spp. (diptera · braconidae) associated with bactrocera spp. (diptera: tephritidae) infesting...

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Dual-target detection using simultaneous amplification of PCR in clarifying

interaction between Opiinae species (Hymenoptera: Braconidae) associated

with Bactrocera spp. (Diptera:...

Article in Arthropod-Plant Interactions · April 2015

DOI: 10.1007/s11829-015-9355-2

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Salmah Yaakop

Universiti Kebangsaan Malaysia

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Arthropod-Plant InteractionsAn international journal devoted tostudies on interactions of insects, mites,and other arthropods with plants ISSN 1872-8855 Arthropod-Plant InteractionsDOI 10.1007/s11829-015-9355-2

Dual-target detection using simultaneousamplification of PCR in clarifyinginteraction between Opiinae species(Hymenoptera: Braconidae) associatedwith Bactrocera spp. (Diptera: Tephritidae)infesting several cropsS. Yaakop, S. Shariff, N. J. Ibrahim,B. M. Md-Zain, S. Yusof & N. MohamadJani

1 23

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ORIGINAL PAPER

Dual-target detection using simultaneous amplification of PCRin clarifying interaction between Opiinae species (Hymenoptera:Braconidae) associated with Bactrocera spp. (Diptera:Tephritidae) infesting several crops

S. Yaakop • S. Shariff • N. J. Ibrahim •

B. M. Md-Zain • S. Yusof • N. Mohamad Jani

Received: 21 July 2014 / Accepted: 12 January 2015

� Springer Science+Business Media Dordrecht 2015

Abstract Simultaneous polymerase chain reaction (PCR)

by using multiplex PCR was conducted to clarify the

interaction between Opiinae associated with Bactrocera

carambolae, B. papayae, and B cucurbitae. We sequenced

and characterized a dual-band target detected on Opiinae

DNA fragments by using two combination pairs of uni-

versal primers on two molecular markers, namely cyto-

chrome oxidase subunit I (COI) and cytochrome b.

Additionally, each Bactrocera species was identified by

amplifying the COI region. Sequence data obtained from

multiplex PCR seemed very effective in confirming spe-

cies-level distinction that has been shown in tree topology.

We conducted phylogenetic analyses prior to clarification

of the interaction among three taxa levels. Interestingly, the

sequences obtained from the simultaneous PCR success-

fully differentiated between six closely related Opiinae

species in three genera, which could potentially be mass-

reared as biocontrol agents of bactroceran fruit flies that

infest several species of fruit. We discovered, proved, and

added molecular data to clarify the interaction between

Opiinae parasitoids, their host (Bactrocera spp.), and

associated plants species. Psyttalia fletcheri parasitizing

Bactrocera cucurbitae, which infests the ridge gourd fruit,

has been added as a new record from Malaysia. This

information would be extremely useful in taxonomic

identification of species as part of an effective method in

biological control program of the targeted fruit fly pests

and their associated crops.

Keywords Opiinae � Parasitoid � Fruit fly � Cytochrome coxidase subunit I � Cytochrome b � Biological control

Introduction

Opiinae are known to be solitary koinobiont endoparasi-

toids of cyclorraphous dipteran larvae (Gimeno et al.

1997). Generally, parasitoid wasps are environmentally

friendly, and widely used for biological control against

various fruit fly species (Garcia and Ricalde 2013). Their

small body size and lack of informative morphological

characters have resulted in the misidentification of the

Opiinae species, and this may limit the successful man-

agement of pests in any biological control program against

fruit flies (Jenkins et al. 2012; Montoya et al. 2009).

Selection of a suitable genetic marker for molecular

identification is very important in confirming the status and

identity of Opiinae individuals not only at higher taxo-

nomic levels, but also for variations within species. How-

ever, not all genes in the mitochondrial genome have

evolved at the same rate (Pesole et al. 1999). In particular,

cytochrome oxidase subunit I (COI) has been frequently

used in species identification because of its ability to show

high nucleotide substitutions (Derocles et al. 2011;

Handling Editor: Rupesh Kariyat.

S. Yaakop (&) � S. Shariff � N. J. Ibrahim � B. M. Md-ZainCytogenetic Laboratory, Faculty of Science and Technology,

School of Environmental and Natural Resource Sciences,

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor,

Malaysia

e-mail: [email protected]

S. Yaakop � S. Shariff � N. J. IbrahimFaculty of Science and Technology, Centre for Insects

Systematics, Universiti Kebangsaan Malaysia, 43600 Bangi,

Selangor, Malaysia

S. Yusof � N. Mohamad JaniHorticulture Research Centre, Malaysian Agricultural Research

and Development Institute (MARDI), 43400 Serdang, Selangor,

Malaysia

123

Arthropod-Plant Interactions

DOI 10.1007/s11829-015-9355-2

Author's personal copy

Mardulyn and Whitfield 1999; Zaldivar-Riveron et al.

