 active worker termites to each test concentration within the test plate. Observation for mortality was made after an exposure period of 6 hours. A termite was confirmed dead when it no longer exhibited movement after being prodded by with a small camel hairbrush.

Data Analysis
Percentage repellency was calculated according to the formula given by McDonald et al., 1970: % Repellency = (NC -NT) x 100 (NC + NT) Where NC is the number of termites in the control/untreated portions and NT, the number of termites on the treated portions.
Percentage mortality = No. of dead termites x 100 Total No. of termites exposed Percentage knockdown is equal to: No of knockdown insects × 100 Total No of insects exposed.
Results were also presented as mean and standard error of the mean. Data were analysed using two-way Analysis of Variance (ANOVA), to determine the significant differences within a group or between different test concentrations. The significant means were separated using Turkey's mean separation procedure. Student t-test was used to determine the more potent of the two oils. All analysis was done using SPSS version 21 (2013).

General Observations
It was observed that on exposure of the test termites to both oils, there were aggressive attempts to escape from the test plates. The mandibles of the termites were also observed to be extended. Reduction in termite activities was also observed, prolonged exposure made the termites weak and sluggish. One of the reactions of the termites to the test oils was their secretion of brownish opaque substance. This was observed in both the repellency and toxicity tests of both oils. Knockdown of some of the termites was observed in all tests using both oils. The number of knocked down termites increased with increase in concentration for all tests. More termites were knocked down on exposure to U. chamae oil than C. odorata oil. Worthy of mention is the case of relatively agile termites making attempts to revive knocked down termites.

Repellent Effect of test oils on M. bellicosus
The test oils used demonstrated repellent effect on the test termites. There was a decrease in repellency with increase in time of exposure of the termites to the various volumes of the oil of C. odorata. Uvaria chamae oil increased percentage repellency of the termites for 0.05 ml and 0.10 ml volumes from 86.70 to 93.30 % and 90.00 to 100 % respectively. At 0.075 ml volume of U. chamae oil, there was a decrease in repellency from 100% at 30 minutes to 90.00 % at 60 minutes exposure period. There was no significant difference (p > 0.05) in the repellency of both oils at the various volumes tested as revealed by t-test analysis. When repellency of both oils was compared at 30-and 60-minutes p-value of 0.842 and 0.212 (p > 0.05) respectively were obtained. The results obtained for repellency are presented in Table 2.   Table 3 shows the knockdown and contact toxicity effects of the oils on M. bellicosus at the 60 th minute of exposure. Results obtained revealed that for both test oils the effect was concentration dependent. At the least volume of 0.05 ml of both oils, 3.33 % mortality was recorded for C. odorata while U. chamae recorded 13.33 % mortality of the termites. The highest oil volume of 0.15ml however resulted in percent mortality of 86.66 and 100 % for C. odorata and U. chamae respectively. There was no mortality in the control experiments. For the knockdown test, 16.65 % of the insects were knocked down on exposure to 0.05 ml of C. odorata oil while 40 % of the termites were knocked down on exposure to the same volume of U. chamae oil. At the highest volume of 0.15 ml of both oils, 56.67 % and 86.67 % knockdown were recorded for C. odorata and U. chamae respectively. There was however no knockdown recorded in the control experiments. Results obtained for knockdown (p = 0.000) and toxicity (p = 0.001) effects revealed significant difference (p<0.05) in the activity of the two oils .

