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Journal of
Entomology and Nematology
Volume 6 Number 9 October, 2014
ISSN 2006-9855
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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: wlrivera@science.upd.edu.ph. 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
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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: tshiberu@yahoo.com.
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 21C, 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.
Among tested botanicals, Nicotiana spp. (local var.) was
effective in reducing population of tef shoot flies after
application of three days. Entomopathogenic fungi B.
bassiana (PPRC-6) was effective in reducing tef shoot
flies population after application of ten days in both tef
sowing methods. Hence, use of this technology
(botanicals and entomopathogenic fungi) can play a vital
role in reducing tef shoot flies under field conditions.
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