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Cilek Florida A & M University 4000 Frankford Avenue, Panama City, Florida 32405 USA Dr. Khan Matiyar Rahaman Bidhan Chandra Krishi Viswavidyalaya AICRP (Nematode), Directorate of Research, BCKV, PO. Kalyani, Dist. Nadia, PIN-741235, West Bengal, India Manas Sarkar Defence Research Laboratory (DRDO, Ministry of Defence, Govt. of India) Post Bag No.2, Tezpur-784001, Assam, India Editorial Board Mehdi Esfandiari Department of Plant Protection College of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran Prof. Dr. Mahfouz M. M. Abd-Elgawad Nematology Laboratory Department of Phytopathology National Research Center El-Tahrir St., Dokki 12622, Giza, Egypt Matthew S. Lehnert Department of Entomology, Soils, & Plant Sciences Clemson University,Clemson, United States Wenjing Pang 3318 SE 23rd Avenue Gainesville, FL 32641 Agronomy and Biotechnological College, China Agricultural University,Beijing, China Dr. G. 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Dr. Mehdi Gheibi Department of Plant Protection, College of Agriculture, Shiraz Islamic Azad University, Shiraz, Iran Hasan Celal Akgul Istanbul Plant Quarantine Service, Nematology Laboratory Halkali Merkez Mahallesi, Halkali Caddesi, No:2, 34140 Halkali, Kucukcekmece-Istanbul/Turkey Dr. Nidhi KakKar University College, Kurukshetra University, Kurukshetra, Haryana, India Dr. Marianna I. Zhukovskaya Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences 44 Thorez Ave, 194223, Saint-Petersburg, Russia Gaurav Goyal University of Florida 282#14 Corry village, Gainesville, FL 32603, USA Gilberto Santos Andrade Universidade Federal de Viçosa Avenida Peter Henry Rolfs, s/n Campus Universitario 36570-000 Vicosa - MG Brazil Joshi Yadav Prasad Gyanashwor Kathmandu, Nepal G P O Box: 8975 EPC: 5519, Kathmandu, Nepal India Baoli Qiu Department of Entomology, South China Agricultural University No 483, Wushan Road, Tianhe, Guangzhou, PR China 510640 J. 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Academic Journals makes no warranty of any kind, either express or implied, regarding the quality, accuracy, availability, or validity of the data or information in this publication or of any other publication to which it may be linked. Journal of Entomology and Nematology International Journal of Medicine and Medical Sciences Table of Contents: Volume 6 Number 9, October, 2014 ARTICLES Molecular profiling of different morphotypes under the genus Rhynchophorus (Coleoptera: Curculionidae) in Central and Southern Philippines Reynaldo G. Abad, Joie Sheen A. Bastian, Rose L. Catiempo, Marian L. Salamanes, Phoebe Nemenzo-Calica and Windell L. Rivera Management of tef shoot fly, Atherigona hyalinipennis (Reg.) (Diptera: Muscidae) on tef at Ambo, West Showa of Ethiopia Abebe Mideksa, Mulugeta Negeri and Tadele Shiberu Vol. 6(9), pp. 122-133, October, 2014 DOI: 10.5897/JEN12.014 Article Number: 432E4D248075 ISSN 2006-9855 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JEN Journal of Entomology and Nematology Full Length Research Paper Molecular profiling of different morphotypes under the genus Rhynchophorus (Coleoptera: Curculionidae) in Central and Southern Philippines Reynaldo G. Abad1, Joie Sheen A. Bastian1, Rose L. Catiempo1, Marian L. Salamanes1, Phoebe Nemenzo-Calica2 and Windell L. Rivera3,4* 1 Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines-Mindanao, Davao City, Philippines. 2 School of Agriculture and Food Sciences, Faculty of Science, The University of Queensland, St. Lucia, Queensland, Australia. 3 Institute of Biology, College of Science, University of the Philippines-Diliman, Quezon City, Philippines. 4 Natural Sciences Research Institute, University of the Philippines-Diliman, Quezon City, Philippines. Received July 21, 2012; Accepted 30 September, 2014 Rhynchophorus ferrugineus Olivier and Rhynchophorus schach Olivier have long been reported as the two most common species of palm weevils in the Philippines. In 2008, surveys conducted in Agusan del Sur, Davao del Sur, Davao City and Cebu revealed more erstwhile unreported palm weevil morphotypes which led to perplexity in taxonomic classification. This study was conducted to determine and ascertain the genetic variation and phylogenetic relationship among 10 collected morphotypes, including R. ferrugineus and R. schach, using sequences of cytochrome oxidase I (COI) gene to resolve their taxonomic status. Results show that there were no marked morphometric differences among the palm weevils as supported by the COI characterization. Nucleotide sequences were subjected to phylogenetic analysis using bootstrapping, namely: neighbor-joining, parsimony and maximum likelihood. Among the phylogenetic trees generated, parsimony and maximum likelihood had similar results. Parsimony yielded low bootstrap values ranging from 2.5 to 10 while the maximum likelihood were from 1 to 7, indicating that these morphotypes can be clustered into one species. In support of these findings, low genetic variation was also observed, from 0 to 5.82% and high genetic similarity of 94.18 to 100% among morphotypes. Based on these results, it can be deduced that the morphotypes are cases of polymorphism, including the synonymy of R. ferrugineus and R. schach. Key words: Morphotypes, polymorphism, Rhynchophorus ferrugineus, Rhynchophorus schach, synonymy. INTRODUCTION Palm weevils under the genus Rhynchophorus have been reported as one of the major pests of coconut (Cocos nucifera L.) in the tropics (Menon and Pandalai, 1960). Two species, namely R. ferrugineus (Olivier) and R. schach Abad et al. (Olivier) were identified as common in India (Menon and Pandalai, 1960) and the Philippines (Blancaver et al., 1977; Gabriel, 1976; Loyola, 1994; Pacumbaba, 1972). Palm weevils have also been reported as major herbivores of sago (Metroxylon sagu Rottb.) in Agusan del Sur (Abad, 1983) and, on the basis of morphological descriptions from literature accounts, identified R. ferrugineus and R. schach as the most frequently encountered species. Further surveys of sago areas in the Caraga Region, Southern Mindanao and Argao, Cebu in 2008 showed weevil collections comprising not only of the two aforementioned species but of 37 other erstwhile unreported morphotypes (Abad et al., 2008). Such situation has created confusion in terms of nomenclatural systematics of weevils at hand. It could