Consumer Sciences, University of Ghana. P. O. Box LG25, Accra, Legon, Ghana. Sunyani, Brong Ahafo Region, Ghana. Accepted 21 October, 2013

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1 International Journal of Agricultural Science Research Vol. 2(10), pp , October 2013 Available online at ISSN Academe Research Journals Full Length Research Paper Performance of Pimenta dioica L. leaf extract as an attractant for Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) in Citrus sinensis (Osbeck) plantations in Ghana Clement Akotsen-Mensah 1 *, Henry Ofosuhene Sintim 2, Collison Francis Brentu 1 and Isaac Amanor 1 1 Forest and Horticultural Crops Research Centre, Kade, Institute of Agricultural Research, College of Agriculture and Consumer Sciences, University of Ghana. P. O. Box LG25, Accra, Legon, Ghana. 2 Department of General Agriculture, School of Applied Science and Technology, Sunyani Polytechnic, P. O. Box 206, Sunyani, Brong Ahafo Region, Ghana. Accepted 21 October, 2013 This study was conducted with the primary objective of comparing the attractiveness of crude aqueous leaf extracts of an exotic spice plant Pimenta dioica L. with two commonly used attractants: methyl eugenol and sweet orange citrus (Citrus sinensis Osbeck) juice for the Africa invader fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) in a citrus plantation. No significant differences were observed among the tested concentrations (5, 10, 15 and 20% wt/vol) for most of the sampling days indicating that none of the concentrations was superior over the other in attracting B. invadens. The performance of the extracts was comparable with the synthetic methyl eugenol, particularly, during the first four days of placing the attractants in the field. Traps baited with synthetic methyl eugenol lure mixed with cypermethrin technical grade in a ratio of 4:1 v/v captured and killed significantly more adult B. invadens compared with traps baited with matured sweet orange citrus juice of about 10% brix. We have therefore shown for the first time that field populations of adult male B. invadens can be attracted to crude aqueous leaf extracts of P. dioica. These results suggest that leaf extracts of P. dioica can be used to detect and control B. invadens in citrus plantations. Key words: Fruit fly, Bactrocera invadens, methyl eugenol, Pimenta dioica. INTRODUCTION The search for effective aromatic lures in insect pest management has been one of the major preoccupations of pest entomologists. The discovery and synthesis of naturally occurring attractants has been the major challenge, particularly in developing countries. Fruit flies belonging to the family Tephritidae have been a major contributing factor to the serious crop losses seen in the horticultural industry in many countries where they are found (Goergen, 2011). The African invader fruit fly Bactrocera invadens (Diptera: Tephritidae) is one of the recently introduced fruit flies species in Africa (Drew et al., 2005; Ekesi and Billah, 2006; Goergen, 2011). In Ghana, B. invadens is believed to have been first introduced in 2005 after it invaded sub-saharan Africa in 2003 (Ekesi and Billah, 2006, 2009; Foba, 2009; Foba et al., 2012 and references therein). The insect is now well established, causing extensive damage to both temperate fruits like apples (Malus domestica Borkh), peaches (Prunus persica (L.) Stokes), almond (Prunus amygdalus Bastch), pear (Persea americana Mill.) and, *Corresponding author. cakotsen@ug.edu.gh.