2010). This gene has often been used in species determi-

nation of braconids because of its higher interspecific

variability but moderate intraspecific variability (Simon

et al. 1994). Additionally, COI is known as a DNA barcode

as it consists of short DNA fragments and has been stan-

dardized for global identification in all animals (Hebert

et al. 2003). Cytochrome b (Cyt b) has been shown to have

higher nucleotide variations (Irwin et al. 1991; Simmons

and Weller 2001) than the COI gene. However, it shows

slow amino acid substitution but rapid nucleotide substi-

tution at the third codon position (Farias et al. 2001; Irwin

et al. 1991). Due to the factors that are mentioned above,

both markers have been chosen to clarify the interaction

between parasitoids, pests, and their associated plants

species.

In Malaysia, heavy losses to fruits and vegetables are

largely caused by the infestation of species of Bactrocera

Macquart, 1835. Potential parasitoids should therefore be

correctly identified for the sustainable control of targeted

fruit fly pests in this region. Several records of parasitoid–

pest–host plant species have been documented based on

morphological identification (Chinajariyawong et al. 2000;

Chua and Khoo 1995; Ibrahim et al. 1995). Opiinae species

have also been identified by Ibrahim et al. (2013) and

Shariff et al. (2013, 2014) using molecular markers to

determine parasitoid species associated with fruit flies

infesting several crop plants. Both researchers showed that

Diachasmimorpha longicaudata (Ashmead 1905), Fopius

arisanus (Sonan 1932), and Psyttalia incisi (Silvestri 1916)

are parasitoid wasps associated with Bactrocera cara-

mbolae Drew and Hancock 1994, which infests star fruits.

Fopius sp. is the parasitoid of Bactrocera papayae Drew

and Hancock 1994, which infests guavas.

Several parasitoids species exhibited specific patterns of

parasitization on Bactrocera species depending on the type

of host fruit. For example, the extensive introduction of

eight parasitoid species to suppress B. cucurbitae Coquil-

lett 1899 has been undertaken in Hawaii (Nishida 1955).

Only P. fletcheri (Silvestri 1916) was found to be an

important biological control agent against B. cucurbitae,

and therefore P. fletcheri was selected as the most suc-

cessful parasitoid to reduce B. cucurbitae populations

(Liquido 1991; Uchida et al. 1990). As such, precise

identification of parasitoid species is extremely important

to improve the biological control program against fruit

flies.

The multiplex polymerase chain reaction (PCR) has

been widely used in identifying parasitoid species in a

single PCR reaction for precise identification and to dis-

criminate species (Muirhead et al. 2012). The advantages

of using multiplex PCR are mainly because it very cost-

effective and can reduce the lengthy process of the PCR

reaction (Greenstone 2006). Furthermore, it has the ability

to amplify multiple targets of DNA with different sizes

simultaneously (Gariepy et al. 2007; Traugott and Sy-

mondson 2008). Therefore, multiplex PCR, which results

in dual-target detection, could provide more information

from different fragments of DNA for species identification

of parasitoid. However, to date, there has been no record on

the use of multiplex PCR with different genes within the

parasitoid species associated with fruit fly species.

The objectives of this study were to sequence two

mitochondrial DNA genes, COI and Cyt b, from a similar

DNA fragment of opiine parasitoids by using multiplex

PCR, to amplify the COI marker from the bactroceran

species, and to use molecular data to investigate the

interaction of three functional taxa groups.

Materials and methods

Collection of samples and insect rearing

Star fruit, guava, wax apple, and ridge gourd fruits infested

by tephritids were collected from several branches of the

Malaysian Agricultural Research and Development Insti-

tute and from Kampung Buah, Dengkil, Selangor. Several

tephritid larvae were also collected and preserved in 98 %

ethanol and stored at 20 �C for molecular analysis. Eachinfested fruit was kept individually by covering it with saw

dust at the base of a transparent plastic container

(24.5 cm 9 13.5 cm 9 13.0 cm) to ensure dry conditions

and to provide a good medium for the development of

larval to pupal stage. The pupae were then reared until

opiine parasitoids adult emerged. All the opiine parasitoids

species emerged (2–9 individuals per species) were col-

lected and preserved in 98 % ethanol for molecular iden-

tification (Table 1). The laboratory culture was maintained

at room temperature of 25 �C and relative humidity of67–69 % (Mohd Noor et al. 2011).