not be determined whether there are distinct species in collections or that all collections are cases of polymorphism; likewise, it casted doubt over the species status of R. ferrugineus and R. schach. While 39 morphotypes were arbitrarily categorized into five (5) major Rhynchophorus groups for convenient classification and reference (Salamanes, 2010), the need to resolve whether or not and if some or all of the phenotypic forms are genotypic in nature was considered to be in order. Pest identity is a basic knowledge that needs to be acquired so that appropriate pest management strategies can be devised in accordance with the nature and habits of the pest species. Outside the Philippines, the synonymy of R. ferrugineus and R. vulneratus has been reported at the molecular level with the aid of RAPD markers and cytochrome oxidase I (COI) sequences (Hallett et al., 2004). In fact, a recent study comparing DNA sequences of the mitochondrial COI gene confirmed R. vulneratus as distinct species from R. ferrugineus (Rugman-Jones et al., 2013). The genetic diversity of Rhynchophorus weevils has not yet been explored in the Philippines, particularly in the Central and Southern areas, and there are no current reports on how far its diversity has reached. New and sophisticated techniques such as molecular characterization can greatly aid in resolving species identification and phylogenetic relationship of Rhynchophorus in the Philippines. The COI gene from the mitochondrial DNA is usually utilized as the source for molecular characterization of different Rhynchophorus morphotypes to determine whether the cause of variations in form is genotypic or a case of polymorphism; with the possibility of looking at the emergence of one or few novel species. This pioneer work in the Philippines was conducted to verify the species/subspecies categories of selected Rhynchophorus weevils 123 found in Central and Southern Philippines using sequences of COI gene to determine the genetic variation among morphotypes of Rhynchophorus; and to ascertain the phylogenetic relationship of the morphotypes within the phylogeny of the genus Rhynchophorus. MATERIALS AND METHODS Sample collection The weevil specimens studied by Salamanes (2010) were used for molecular characterization. Samples were obtained from different areas in Central and Southern Philippines (Figure 1). Laboratory procedures were conducted from December 2010 to January 2011 at the Molecular Biology Laboratory of the University of the Philippines Mindanao, Davao City. Rhynchophorus species verification Species verification of Rhynchophorus was done based on morphological characteristics according to the parameters set by White and Borror (1970). They were classified according to the following features: distinct coloration on its pronotum and elytra; clubbed antennae which rise near the eyes, the first segment of its antennal club being enlarged and shiny; and an exposed pygidium. Members of the genus have sizes ranging from 3 to 31 mm. Morphometrics The 140 weevils collected, comprising 39 morphotypes, were arbitrarily classified into five categories, namely: striped, dotted, maple leaf-shaped, black-shaded and bilateral symmetry (Salamanes, 2010). Two variants were selected from each category for a total of 10 morphotypes, which were then subjected to DNA extraction. The samples used are shown and described in Figures 2-11. The morphometric data gathered by Salamanes (2010) on the 10 selected palm weevils were summarized and incorporated in the study to reconcile it to the results obtained from the molecular characterization of Rhynchophorus variants using COI gene. Measurements of antenna, rostrum, head, thorax, legs, and abdomen of each weevil sample were also included. Sexes of the samples were determined by examining the presence or absence of hair-like structures at the anterior part of the rostrum. Presence of these structures indicates that the weevil is a male; otherwise, the specimen is a female. DNA extraction DNA samples from 10 palm weevils in storage were processed following the protocol of Bastian et al. (2008). Briefly, the genomic DNAs were extracted from the leg muscle tissues of each weevil *Corresponding author. E-mail: [email protected]. Tel./Fax: No.+63-2-9205471. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 124 J. Entomol. Nematol. Figure 1. Map of the Philippines showing the sites where samples were obtained: (A) Argao, Cebu; (B) Davao City, Davao del Sur, Agusan del Norte and Agusan del Sur (Google Earth, 2011). and preserved in 95% ethanol. Three out of six legs of each weevil were prepared by washing six times with 100 mM EDTA pH 8.0 solution prior to extraction. Proteinase K digestion, phenol/chloroform extraction and ethanol precipitation were then employed. annealing at 45°C for 1 min, extension at 72°C for 1 min and a final elongation at 72°C for 5 min and finalized with a holding temperature of 4°C. PCR products were then subjected to electrophoresis and DNA bands were visualized under ultraviolet transilluminator (UVITEC Limited, UK). Polymerase chain reaction DNA sequencing and phylogenetic analysis PCR conditions were optimized using Veritti Thermal Cycler (Applied Biosystems) and the PCR mix was prepared according to the instructions stated in the PCR GoTaq® PCR Core System I manual (Promega, USA). The primers used were short F and short R, to amplify the COI gene with approximately 220 bp product (Gilbert et al., 2007). The final optimized conditions used were 94°C for 1 min for pre-denaturation, 35 cycles of denaturation at 94°C for 1 min, PCR products were purified using Purification Wizard SV Gel and PCR Clean-up System (Promega, USA) following the manufacturer’s protocol. Purified PCR products were shipped to Macrogen Inc., Korea for DNA sequencing services. Alignment of DNA sequences was done to identify regions of similarity on Rhynchophorus species using the WSSADI program (Polinar, 2005). After the alignment, Abad et al. Figure 2. Striped B Agusan del Norte. Figure 3. Striped C Norte. ♀ (Arl4-16) collected from Lansones, Rosario, ♀ (Anli-8) collected from Libas, Agusan del 125 Figure 4. Dotted B ♀ (Arl6-D5) collected from Lansones, Rosario, Agusan del Norte. Figure 5. Dotted J ♀ (Ala3-33) collected from Lamacan, Argao, Cebu. 126 J. Entomol. Nematol. Figure 6. Maple leaf-shaped E ♀ (Alu3-23) collected from Luwak, Argao, Cebu. Figure 