2 Int. J. Agric. Sci. Res. 299 tropical and sub-tropical fruits like guava (Psidium spp.), mango (Mangifera spp.), and citrus (Citrus spp.), and the wild hosts tropical almond (Terminalia catappa L.), African wild mango (Irvingia gabonensis (Aubry-Lecomte) Baill.), and sheanut (Vitellaria paradoxa Gaertn.) (COLEACP and CIRAD Information Letter, 2008; Goergen, 2011). The insect is also competing well with other endemic fruit fly species such as Ceratitis cosyra Walker (Ekesi et al., 2010); Mediterranean fruit fly Ceratitis capitata Wiedemann and C. ditissima Munro (Akotsen-Mensah et al., unpublished). Although B. invadens is very important in mango production, there is evidence that they can also damage other fruit crops. B. invadens is reported to be the most devastative among both the indigenous and exotic species, and it is responsible for the extensive economic losses seen throughout sub-saharan Africa with damage levels exceeding 80% (Ekesi, 2010, Ekesi et al., 2010). It is estimated that where plantations are not managed there could be total fruit loss, although losses less than 30% can be salvaged if control measures are applied. Damage is caused by the adult female when eggs are deposited in the fruit of an ideal maturity stage. The larva which develops from the egg causes direct loss to the fruit through its feeding. In many cases, the damage predisposes the fruit to infection by other opportunistic organisms like fungi and bacteria. Because most countries regard fruit flies as quarantine pests, effective control usually requires international collaborations to prevent entry and establishment of flies in new areas (Ekesi and Billah, 2006, 2009). Management of B. invadens in many regions where they are a major pest has in the past relied extensively on insecticides (Afreh-Nuamah et al., 1985, 2007; Obeng- Ofori and Afreh-Nuamah, 2007) like dimethoate (organophosphate) and cypermethrin (pyrethroid). However, due to the adverse effects of these chemicals on the environment and also the quick development of resistance and the health risk insecticides generally pose to humans and other organisms, there is a shift from the sole use of insecticide to more sustainable strategies based on targeted application of novel attractants from either insects or plant sources. The use of these attractants has become more important following the renewed interest in organic farming which accepts only listed pesticides such as neem (Azadirachta indica A. Juss), pyrethrins, and spinosyns. These insecticides are mostly not effective against fruit flies. In recent times, several techniques such as the bait application technology (BAT), male annihilation technology (MAT) (Stark and Vargas, 1992; Vargas et al., 2005) and waste brewer s yeast have been developed as alternatives to the complete cover spray of high risk insecticides. For example, the MAT with a sprayable Specialized Pheromone and Lure Application Technology (SPLAT), in combination with methyl eugenol (ME) (4- allyl-1, 2-dimethoxybenzene-carboxylate), cue-lure (C-L) (4-(p-acetoxyphenyl)-2-butanone), and raspberry ketone (RK) (4-(p-hydroxyphenyl)-2-butanone) and the reducedrisk insecticide, spinosad, were developed for area-wide suppression of fruit flies in Hawaii (Vargas et al., 2005) although spinosad has been reported to have effect on the tephritid parasitoid Diachasmimorpha longicaudata (Ashmead) (Ruiz et al., 2008) and other natural enemies (Tillman and Mulrooney, 2000; Galvan et al., 2005). Also, field studies have revealed that the behavioral response of female fruit flies, in particular, the melon fly Bactrocera cucurbitae (Coquillett) and C. capitata to yellow-painted bait stations sprayed with GF-120 NF Naturalyte Fruit Fly Bait (BAT technique), was significantly enhanced in a papaya Carica papaya orchard (Piñero et al., 2009a). Some of these attractants have been used successfully in both conventional and organic fruit production and also in integrated pest management (IPM) programmes as documented in many parts of the world using the principle of attract and kill (Mau et al., 2007; Vargas et al., 2008a, b; Piñero et al., 2009b). Although these strategies were mainly developed and found to be very effective in the U.S., they are currently available in other parts of the world including Ghana and other African countries. It has, however, been observed that many small-holder farmers, particularly those in Ghana, may find