DISCUSSION
The significant roles played by some plant essential/volatile oils in the management of insect vectors and pests, including some termite species have been well documented (Okweche et al., 2015;Nta et al., 2017). The escape attempt and loss of agility/weakness observed in this study is corroborated by the report of Mehmood and Shahzadi (2016), who made similar observation when they tested the essential oil of Boenninghausenia albiflora against black garden ants. The mandible extension of termites on exposure to test oils was described by Seid et al (2007) as "mandible strike", a form of defense mechanism. The brownish secretion observed in this study was described earlier by Prestwich (1979) as spray from the frontal gland of the termites that acts as defense mechanism when either soldier or worker termites feel threatened. Termites damage to buildings in tropical countries is a serious concern. Ugbomeh and Diboyesuku (2019) carried out a study on termite infestation of buildings in Aso, a rural community in the Niger Delta of Nigeria. They reported that of the 106 houses inspected, 35.85% were infested with termites. A study on the incidence and severity of termites' infestations on Azadirachta indica used as avenue trees in University of Port Harcourt, Nigeria was conducted by Adedeji et al. (2015). Two termite species incriminated in the infestations of these trees were Amitermes evuncifer and M. bellicosus. They further added that because of this infestation A. indica could not sustain the expected environmental service functions of avenue trees over time. The activity of mound-building M. bellicosus around Kwara State University Campus, Nigeria, was investigated by Ajao et al., (2018). Results obtained revealed the abundance of M. bellicosus mounds and called for precautionary measures to be taken to ensure the protection of the buildings in the campus. Earlier, Aisagbonhi (1989), carried out a survey of the destructive effect of M. bellicosus on coconut seedlings in the Nursery of Nigerian Institute for Oil palm Research (NIFOR), Benin, Nigeria. He found out that 10.8 % of 1300 coconut seedlings were attacked by termites and M. bellicosus was implicated as the main termite species. Also, nest-mounds of termites including those of M. bellicosus often obstruct agricultural machinery and have to be removed with explosives or bulldozers, thereby increasing the cost of mechanized farming, road construction and building site clearance (Oyedokun et al., 2011). Thus, Macrotermes is an important agricultural, forestry and household pest (Ojianwuna et al., 2016). Serious damage to crops such as maize, sugar cane, millet, rice, yam, groundnut, etc by M. bellicosus has been reported (Ito and Ighere, 2017 (Udebuani et al., 2015;Nyamador et al., 2017). Similarly, the efficacy of U. chamae in insect pest management have been documented by Negbenebor et al., (2018). They reported the insecticidal effect of the powdered stem bark and ethanol extract of U. chamae against Callosobrunchus maculatus, Rhizopertha dominica and Sitophilus zeamais. Chemical analysis of these oils revealed the presence of some chemical constituents that possess insecticidal properties. For instance, monoterpenes that were detected in both oils have been reported by Erol et al. (2013) to possess insecticidal property. Jija and John (2011) also detected sesquiterpenes in the essential oils of four species of Salvia (Salvia splendens, Salvia schult scarlet, Salvia elegans and Salvia dorisiana) that they studied. They attributed the insecticidal activities observed to the presence of sesquiterpenes in the oils. Benzyl benzoate that was detected in the oil from the roots of U. chamae has notable potency against the human itch mite, Sarcoptes scabiei. Its insecticidal efficacy is also well known. Walton et al., (2000) in their in vitro study reported the efficacy of benzyl benzoate in the control of Sarcoptes scabiei. Thus, the repellent, knockdown and contact toxicity of the test oils against M. bellicosus as reported in this research are attributable to these chemical constituents. Their effects which may have been additive or synergistic or otherwise require further evaluation. In terms of knockdown and toxicity efficacy, U. chamae was more potent than C. odorata. Similar differential potency was documented by Saeidi and Pezham (2018), when they tested essential oils of Eucalyptus globulus and Eucalyptus camaldulensis against the maize weevil, Callosobrunchus maculatus. They observed that E. globulus was more potent than E. camaldulensis against their test insect.

CONCLUSION
The need for the control of termites has become imperative owing to their devastating damage in agriculture and destruction of properties. Synthetic insecticides have remained the commonest method of control of these termites over the years. However, the continuous use of synthetic insecticides has given rise to problems such as resistance, persistence in the environment and high mammalian toxicity. Current research efforts on product development should focus more on ecologically tolerable control measures such as the use of plants products. Results obtained from this preliminary study reveal that essential oils from C. odorata and U. chamae could be used by resource poor farmers in the management of M. bellicosus. Adequate formulations of these oils could thus be prepared as eco-friendly repellents and also as contact insecticides for the control of this pest species.

CONFLICT OF INTEREST
Authors have no conflict of interest to declare.

ACKNOWLEDGEMENT
The GC-MS analysis of the oils was facilitated by Dr. Emmanuel Essien of the Faculty of Science, University of Uyo, Nigeria, while on his post-doctoral fellowship at the University of Karachi, Pakistan, Authors owe a debt of gratitude to him. Pharmacist Paul S. Thomas of the Faculty of Pharmacy, University of Uyo, Nigeria, is appreciated for his technical assistance.