7. Maple leaf-shaped G Luwak-Lamacan, Argao, Cebu. ♀ (Alula-21) collected from Figure 8. Black-shaded B ♀ (Ala2-25) collected from Lamacan, Argao, Cebu. Figure 9. Black shaded H♂ (Ala3-35) collected from Lamacan, Argao, Cebu. Abad et al. 127 mutations that might be useful in the analysis of the phylogenetic tree of the species Rhynchophorus were identified. Aligned nucleotide sequences were subjected to SEQBOOT with 1000 bootstrap replications. To obtain the genetic similarity (percentile) matrix, the bootstrap replicates were subjected to a program called DNADIST software using the two-parameter model (Kimura, 1980). For the phylogenetic tree regeneration, the programs DNAPARS, DNAML and NEIGHBOR were used for parsimony, maximum likelihood and neighbor-joining, respectively. All softwares are included in the Phylogenetic Inference Package (PHYLIP) v3.68. DNA sequences of the COI gene of different weevil samples were processed and submitted to the DNA databank. RESULTS Morphotypes of Rhynchophorus Figure 10. Bilateral symmetry B♂ (Ala2-28) collected from Lamacan, Argao, Cebu. Figure 11. Bilateral symmetry E♂ (Ata1-11) collected from Talaytay, Argao, Cebu. The 10 samples, two from each morphotype groupings, included in this study are presented in Table 1 and described in Figures 2-11. These palm weevils, bearing their own unique markings on the thorax region, were collected from areas in Central and Southern Philippines. Palm weevils categorized under this morphotype are characterized by having a bottle like stripe at the center of its thorax. The stripe is smoothly curved at the mid and upper portion. Forty specimens of both sexes were collected (Figure 2). This morphotype group has a central stripe with curvatures on both sides of its base with a distinct bulge at the middle. The anterior part of the stripe near the rostrum has a tapered like appearance which resembles a bottle neck. A total of four individuals comprising both sexes were collected (Figure 3). They are characterized as those having two pairs of dots placed symmetrically, the upper pair of dots smaller than the lower diamondshaped dots. A pair of faint black vertical lines can also be observed along the center of the thorax; and its elytra have colored striations. A total of two female individuals were collected (Figure 4). Weevils under this group have similarities with the Dotted B except that it has a distinct marking at the mid right portion of its thorax. Its two pairs of dots are located symmetrical to one another with the lower pair bigger and shaped like a diamond compared to the upper pair. Colored striations can be observed on its elytra. Only one female weevil was collected (Figure 5). This group of weevils has a pair of blotches at the upper periphery of the thorax. Its maple leaf-shaped pattern is smooth and rounder at the side with the tip of the central blotch being sharp. A small streak of red-orange can be observed at the center of the thorax from posterior to mid part. Only one female weevil was collected (Figure 6). Palm weevils under this group have a maple leaf-like outline and bean-shaped markings on their upper peripheral part; a slit can also be seen on the posterior end to the mid portion of the thorax. Colored striations on the elytra are also present. One female weevil was collected (Figure 7). 128 J. Entomol. Nematol. Table 1. Rhynchophorus morphotypes collected from Agusan del Norte and Cebu, Philippines. Morphotype ♀ Striped B ♀ Striped C ♀ Dotted B ♀ Dotted J Maple leaf-shaped E♀ Maple leaf-shaped G♀ ♀ Black-shaded B Black-shaded H♂ ♂ Bilateral symmetry B Bilateral symmetry E♂ Code Location Arl4-16 Anli-8 Arl6-D5 Ala3-33 Alu3-23 Alula-21 Ala2-25 Ala3-35 Ala2-28 Ata1-11 Lansones, Rosario, Agusan del Norte Libas, Agusan del Norte Lansones, Rosario, Agusan del Norte Lamacan, Argao, Cebu Luwak, Argao, Cebu Luwak-Lamacan, Argao, Cebu Lamacan, Argao, Cebu Lamacan, Argao, Cebu Lamacan, Argao, Cebu Talayatay, Argao, Cebu Source: Salamanes (2010). The thoraces of the weevils that belong to this group have a paired pattern that appear like a stalactite with a thin flare-like orange marking around it and an arch-like pattern prevailing it. No colored striations were observed. Only one female weevil was collected (Figure 8). A pair of boot-like blotches on the anterior region of the thorax was observed. The blotches resemble the land outline of Italy. A minute diamond-shaped space among the black markings at the mid portion of the thorax and a faint orange vertical line at the posterior region can be observed. Six weevils of both sexes were collected (Figure 9). Weevils under this group have a pair of markings, symmetrically placed on both sides and resemb-ling foliage on their thoraces. Another pair of elongated blotches is present at the mid upper portion of the thorax. Its elytra have colored striations. Two indivi-duals of both sexes falling under Bilateral Symmetry B were collected (Figure 10). This group of weevils is charac-terized with a pair of black markings superimposed on their prominently orange colored thorax. These markings give an orange-colored stripe along the mid-thoracic region. A total of four individuals, male and female were collected (Figure 11) Morphometrics No remarkable differences were observed on the sizes of the head, thorax, abdomen, rostrum and antennae among the 10 Rhynchophorus morphotypes, including R. schach and R. ferrugineus (Table 2). The same is true for the femur, tibial and tarsal lengths (Table 3). Genetic variation of Rhynchophorus morphotypes The amplified COI genes of the 10 Rhynchophorus morpho- types were 220 bp and produced low genetic variation among the 10 samples (Figure 12 and Table 4). Genetic variation and genetic similarity were then determined based on the sequences. The highest genetic variation yielded only 5.82% between Anli-8 and Ala2-25 while the lowest genetic variation yielded 0.88% between Arl4-16 and Anli-8. Arl4-16 and Ata1-11, Anli-8 and Ata1-11, Anli-8 and Alula-21, Arl6-D5 and Alu3-23 showed no genetic variation. Low genetic variation among 10 Rhynchophorus morphotypes suggests that there may have been insufficient time for these morphotypes to completely diverge into two significantly different species. Genetic similarity ranged from 94.18 to 100% which is very high in terms of likeliness for specific morphotypes to be considered as distinct species