the technologies expensive and thus be unwilling to use it to control fruit flies. This observation was also made by Manrakhan et al. (2013) who reported that GF-120 NF, a product similar to SUCCESS Appat (Vayssières et al., 2009; Bulley, 2011), can reduce fly infestation by over 80%, although citrus farmers in South Africa could not afford it. Recent field observations made at the University of Ghana s Forest and Horticultural Crops Research Centre, Kade, located in the forest transition agroecological zone showed that mechanically damaged leaves of an exotic spice plant Pimenta dioica could attract B. invadens in large numbers. The major compound in P. dioica oils is eugenol (70 80%) (Jirovetz et al., 2007; Rao et al., 2012). Also, leaf oils of the plant have 1, 8-cineole, α- humulene, β-caryophyllene and cadinene-derivatives which are also important. Given our initial results we hypothesized that crude extracts of P. dioica will attract and perform similarly to the commercially available methyl eugenol attractant for fruit flies because of the presence of methyl eugenol and thus can be used in conjunction with or replace the existing commercially available methyl eugenol used to manage B. invadens in Ghana. As part of our ongoing research efforts to develop cost effective attractants for monitoring and control of B. invadens and other fruit fly species, we initiated this field study to evaluate the performance of crude aqueous leaf extracts of P. dioica in attracting field populations of

3 Akotsen-Mensah et al. 300 B. invadens in citrus plantations. The ultimate goal was to develop an alternative, cost effective attractant similar in performance to the synthetic methyl eugenol which is not economically viable for small citrus farmers. MATERIALS AND METHODS Location and site description The study was conducted at the University of Ghana s Forest and Horticultural Crops Research Centre, Kade, Eastern Region, Ghana. The Centre is located (06º 09' 26N; 000º 55' 00W) in the forest transition ecological zone which is characterized by humid climate associated with two wet seasons and a dry spell with average elevation of 150 m. The annual temperature range at this location is C. Relative humidity is around 60-80% most of the year. This provides the appropriate environmental conditions for citrus varieties of both local (Citrus sinensis L.: Anomabo, Asuansi, Obuasi, Subi, etc.) and exotic (Citrus sinensis: Late Valencia, Sweet Mediterranean and Citrus unshiu Marcovitch: Satsuma, etc.) to grow well and produce fruit all year round, thus providing the necessary conditions for fruit flies to develop overlapping generations. Experimental set-up The experiments were conducted in an experimental citrus orchard (herein referred to as orchard) located at the Centre during the latter part of the 2012 major citrus season (January to April 2013) and 2013 minor citrus season (May-August). The two seasons were chosen because most fruit fly species found at the Centre peak during these seasons when ripened fruit which are the most vulnerable stage for fruit fly attack are in abundance (Akotsen-Mensah, 1999; Appiah, 2007; Appiah et al., 2009). The orchard has a history of high infestations of various fruit fly species. Three separate experiments were conducted. In experiment 1, two trap types and two attractants were compared. The trap types used were Pherocon trap (Great Lakes IPM, Vestuburg, MI) (Figure 1A) and a locally made Lynfield bottle trap (LMLT) (Figures 1B and C) made from a recycled transparent 500-ml cylindrical plastic bottle (Voltic Water, Ghana) with two equidistant holes created opposite each other in the uppermost part, a lid and a small metal string which was used as a hanger for one of the attractants. The attractants used were methyl eugenol (ME) and matured Late Valencia citrus juice of brix 10 (herein referred as citrus juice). In the Pherocon trap, the methyl eugenol and citrus juice were applied to a rubber septum before they were placed at the center of the trap s sticky base. No killing agent was added to the attractants but rather the sticky material at the base of the trap served as the capture medium. In the LMLT, the methyl eugenol was mixed with a killing agent (Cypermethrin technical grade) in a ratio of 4:1 v/v after which a cotton wool of ~10 cm diameter was dipped in the methyl eugenol-insecticide mixture. With the aid of the metal string and rope, the treated cotton wool was hung in the bottle. For the citrus juice, 200 ml of the juice with no killing agent was poured into the container. The killing mechanism in this case was by drowning as the adults entered the container to feed on the citrus juice. Based on the trap with lures, the mechanism of capture of the insect, and in-situ visual observation, the following treatments were evaluated: 1) Pherocon trap baited with methyl eugenol only (PT + ME), 2) Pherocon trap with citrus juice in cotton wool only (PT + CJ), 3) Pherocon trap with only rubber septum (PT + RUBB), 4) locally made Lynfield bottle trap baited with methyl eugenol and cypermethrin (LMLT + ME + CYP), 5) locally made Lynfield bottle trap containing Late Valencia citrus juice (LMLT + CJ), 6) a control consisting of the locally made Lynfield bottle trap and cotton wool (LMLT), and 7) visual observation (VO) on citrus plant for 10 min. Each of the treatments was replicated three times. Treatments except the VO were exposed for 24 h per month between January and April Placement of traps was done between the hours of GMT. Other beneficial insects such as the predacious lacewings (Chrysoperla spp) and bees (Apis spp) when present, were counted and recorded in order to determine the impact of the different treatments on these insects. In experiment 2, we examined the performance of two crude aqueous extract preparations, that is, dry or fresh leaves of P. dioica which we found in our preliminary test to attract B. invadens. Leaves used were dried and kept at room temperature for 48 h. Leaves were collected from two P. dioica plants located at the Centre and pounded in a wooden mortar with a pestle until a coarse paste was obtained. Known weights (50, 100, 150 and 200 g) of the paste was extracted with 1000 ml tap water to give an estimated 5, 10, 15 and 20% wt/vol suspensions, respectively. The mixtures were decanted using a sieve of mesh size 0.1 mm into separate open top plastic containers and used immediately. The container, measuring 12 cm high and 15.5 cm diameter was made of aluminum. The containers with the extracts (Figure 2B) were randomly placed in the orchard and monitored daily for a period of 8 days. During each sampling day, the contents of the containers were individually sieved and all insects captured were recorded and subsequently used for data analysis. Each treatment was replicated three times and arranged in a randomized complete block design. The sex of representative B. invadens adults captured in the traps baited with methyl eugenol or extracts were determined visually and also with the aid of a dissecting microscope (Leica) at 10X.

4 Int. J. Agric. Sci. Res. 301 A B C Figure 1. The baited traps. (A) Pherocon trap and (B and C) locally made modified Lynfield trap. In experiment 3, the best traps based on performance in previous experiments, material availability, and the best attractant or extract from experiment 1 and 2 were compared. Thus, locally made Lynfield bottle trap with methyl eugenol plus cypermethrin was compared with 10% wt/vol fresh aqueous leaf extract of P. dioica. The experiments were replicated four times. Statistical analyses All data were tested for the assumptions of ANOVA. The distribution of the data was performed using the Shapiro- Wilks W test of goodness-of-fit (JMP version 7.0.1, SAS Inc. Cary, NC, 2007). Where any of the assumptions were not met the data were subjected to ( X + 0.5) before analyses. Data were further analyzed by using standard least squares analysis of variance (two-way ANOVA) (P < 0.05; JMP version 7.0.1, SAS Inc. Cary, NC, 2007) to test for the effects of sampling date, treatment, extract type (dry vs. fresh) and interactions among the factors on B. invadens capture. Captures of B. invadens were then calculated for effects of factors that showed significant effect. Factor effects which showed significant difference were analyzed using one-way ANOVA using number of insect captured as the independent variable after which means were separated by the Tukey-Kramer HSD test (P < 0.05; JMP version 7.0.1, SAS Inc. Cary, NC, 2007). For other insects like predators, the numbers were very low for statistical analyses to be performed and thus data were not shown in result. RESULTS In experiment 1, the standard least squares ANOVA showed significant effect of treatment (F = 9.81, df = 6, P < ), but no significant differences for sampling date (F = 1.71, df = 