belonging to R. ferrugineus or R. schach (Table 5). Furthermore, samples with genetic variation ranging from 3 to 5% suggest that these morphotypes may be exemplifying the emergence of a novel subspecies. Phylogenetic tree of Rhynchophorus morphotypes To ascertain the phylogenetic relationship among Rhynchophorus morphotypes, three methods were used: neighbor-joining, parsimony and maximum likelihood. Among the three, parsimony and maximum likelihood presented similar results thus, they were subjected to further analysis and the outcomes were compared. Drosophila melanogaster, a member of the class Insecta was used as outgroup for the phylogenetic tree reconstruction (Figure 13). An outgroup was needed for the first outbranching of the tree. Bootstrap values, as indicated at each node of the tree were the basis for determining the relatedness of the 10 morphotypes. For Abad et al. Table 2. Mean length and width of head, thorax, abdomen, rostrum and antennae of the different Rhynchophorus morphotypesz. Morphotypes Arl4-16 Anli-8 Arl6-D5y Ala3-33 y y Alu3-23 Alula-21 y Ala2-25 y Ala3-35 Ala2-28 y Ata1-11 H 2.53 (±0.50) 1.85 (±0.15) 2.90 1.70 2.40 2.00 2.20 1.72 (±0.22) 1.90 1.85 (±0.23) T 12.43 (±1.65) 10.75 (±1.58) 12.80 11.50 12.00 10.30 11.20 10.88 (±0.80) 10.60 10.75 (±0.58) Length (mm) Ab 19.04 (±2.73) 16.08 (±1.67) 20.20 16.50 17.90 15.30 16.60 16.06 (±1.68) 15.80 15.80 (±1.27) z R 10.59 (±1.24) 9.30 (±1.64) 11.90 9.90 9.80 8.80 9.90 8.83 (±1.05) 8.80 8.45 (±0.41) An 7.81 (±1.51) 6.90 (±0.78) 8.10 6.20 6.80 6.10 6.10 6.58 (±0.57) 6.40 6.25 (±0.17) H 3.85 (±0.49) 3.50 (±0.22) 4.60 3.30 3.50 3.50 3.60 3.33 (±0.18) 2.90 3.13 (±0.16) Width (mm) T Ab 10.83 (±1.42) 13.48 (±1.69) 9.23 (±1.46) 11.33 (±1.86) 11.70 14.30 9.90 12.20 10.20 12.40 9.10 11.10 9.70 11.80 9.53 (±0.85) 11.74 (±1.08) 9.30 11.30 9.13 (±0.57) 11.33 (±0.69) R 1.61 (±0.21) 1.43 (±0.15) 1.80 1.30 1.40 1.30 1.40 1.37 (±0.11) 1.50 1.33 (±0.11) y H, Head; T, thorax; Ab, abdomen; R, rostrum; An, antennae. . Source: Salamanes (2010). only 1 or 2 specimens. Table 3. Mean length of femur, tibia and tarsus of the different Rhynchophorus morphotypes according to segments z. Morphotypes Arl4-16 Anli-8 Arl6-D5 y y Ala3-33 Alu3-23 y Alula-21 y Ala2-25 y Ala3-35 y Ala2-28 Ata1-11 Femur Length (mm) FL ML 6.66 (±0.96) 6.72 (±0.94) 5.75 (±0.90) 5.83 (±1.20) 7.10 7.10 5.10 5.40 4.90 5.70 5.10 5.00 5.00 5.20 5.15 (±0.43) 5.38 (±0.50) 5.50 5.40 5.25 (±0.32) 5.40 (±0.12) z HL 7.44 (±1.06) 6.48 (±0.99) 8.00 6.30 6.80 6.10 5.60 5.80 (±0.48) 6.20 5.90 (±0.12) y Tibia Length (mm) FL ML 6.76 (±1.20) 5.37 (±1.10) 5.73 (±1.31) 4.28 (±1.02) 6.70 5.40 5.10 4.50 5.70 4.70 5.00 4.40 4.80 4.10 5.35 (±0.37) 4.48 (±0.50) 5.60 4.10 5.15 (±0.15) 4.30 (±0.12) FL, Foreleg; ML, Middle leg; HL, Hind leg. Source: Salamanes (2010). only 1 or 2 specimens. HL 6.37 (±0.93) 5.80 (±1.10) 6.00 5.40 6.20 5.00 5.10 5.23 (±0.50) 5.10 5.25 (±0.25) Tarsus Length (mm) FL ML HL 5.69 (±0.92) 5.44 (±1.00) 5.57 (±0.83) 4.68 (±0.87) 4.68 (±1.02) 4.73 (±0.59) 5.90 6.00 5.80 4.30 4.20 --5.10 5.10 5.00 4.40 4.40 4.40 3.90 --4.50 4.50 (±0.59) 4.42 (±0.47) 4.48 (±0.52) 4.30 4.60 4.40 4.60 (±0.22) 4.63 (±0.23) 4.58 (±0.22) 129 130 J. Entomol. Nematol. Figure 12. The amplified COI genes of the 10 Rhynchophorus morphotypes: 1, Arl4-16; 2, Anli-8; 3, Arl6-D5; 4, Ala3-33; 5, Ala2-28; 6, Ata1-11; 7, Ala2-25; 8, Ala3-35; 9, Alu3-23; 10, Alula-21 using the short F and short R primers (Gilbert et al., 2007). Table 4. Genetic variation (%) of 10 morphotypes of Rhynchophorus from different locations based on COI gene sequences. Arl4-16 Anli-8 Arl6-D5 Ala3-33 Ala2-28 Ata1-11 Ala2-25 Ala3-35 Alu3-23 Alula-21 1 0.000000 0.878470 4.312502 4.641839 3.596298 -1.000000 4.627843 4.848904 4.779708 4.812748 2 3 4 5 6 7 8 9 10 0.000000 4.188519 4.922578 4.136224 -1.000000 5.820000 3.891408 4.908820 -1.000000 0.000000 2.741262 3.618501 2.128683 3.368906 3.323647 -1.000000 4.750243 0.000000 3.744180 3.532978 4.177738 4.194904 4.708742 1.315442 0.000000 5.092266 3.680079 4.187688 4.987939 3.666670 0.000000 4.944946 2.409259 4.679949 4.958101 0.000000 4.594211 5.267461 2.036880 0.000000 5.058018 3.571055 0.000000 4.009349 0.000000 (1), Arl4-16; (2), Anli-8; (3), Arl6-D5; (4), Ala3-33; (5), Ala2-28; (6), Ata1-11; (7), Ala2-25; (8), Ala3-35; (9), Alu3-23; (10), Alula-21. morphometrics between the two Rhynchophorus “species”, R. schach (categorized under striped group) and R. ferrugineus (under the dotted group), except for the body coloration or markings along with the other morphotypes, the characterization of COI gene of the 10 morphotypes revealed the relatedness of these weevils. the parsimony, low bootstrap support was observed with values ranging from 2.50 to 10.00. The same was observed for maximum likelihood with bootstrap values ranging from 1.00 to 7.00. Since trees generated from the two methods presented bootstrap values lower than 50, all of the 10 morphotypes of Rhynchophorus can be clustered into one group thus, supporting opinions that these morphotypes belong to one species of Rhynchophorus. DISCUSSION Variations in the morphology, specifically on thoracic markings of Rhynchophorus may be attributed to genetic Abad et al. 131 Table 5. Genetic similarity percentile matrix among Rhynchophorus morphotypes using COI gene sequences. Arl4-16 Anli-8 Arl6-D5 Ala3-33 Ala2-28 Ata1-11 Ala2-25 Ala3-35 Alu3-23 Alula-21 1 100.00 99.12 95.69 95.36 96.40 100.00 95.37 95.15 95.22 95.19 2 3 4 5 6 7 8 9 10 100.00 95.81 95.08 95.86 100.00 94.18 96.11 95.09 100.00 100.00 97.26 96.38 97.87 96.63 96.68 100.00 95.25 100.00 96.26 96.47 95.82 95.81 95.29 98.68 100.00 94.91 96.32 95.81 95.01 96.33 100.00 95.06 97.59 95.32 95.04 100.00 95.41 94.73 97.96 100.00 94.94 96.43 100.00 95.99 100.00 (1), Arl4-16; (2), Anli-8; (3), Arl6-D5; (4), Ala3-33; (5), Ala2-28; (6), Ata1-11; (7), Ala2-25; (8), Ala3-35; (9), Alu3-23; (10), Alula-21. Figure 13. Phylogenetic tree derived from (A) parsimony and (B) maximum likelihood for 10 Rhynchophorus morphotypes with Drosophila melanogaster as an outgroup. Bootstrap values are indicated at each node. drift, polymorphism or phenotypic plasticity wherein geographical distribution may have contributed to its distinct modification. Sexual dimorphism cannot be a factor among these morphological transformations since both male and female sexes were observed on the majority population of the five main categories of Rhynchophorus morphotypes. Nonetheless, several groups exemplified genetic variation percentile ranging from 3 to 5% suggesting an emerging of a subspecies. Based on the results obtained, the synonymy of R. ferrugineus and R. schach may be deduced. The findings parallel that of Hallett et al. (2004) who, by using RAPD markers and COI sequences, showed that the R. ferrugineus and R. vulneratus from Java are of the same 132 J. Entomol. Nematol. species. Thus, molecular characterization was a defining work to elucidate the species status of the morphotypes. With the trivial differences in Molecular characterization utilizing the COI gene was successfully carried out. Among the subunits of cytochrome oxidase, COI was preferred due to its highly conserved yet variable regions closely associated to the mitochondria (Lunt et al., 1996). The COI is unique in its ability to discriminate closely related species in all animal phyla except Cnidaria (Hebert et al., 2003). Phenotypic plasticity is a phenomenon that covers all types of environmentally- induced phenotypic variation (Stearns, 1989). It is the ability of a single genotype to produce more than one phenotype, be it morphological, physiological or behavioral in response to environmental conditions (West-Eberhard, 1989). However, epigenetic systems generally drive such changes. Mechanisms other than DNA sequences are responsible for the phenotypic variation in epigenetics. Polymorphism occurs when a species have more than one morph existing in the same population that inhabits the same time and space. These morphs must belong to a panmictic population (Ford, 1965). Such population assumes random mating and that every individual is a potential mate. Polymorphism can be passed on to progenies with modifications by natural selection. The genetic make-up or DNA sequences determine which morphs will be expressed by a population. From the characterization of the COI gene of the 10 different Rhynchophorus morphotypes, it is more likely that polymorphism could be the main factor which caused such morphological modifications. Genetic variation among the morphotypes is too high to be considered as due to phenotypic plasticity driven by epigenetic systems whereas, its high similarity with one another still indicates that they belong to one species. Geographical barrier leading to reproductive isolation is speculated to be a factor for the emergence of different morphotypes. Cebu, has only small patches of sago plantings in the sites visited, the biggest area of which was just about two hectares. This may have limited the breeding area of the weevils leading to more inbreeding thus giving greater chances for rare morphotypes to emerge. Consequently, large sago areas in Mindanao, especially in the provinces of Agusan, are common. This should allow gene flow over large populations of weevils to operate where rare traits could seldom emerge. It is of interest to note that the photographs of the collections by Rugman-Jones et al. (2013) from the northern island of the Philippines contained both the erstwhile considered “ferrugineus” type (with black dots on the thorax region) and the “shach” type (with orange to red stripe on the thorax region) and now classified on the basis of results of their molecular study as all belonging to the R. ferrugineus species. While the outcome of this study concurs with their findings, Rugman-Jones et al. (2013) found no genetic evidence of R. ferrugineus in the 150 palm weevils collected from Java. They added that Hallett et al. (2004) may not have encountered R. ferrugineus in Java at all and instead worked only with a R. vulneratus population showing high levels of color variation. Rugman-Jones et al. (2013) also hinted the possibility of finding R. vulneratus in Mindanao, obviously due to the relative proximity of the southern Philippine island to Java. The revealed synonymy of R. ferrugineus and the commonly referred to as R. schach in the Philippines has great implications for farmers specifically those who grow crops such as C. nucifera and M. sagu, plants that are both susceptible to palm weevils. Preventive and mitigating procedures for their destructive effects can now be formulated with relative ease as both morphotypes are of the same species and have the same biological properties. As a result, an applicable pest management strategy can now be devised to both morphotypes. This could also mean that in the long run, less input would be entailed in dealing with Rhynchophorus infestation. Conflict of Interests The author(s) have not declared any conflict of interests REFERENCES Abad RG (1983). Coconut pests and diseases in the Philippines. Coconuts Today United Coconut Association of the Philippines 1:119-152. Abad RG, Ligad MV, Aterrado ED (2008). Notes on two herbivorous palm weevil species associated with sago palms (Metroxylon sagu Rottb.) in the Philippines. Banwa 5:24-29. Bastian ST Jr., Malcampo RJA, Yamagata T, Namikawa T (2008). Phylogenetic affinity of poorly-known species of microbats in Mindanao based on complete cyt B gene sequences. J. Nat. Stud. 7:19-34. Blancaver RC, Abad RG, Pacumbaba EP, Mordeno JC (1977). Guidebook on Coconut Diseases. Philippine Coconut Authority, Davao City, Philippines. Ford EB (1965). Genetic polymorphism. Faber & Faber, London. Gabriel BP (1976). Pests of coconut in the Philippines. Phil. J. Cocon Stud. 1:15-26. Gilbert MT, Moore W, Melchior L, Worobey M (2007). DNA extraction from dry museum beetles without conferring external morphological damage. PloS One 2:e272. Hallett RH, Crespi BJ, Borden JH (2004). Synonymy of Rhynchophorus ferrugineus (Olivier), 1790 and R. vulneratus (Panzer), 1798 (Coleoptera, Curculionidae, Rhynchophorinae). J. Nat. Hist. 38:2863– 2882. Hebert PD, Ratnasingham S, deWaard JR (2003). Barcoding animal Abad et al. life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol. Sci. 270 Suppl 1:S96-99. Loyola AA (1994). A Guide to Coconut Pests and Diseases. Philippine Coconut Authority, Davao City, Philippines. Lunt DH, Zhang DX, Szymura JM, Hewitt GM (1996). The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol. Biol. 5:153-165. Menon KPV, Pandalai KM (1960). Pests. Indian Central Committee, Inrankulam, South India. Pacumbaba EP (1972). Biology of Rhynchophorus schach Olivier (Curculionidae : Coleoptera.). Third Annual Conference of the Crop Science Society of the Philippines, Cagayan de Oro City. Rugman-Jones PF, Hoddle CD, Hoddle MS, Stouthamer R (2013). The lesser of two weevils: molecular-genetics of pest palm weevil populations confirm Rhynchophorus vulneratus (Panzer 1798) as a valid species distinct from R. ferrugineus (Olivier 1790), and reveal the global extent of both. PloS One 8:e78379. 