3, P = ) and sampling date*treatment (F = 1.18, df = 18, P = ). The results on the performance of the two trap types and two attractants for each sampling date are summarized in Table 1. The results showed there were significant differences among the treatments during all the sampling dates except on 15 April (Table 1). The mean total number of B. invadens captured by the treatments on January 14 showed no significant difference among the locally made Lynfield trap + methyl eugenol + cypermethrin (mean ± SE: 97.7 ± 36.9), Pherocon trap + methyl eugenol only (45.0 ± 12.7) and Pherocon trap + citrus juice only (35.3 ± 12.9). But these three treatments were significantly different from the treatments which had citrus juice or during visual observation. The highest numbers of dead adult B. invadens were consistently recorded in the locally made modified Lynfield trap + methyl eugenol + cypermethrin treatment throughout the sampling periods. In experiment 2, the standard least squares ANOVA showed significant effects of sampling date (F = 41.06, df = 7, P < ), treatment (F = 3.23, df = 3, P = ) but no significant difference was recorded for the extract type (dry vs. fresh) (F = 2.58, df = 1, P = ) (Table 2). Also, there were significant two-way interactions between sampling date * treatment (F = 1.68, df = 21, P = ) and extract type * treatment (F = 3.09, df = 3, P = ) but no significant two way interaction between sampling date * extract type (F = 0.42, df = 7, P = ). There was also significant three-way interaction among sampling date * extract type * treatment (F = 2.71, df = 21, P = ). Since significant three-way interacttion among sampling date * extract type * treatment was recorded, captures of adults were compared using oneway ANOVA for each sampling date. The results showed no significant differences among the different concentrations for the number of adult B. invadens captured

5 Akotsen-Mensah et al. 302 Table 1. Mean (± SE) number of B. invadens captured in baited traps in a citrus plantation within 24-h sampling duration in later part of 2012 major citrus season. Treatment a Mean number of B. invadens recorded 14-Jan 15-Feb 15-Mar 15-Apr PT + ME 45.0 ± 12.7 ab 26.7 ± 6.5 ab 45.0 ± 5.6 a 27.7 ± 12.4 PT + CJ 35.3 ± 12.9 ab 0.0 ± 1.4 c 9.3 ± 1.4 b 6.7 ± 11.6 PT + RUBB only 4.3 ± 4.3 b 0.0 ± 1.4 c 0.3 ± 1.0 b 0.7 ± 10.1 LMLT + ME + CYP 97.7 ± 36.9 a 35.0 ± 9.6 a 33.3 ± 5.0 a ± 65.9 LMLT + CJ 7.7 ± 5.4 b 9.0 ± 2.5 bc 0.0 ± 0.8 b 13.7 ± 14.7 LMLT only 0.0 ± 3.8 b 0.0 ± 1.4 c 0.0 ± 0.8 b 0.0 ± 10.2 VO 2.6 ± 4.0 b 5.3 ± 0.8 bc 10.3 ± 3.2 b 6.7 ± 7.5 F-value P-value * * <.0001* df 3, 6 3, 6 3, 6 3, 6 Means in the same column for each sampling date with the same letters are not significantly different from each other (P = 0.05; Tukey-Kramer HSD test). * means significant at P < a Treatments are defined as: 1) Pherocon trap baited with methyl eugenol only in rubber septum (PT + ME), 2) Pherocon trap with citrus juice in cotton wool only (PT + CJ), 3) Pherocon trap with only rubber septum (PT + RUBB), 4) locally made Lynfield bottle trap baited with methyl eugenol and cypermethrin (LMLT + ME + CYP), 5) locally made Lynfield bottle trap containing 200-ml Late Valencia citrus juice (LMLT + CJ), 6) a control of only the locally made Lynfield bottle trap and cotton wool (LMLT), and 7) visual observation (VO).for 10 min. during the 8 consecutive sampling days except on day 4 (July 30) for extracts which were prepared from dry leaves (Figure 3A: F = 5.19, df = 3, 6, P = ) and day 1 (July 27) for extracts prepared from fresh leaves (Figure 3B: F = 7.83, df = 3, 6, P = ). Comparing the means on the sampling day in which the treatments showed significant differences for dry leaves extracts, the results showed that significantly fewer adult B. invadens were captured by the 10% (5.33 ± 4.01), 15% (8.33 ± 3.28) and 20% wt/vol (17.33 ± 4.69) treatments than the 5% wt/vol (31.00 ± 5.28) treatment. Similarly, the results on the sampling day in which the treatments showed significant difference for fresh leaves extracts showed that significantly fewer adult B. invadens were recorded on 15% (19.00 ± 7.23) and 20% wt/vol (22.33 ± 7.37). However, since majority of the sampling days showed no significant differences, it was considered that none of the concentrations was superior. Also, the number of B. invadens captured on extracts from both dried and fresh leaves declined with time indicating that the potency of the extract reduced over time. In experiment 3, the 10% wt/vol extract of fresh leaves was selected and compared with the locally made Lynfield bottle trap + methyl eugenol and cypermethrin. Standard least squares ANOVA showed significant effect of sampling date (F = 21.25, df = 8, P < ), treatment (F = 21.25, df = 8, P < ), but no significant difference in sampling date*treatment (F = 1.19, df = 1, P = ). Since significant sampling date*treatment was not recorded, captures of adults were compared using one-way ANOVA for each sampling date. The results showed there were significant differences in the number of adult B. invadens captured on sampling day 2 (F = 25.69, df = 1, 3, P = ), sampling day 5 (F = 18.36, df = 1, 3, P = ), sampling day 7 (F = 26.49, df = 1, 3, P = ), and sampling day 9 (F = 35.10, df = 1, 3, P = ). Where significant differences were observed between the treatments, the locally made Lynfield bottle trap + methyl eugenol + cypermethrin had the higher number of B. invadens compared with the 10% aqueous extract of the fresh leaves of P. dioica (Figure 4). In general, the number of insect captures declined over time (Figure 4). DISCUSSION We have shown for the first time that leaf extracts of P. dioica can attract field populations of B. invadens in citrus plantations in Ghana. Although the determination of the chemical composition of the extracts was beyond the scope of this study, we anticipate that the attractiveness of the extracts is mainly because of the presence of methyl eugenol, which has been reported and shown to mainly attract B. invadens (Ekesi and Billah, 2009 and references therein) and many other Bactrocera species (Vargas et al., 2009; Vargas et al., 2012). The fact that all the adults captured in the P. dioica extract and methyl eugenol baited traps were males also confirms the presence of methyl eugenol in the extracts since methyl eugenol is a known male-specific attractant for B. invadens

6 Int. J. Agric. Sci. Res. 303 Table 2. Standard least squares analysis of variance testing for effects of sampling date, extract type (dry vs. fresh) treatments (concentrations), and interactions of these variables on B. invadens capture. Source of variation df F-value P-value Sampling date < Extract type (dry vs. fresh) Sampling date extract type Treatment * Sampling date treatment * Extract type treatment * Sampling date extract type treatment * *Significant variables. Figure 2. Mechanically injured young P. dioica seedling with B. invadens (A) and extract with numerous B. invadens drowned in it (B). and other fruit fly species (Shelly et al., 2012). The leaves and berries of P. dioica have been shown to contain methyl eugenol and eugenol as the main constituents (Rao et al., 2012). All four of the concentrations we tested attracted B. invadens and thus can be used to manage the insect. We also found that large numbers of B. invadens can be attracted and drowned by trapping them with crude aqueous extract of the plant in open containers placed in a citrus plantation. This is in contrast with the commercially available synthetic methyl eugenol when placed on a cottonball in a LMLT, which requires an additional killing agent. The results showed that the number of captures of B. invadens reduced as the attractants aged in the field which suggests reduced potency of the active compound over time, particularly, in the crude extracts compared with the synthetic methyl eugenol. This may be perceived as a limitation of the extract. This limitation would likely be overcome if the extracts can be better preserved by adding a stabilizing agent. Despite this limitation, the crude extract in open containers has an advantage over the modified Lynfied trap because it can easily be prepared and used by farmers without any technical difficulties. Also, given that most farmers in Ghana mishandle insecticides (Essumang et al., 2008), by this method they will avoid exposure to harmful insecticides. In conclusion, our results show that crude extracts of P. dioica can attract B. invadens in citrus plantations. There were no indications of the extracts being detrimental to the environment by either attracting non-target insects or requiring the addition of a killing agent like insecticide. For now, the extract could be used for detection, monitoring, and control of B. invadens in citrus plantations. Also, the observation that mechanically damaged leaves (Figure 2A) on the plant can attract the insect could mean that citrus growers can plant seedlings of this plant around their orchards to serve as trap crops. Further studies are, however, required in this. We are therefore recommending further studies that would eventually identify the active ingredient of the extracts prepared from the P. dioica found in Ghana. This could further be developed into a commercial product if