133 Salamanes ML (2010). Morphological characterization of the Palm weevils associated with Sago in the Philippines. Undergraduate Thesis. University of the Philippines Mindanao, Davao City, Philippines, pp. 12-73. Stearns SC (1989). The Evolutionary Significance of Phenotypic Plasticity. BioScience 39:436-445. West-Eberhard MJ (1989). Phenotypic Plasticity and the Origins of Diversity. Annu Rev Ecol Syst 20:249-278. White RE, Borror DJ (1970). Insects. Houghton Mifflin Company, USA. Vol. 6(9), pp. 134-139, October, 2014 DOI: 10.5897/JEN2014.0101 Article Number: 18D0BCB48077 ISSN 2006-9855 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JEN Journal of Entomology and Nematology Full Length Research Paper Management of tef shoot fly, Atherigona hyalinipennis (Reg.) (Diptera: Muscidae) on tef at Ambo, West Showa of Ethiopia Abebe Mideksa1*, Mulugeta Negeri2 and Tadele Shiberu2 1 Ambo district, West Shawa Zone Agricultureal and Rural Development office, Ambo, Ethiopia Department of Plant Sciences, College of Agriculture and Veterinary Sciences, Ambo University, Ethiopia. 2 Received 30 June, 2014; Accepted 4 September, 2014 Tef [Eragrostis tef (Zucc.) Trotter: Poaceae] is one of the major cereal crop and stable food crop of Ethiopia where it originated and diversified. Tef shoot fly (Atherigona hyalinipennis) is a serious pest of tef grown on black clay soils. Therefore, the present study was taken to assess the status of tef shoot fly on both row planted and broadcasted tef at small-scale farm. The experiment was laid out in randomized complete block design (RCBD) with three replications. Kuncho (Dz-Cr-387) variety was used for the experiment. Six treatments [two botanical extracts (Nicotiana sp. and Azadirachta indica) and two entomopathogenic fungi, (Beauveria bassiana and Metarhizium anisopliae) were evaluated against tef shoot flies. The result shows that all the treatments were significant (P≤ 0.05) from untreated control. Nicotiana sp. exhibited high mortality rate on both broadcast and row planted tef to a level of 80.09 and 82.59%, respectively. However, A. indica showed high mortality rate only for tef planted in rows (77.7%). Metarhizium anisopliae caused high mortality rate only in tef planted in rows. The highest yield loss (9.03%) was recorded in broadcasted on M. anisophliae followed by untreated control and in row planted B. bassiana and M. anisophliae were estimated as 8.03 and 8.23%, respectively. Hence, all treatments gave promising efficacy percent and can be used for tef shoot fly management under field conditions. Key words: Botanicals, Metarhizium anisopliae, Beauveria bassiana, tef shoot fly, management. INTRODUCTION Tef [Eragrostis tef (Zucc.), Trotter: Poaceae] originated and diversified in Ethiopia. In Ethiopia tef is grown as a staple food crop for centuries. Over 2.8 million hectares of land is covered with tef every year with its mean pro- ductivity at national level predicted at 1228 kg ha-1 (CSA, 2011). During cropping season, tef occupied 22.6% of the cultivated lands under cereals, while maize occupied 17%, sorghum 15.92%, wheat 11.89% (CSA, 2012). *Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Mideksa et al. Although, tef is annually cultivated in a large area of lands in Ethiopia, it gives relatively low yield compared to the other cereals (CSA, 2010). One of the main reason for the low yield of tef is biotic factors (insects, diseases and unimproved seeds). In addition, inappropriate crop manage-ment practices that mainly include sowing methods, weeding practices, harvesting stage, cropping system, and fertilization contributed for the low productivity of tef in Ethiopia. Tef crop management practices such as tillage and cropping systems were studied and reported in order to develop improved practices for various tef producing regions in Ethiopia (Worku et al., 2005). Major insect pests recorded on this crop include, Atherigona hyalinipennis (tef shoot fly), Delia arambourgis (Barley fly), Decticoides brevipennis (Wello bush cricket), Eriangerius niger (Black tef beetle), Macrotermes subhyalinus (Mendi termes), and Mentaxya ignicollis (red tef worm). Others such as Acrotylus spp., Aiolopus longicornis, Carbula recurva and Odontotermes sp. also cause damage to tef (HARC, 1989). The species composition of tef shoot fly in Haramaya area includes: Elachiptera simplicipes (Becker), Mlanochaeta vulgaris (Adams), Osicinella nartschukiana (Beschovsky), O. acuticornis (Becker) and Rhopalopterum sp. in the Chloropidae and Atherigona hyalinipennis and Atherigona sp. of the family Muscidae (Sileshi, 1994, 1997). The status of tef shoot fly, A. hyalinipennis as a major pest of tef was reported in five regional state of the country (Tadesse, 1987). It caused 4.9 up to 24% yield loss in Tigray Region, East and South West Showa zone (DZARC, 2000; Bayeh et al., 2008) with more than 90% of panicle damage (Sileshi, 1997). Similarly, Bayeh et al. (2009) also reported that shoot fly cause serious damage on seedlings and panicle stages of tef. The control of tef shoot fly using chemical and cultural practices has been attempted earlier to some extent. However, they were not adequate to minimize the density of tef shoot fly and thereby alleviating the yield loss caused by the pest, hence it is necessary to develop Integrated Pest Management (IPM) tactics. Therefore, the present study was designed to evaluate bio-pesticides, and to compare the infestation level of shoot flies on row planting and traditional broadcasting system of tef crop. MATERIALS AND METHODS 135 is about 500-900 mm; the soil type is Vertisol and consists of 60%; Clay, 15%, Silt, 20% and Sand 5% (AWAB, 2012). The farming system of the study site is cereal crops dominated. Crop establishment A seed of tef, Kuncho variety (DZ-cr-387) was obtained from Ambo District Agricultural Office. Each plot was (3 x 2 m) = 6 m2 with in row planting (20 cm between row) and broadcast. All necessary agronomic practices and fertilizers applications were carried out. Experimental design and treatments The experiment was carried