7 Akotsen-Mensah et al. 304 Figure 3. Mean (± SE) number of B. invadens captured in different traps and bait combinations on citrus trees at each sampling day. (A) Dried leaf extract and (B) fresh leaf extract. the economics are better than the methyl eugenol currently available on the market and made available to citrus growers. P. dioica can be grown commercially for extract production. The trees might also be used to develop a dead end trap tree approach similar to that which use pure compounds of semiochemical cues to aggregate certain insects to a particular tree to make management decisions (Prokopy et al., 2003). As it stands now, citrus growers who would want to control B. invadens could consider using P. dioica crude leaf extract in the absence of the synthetic methyl eugenol. The possibility of including P. dioica in existing traps and mass trapping techniques so that we can develop attractand-kill systems including bait stations for fruit fly control is encouraged. This will be in agreement with current priority research areas in several regions of the world (IAEA, 2007 cited in Piñero et al., 2009a). ACKNOWLEDGEMENTS The authors want to thank the 2013 Leventis Foundation students of University of Ghana for their help in extract preparation and also data collection. We also express our gratitude to Mr. Badu-Darkwa, Mr. Charles Adu-Gyamfi

8 Int. J. Agric. Sci. Res. 305 Figure 4. Mean (± SE) number of B. invadens captured in locally made Lynfield bottle trap and 10% wt/vol of aqueous extract of fresh P. dioica leaves on citrus plants over time. and Mr. Isaac Amoh for their support in field work. REFERENCES Afreh-Nuamah K (1985). Important pests of citrus fruits in the Eastern Region of Ghana. Legon Agric. Res. Bull., 1: Afreh-Nuamah K (2007). Pests of citrus species- Rutaceae, pp In: Obeng-Ofori, D. (ed.). Major pests of food and selected fruit and industrial crops in West Africa, City Publishers Ltd, Accra, Ghana. Akotsen-Mensah C (1999). Management of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) using pheromone traps and neem seed extract. MPhil. Thesis, University of Ghana, Legon, 78 pp. Appiah EF (2007). Relationship between fruiting phenology of Late Valencia citrus (Citrus sinensis) and abundance and distribution of Mediterranean fruit fly, Ceratitis capitata. M. Phil thesis, University of Ghana, Legon, 85 pp. Appiah EF, Afreh-Nuamah K, Obeng-Ofori D (2009). Abundance and distribution of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) in Late Valencia citrus orchards in Ghana. Intern. J. Trop. Insect Sci., 29: Bulley F (2011). Comparative study of the effectiveness of Mazoferm, Success Appat (GF-120) and Waste Brewers Yeast (WBY) against fruit flies (Diptera: Tephritidae) in citrus orchard. M. Phil. Thesis, University of Ghana, Legon, 105 pp Drew RAI, Tsuruta K, White IM (2005). A new species of pest fruit fly (Diptera: Tephritidae: Dacinae) from Sri Lanka and Africa. Afr. Entomol., 13: Ekesi S, Billah MK (2006). A Field Guide to the management of economically important tephritid fruit flies in Africa. ICIPE Science Press. Nairobi, Kenya. Ekesi S, Billah MK (2009). A field guide to the management of economically important tephritid fruit flies in Africa (2nd edition). ICIPE Science Press. Nairobi, Kenya. Ekesi S, Billah MK, Nderitu PW, Lux SA, Rwomushana I (2010). Evidence for competitive displacement of Ceratitis cosyra by the invasive fruit fly Bactrocera invadens (Diptera: Tephritidae) on mango and mechanisms contributing to the displacement. Vitam Horm., 83: Ekesi S (2010). Development and implementation of a sustainable IPM program for major mango pests and opportunity for improving market and processing information in sub-saharan Africa. Progress Report, Deutche Gessellschaft fur Techniche Zusammenarbeit (GTZ) Gmbh, Eschborn, Germany. Essumang DK, Dodoo DK, Adokoh CK, Fumador EA (2008). Analysis of Some Pesticide Residues in Tomatoes in Ghana. Hum. Ecol. Risk Ass., 14:

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