out using randomized complete block design (RCBD) arranged factorially with three replications at farmers’ field conditions. Factor “A” (treatments) was in the main plot (Figure 2), while Factor “B” (Row planting/Broadcast) in the sub plot (Figure 1). The treatments were: tobacco leaf and stalk aqueous extract (Nicotiana spp., local variety), neem seed powder aqueous extract (A. indica), Beauveria bassiana (PPRC-6), M. anisopliae (PPRC-56), Endosulfan 35% E.C and untreated check. Extraction and preparation of botanicals Nicotiana sp. (tobacco) Tobacco leaves and stalks were collected from the gardens of farmers around Ambo areas, Ethiopia. The collected materials were dried under shade, 10.8 g of dried leaves and stalks were crushed and mixed with 0.54 L of water and 10 mg of soap flake was added as adhesive agent (Tadele et al., 2013). The prepared extract was showed in (Figure 5). Azadirachta indica (neem) The neem seeds were collected from Melka Werer Agricultural Research Center, Ethiopia. The seeds were ground and then 18 g of neem powder was mixed with 0.72 L of water, and extracted over night (Tadele et al., 2013). The next day, it was filtered with the help of cheese cloth and mixed with liquid soap at the rate of 1 mL/L of extract (Figure 4). Then the solution was ready for spray on infested shoot fly (Stoll, 2000). Rate of botanicals and chemical used against tef shoot fly The rate of botanicals Nicotiana spp. (local var.) leaves and stalks, A. indica (seeds) and Endosulfan 35% EC were 3 and 5 kg and 2 L per hectare and mixed with 150, 200 and 100 L amount of water required per hectare, respectively. Description of the study area Entomopathogenic fungi (EPF) The experiment was conducted at Ambo district, Boji Gebissa Kebele, West Showa of Ethiopia, during the main cropping season of 2013. The experiment site is located about 116 km West of Addis Ababa at an altitude of 2184 m.a.s.l., latitude of 37 49’E and longitude of 8 56’N. The annual mean minimum and maximum temperature is about 15 and 21C, respectively. The annual rainfall Two indigenous EPF viz. B. bassiana (PPRC-6) and M. anisopliae (PPRC-56) were obtained from Ambo Plant Protection Research Center, Ambo, Ethiopia. Sabouraud Dextrose Yeast Agar (SDYA) media was used for sub-culturing of both EPF. The cultures were incubated at 28°C for 10 days in the dark. On each culture plate, 136 J. Entomol. Nematol. Yield loss (%) = X-Y X x 100 Where, X = Mean grain yield of treated plot; Y = Mean grain yield of untreated plot. Data were analyzed using Statistically Analysis Software (SAS, 2000). The mean comparisons were carried out using Duncan’s Multiple Range Test (DMRT). Efficacy data was analysed after being transformation to arcsine (Gomez, 1984). RESULTS AND DISCUSSION Effects of botanicals on shoot fly in broadcasted tef field Figure 1: Row planted tef Figure 2: Broadcasted tef spores were harvested by flooding 10 mL of distilled sterilized water and 0.01% Tween 20 on plates. The spore suspensions were again filtered through cheese cloth and diluted (1:10) in sterile water. The suspensions were vortexed for 8 min to avoid clumping of the spores. The conidial concentration of each isolate was adjusted to 1×107 conidia/ml using haemocytometers (Seneshew et al., 2003) and then made ready for foliar application by hand sprayers. Data collection and statistical analysis Germination and number of tef plants per plot were recorded up to its economic threshold level. The number of dead heart per plot and number of larvae per plant were recorded before and after application. Yield loss percentage was calculated by comparing the weight of protected grain yield with other treatments. The effect of botanicals on tef shoot flies population in the broadcasting plots is given in Table 1. The infestation was high with increasing trend of damage on the crop during mid September to early October. Non significant differences were observed between both treatments and the three and ten days after application a percent mortality of tef shoot flies. All treatments were significantly (P≤0.05) different from the untreated control. The rate of mortality in all of the treatments were recorded within three and ten days after application ranged from 0.01 - 85.89%. The plot which was treated with Nicotiana sp. (leaf and stalk) showed significantly high mortality rate (80.09%). Tobacco extract provided the best control of tef shoot flies than the plots treated with A. indica. The better treatment effect was recorded within three days after application and the analysis of variance also indicated significant (p≤0.05) differences from that of untreated control. Similarly, Tadele et al. (2013) reported that the tobacco aqua extract solutioncontrolled onion thrips which reduces the population number of nymph and adults stage after three days of application. Effects of entomopathogenic fungi on shoot fly in broadcasted tef field The data presented in Table 1 shows that B. bassiana and M. anisopilae caused 77.92% and 67.56% mortality on tef shoot flies after ten days applications, respectively. It was observed that the effect of B. bassiana on the mortality rate of tef shoot flies within ten days after application was significantly higher when compared with that of the treatment of M. anisopilae. Three days after application of treatments, entomopathogenic fungi and untreated control showed no significant differences. The effectiveness of both treatments were recorded within ten days after application and indicated significant (p≤0.05) differences from that of the untreated. Daglish (1998) reported that the efficacy of B. bassiana Mideksa et al. 137 against the larvae, pupae, and adult females of the Mexican fruit fly, Anastrepha ludens (Loew) showed high levels of mortality for adult flies. Some of the major economic insect pests are susceptible to this fungus (Tanada and Kaya, 1993). In experimental field condition, there was no side effect due to the application of botanicals and entomopathogenic fungi on the crop. The treated crops were not different in appearance and growth performance from that of standard check on both row planted and broadcasted tef. Effects of botanicals on shoot fly in row planted Effects of botanicals and entomopathogenic fungi on yield The effect of botanicals on tef shoot flies populations that were planted in rows showed that the infestation level was low when compared with broadcasting method. The damage on the crop during mid September to early October was very low. All the treatments gave significant (P≤0.05) differences when compared with untreated plots. Both exhibited no significant difference between the two treatments and three and ten days after application and percent of mortality tef shoot flies. The rates of mortality in all of the treatments were recorded three and ten days after application which ranged from 0.01 - 83.86%. The highest mortality rate of tef shoot flies three days after application in the plots treated with Nicotiana spp. (leaf and stalk) and A. indica (seed) were 82.59 and 77.71%, respectively. The effect of Nicotiana spp. and A. indica on tef shoot flies population were significantly increased three and ten days after application when compared to untreated plots and the analysis of variance indicated significant (p≤0.05) differences from untreated control. Singh and Batra (2001) studied the bio-efficacy of different neem formulations along with recommended insecticide endosulfan in forage sorghum against shoot fly. Rao and Panwar (1992) reported that the efficacy of neem reduced the infestation level of shoot fly, A. socata and A. naquii in maize. Juneja et al. (2004) reported that neem seed kernel suspension were the most effective in the reduction of shoot fly (A. approximate) infestation. Effects of entomopathogenic fungi on shoot fly in row planted The data presented in Table 1 shows that B. bassiana and M. anisophliae caused 70.85 and 63.45% mortality of tef shoot flies after ten days application, respectively. The results show that the mortality rate of tef shoot flies, B. bassiana and M. anisophliae was significantly (p≤0.05) different from the untreated control. After three day application of treatments, entomopathogenic fungi and untreated control had no significant difference. The result was confirmed with the previous work of Booth and Shank (1998) that found that the M. anisopliae was extensively used for the biological control of insect pests and various other soil borne Popillia japonica was controlled by using B. bassiana and M. anisopliae. Broadcasting methods The tef crops were harvested on November, 2013, threshed and the yield was measured and presented in Table 2. The treatments included Nicotiana sp., A. indica and B. bassiana; recorded statistically no significant differences on yield. There was significant increase in tef yield in the plots treated with Nicotiana spp. (local var.) when compared to untreated control. The highest yield loss presented in the Table 2 on the treatment of M. anisophliae was estimated as 9.03% followed by untreated control (17.13%). This showed that the yield loss due to tef shoot fly infestation was high and significantly (P≤0.05) different among the treatments. The tef yield loss due to tef shoot fly in different areas of the country varies depending on certain environmental condition. In East and South West Shewa Zones was less than 5%. Similarly, in Gojam area tef shoot fly did not cause yield losses (AARC, 2002). On the other hand, in Tigray Region where precipitation is low and the soil was degraded (Tesfaye and Zenebe, 1998), the tef shoot fly population that built- up on sorghum might have caused several damage (378 to 522 kg ha-1) (Sileshi, 1997). Row planting methods Nicotiana spp., A. indicia and B. bassiana showed a non significant difference. There were an increased tef yields in those treated with Nicotiana spp. (local Var.) when compared to untreated plot. Row planted tef produced higher and stronger tiller, and increased number of seeds /panicle. The losses of tef yield presented in Table 2 shows that tef yield loss attributed to tef shoot fly infestation (Figure 3). Treatments B. bassiana and M. anisopliae showed no significant differences. The highest yield loss recorded from the treatments of B. bassiana and M. anisopliae was estimated as 8.03 and 8.23% compared to the untreated control. This shows that the yield losses due to tef shoot fly infestation was very high and significantly (P≤0.05) different among the treatment. Row planted tef yield losses due to tef shoot fly infestation was low compared 138 J. Entomol. Nematol. Table 1. Percent mortality of tef shoot flies under field condition in planted tef at Ambo, West Shawa Ethiopia in 2013/14. Treatment Nicotiana spp. (local var.) Azadirachta indica (seed) Beauveria bassiana Metarhizium anisophliae Endosulfan 35% EC Control (untreated) MSE Days after treatment application a percent mortality of tef shoot flies Broadcasted tef Row planted tef rd th rd th 3 day 10 day 3 day 10 day ab ab a 95.58(80.09±1.39) 95.58(80.09±1.39) 95.24(82.59±1.44) 95.24(82.59±1.44)a b b a a 89.66(71.35±1.25) 89.66(71.35±1.25) 93.33(77.71±1.36) 93.33(77.71±1.36) c ab b ab 0(0.01±0) 93.52(77.92±1.36) 0(0.01±0) 88.68(70.85±1.23) c b b 0(0.01±0) 85.25(67.56±1.18) 0(0.01±0) 77.66(63.45±1.08)b a a a a 98.49(85.89±1.49) 98.49(85.90±1.49) 96.67(83.86±1.46) 96.67(83.86±1.46) c c b c 0(0.01±0) 0(0.01±0) 0(0.01±0) 0(0.01±0) 5.14 6.66 6.98 7.40 CV (%) 12.99 10.44 16.98 Means with the same letter are not significantly different (P≤0.05), and Figures in the brackets are Arcsin 11.80 transformation value. Table 2. Mean yield and yield loss of tef against tef shoot flies on broadcasted and row planted of tef at Ambo, 2013 /14. Treatments Nicotiana spp.(loca var) Azadirachta indica Beauveria bassiana Metarhizium anisophliae Endosulfan 35% EC Control (untreated) MSE CV (%) Broadcasted tef Mean yield/Plot Percentage yield loss (%) (kg) ab d 1.25 3.73 bc bc 1.23 7.83 1.09bcd 7.03c cd 1.05 9.03b a 1.42 0.83e d a 0.91 17.13 0.09 0.82 8.6 10.82.82 Row planted tef Mean yield/plot Percentage yield (kg) loss (%) ab 1.62 1.55bc 1.57bc 1.53c 1.67a d 1.40 0.04 2.56 Means with the same letter are not significantly different (p≤0.05). Figure 3: Tef infected by tef shoot fly Figure 4: Neem seeds extract d 3.30 7.07c 8.03b 8.23b 0.74d a 12.67 0.46 6.87 Mideksa et al. Figure 5: Tobacco leave and stalk extract to broadcasted tef (Table 2). The yield loss ranged from 0 to 12.67% during the study period. Conflict of Interests The author(s) have not declared any conflict of interests. CONCLUSIONS AND RECOMENDATION Botanical and entomopathogenic fungi were effective to reduce tef shoot flies population infesting tef crops. 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