DNA isolation

DNA was extracted from adult Opiinae and tephritid larva

with freezing method by using DNeasy Blood and Tissue

Kit (Qiagen, Valencia, CA, USA) The freezing method

with some modification following Yaakop et al. (2013). It

was started by adding 180 ll of buffer ATL and 20 ll ofproteinase K to the samples. These samples were then

incubated at 55 �C and kept overnight in a freezer at-22 �C; subsequent steps remained the same as the givenprotocol. The advantage of using this method is that the

voucher specimens still exist, which is useful for mor-

phological re-examination purposes.

S. Yaakop et al.

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8–

Dual-target detection using simultaneous amplification of PCR

123


PCR amplification, DNA purification, and DNA

sequencing.

For the braconid species, two pairs of primers designed by

Folmer et al. (1994) and Simon et al. (1994) were used to

amplify the COI and Cyt b genes in multiplex PCR

(Table 2). For tephritid species, a portion of the mito-

chondrial DNA, the COI gene, was amplified by singleplex

PCR using a primer designed by Han and Ro (2005). PCR

was carried out using MyGene MG96G Thermalcycler.

Multiplex PCR with the following cycles for braconids was

performed in order to amplify two DNA fragments

simultaneously: initial denaturation at 94 �C for 3 min,followed by 40 cycles at 94 �C for 1 min, 47 �C for 1 min,and 72 �C for 2 min. Final extension was at 72 �C for10 min. For COI tephritids, PCR conditions were as fol-

lows: 94 �C for 3 min as the initial denaturation, followedby 40 cycles at 93 �C for 1 min, 56 �C for 1 min, 72 �C for2 min, and the final extension step of 72 �C for 15 min. Forbraconids, multiplex PCR was performed in a 50-ll reac-tion volume containing 33.0 ll ddH2O, 5.0 ll PCR buffer10X (Vivantis), 2.5 ll 50 mM MgCl2, 1.0 ll 10 mMdNTPs, 0.5 ll forward and reverse primers (10 pmol/ll),0.5 ll Taq DNA polymerase (5 U/ll) (Vivantis), and 6 llDNA template (1–5 ng/ll). For amplification of COI te-phritids, PCR was performed in a 25-ll reaction volumecontaining 16.50 ll dd H2O, 2.5 ll PCR buffer 10X (Vi-vantis), 1.30 ll 50 mM MgCl2, 0.5 ll 10 mM dNTPs,0.5 ll forward and reverse primers (10 pmol/ll), 0.2 llTaq DNA polymerase (5U/ll) (Vivantis), and 3 ll DNAtemplate (10–15 ng/ll). The PCR products were electro-phoresed on a 1.5 % agarose gel. A negative control (no

DNA template) was also included in the reactions to trace

potential contamination in the reaction mixture; no PCR

products were generated in this sample. The bands corre-

sponding to the target PCR products were purified using

the Geneaid Purification Kit (Axon Scientific, Malaysia).

All PCR products were then sent to the sequencing service

company, First Base Sdn. Bhd., Petaling Jaya, Selangor, for

sequencing.

Pairwise alignment and basic local alignment search

tool analyses

All the sequences were aligned using Clustal W to deter-

mine the similarity of characters between sequences

(Thompson et al. 1994). In order to optimize the pairwise

alignment results, these sequences were manually edited

using BioEdit version 7.0.4 (Hall 2005). To narrow down

the classification and to confirm no DNA contamination,

the basic local alignment search tool (BLAST) was applied.

This approach is simple and robust for rapid sequence

comparisons of query sequences to database sequencesTa

ble

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6–

S. Yaakop et al.

123


(Altschul et al. 1990; Mittler et al. 2010). The BLAST

search shows a maximum hit for the respective species

only, which is available in GenBank (http://www.ncbi.nlm.

nih.gov/genbank). Several criteria are involved when

measuring the similarity of the sequences, including

expected value, maximum identical, query coverage, and

maximum score. Thus, species identity of requested sam-

ples can be determined. All sequences of braconids and

tephritids have been deposited in GenBank under accession

numbers JX240364–JX240393, KC753508–KC753522,

and KC662198–KC662221 (Table 1).

Phylogenetic analyses

Phylogenetic analysis of maximum parsimony (MP) and

Bayesian inference (BI) was performed using Phylogenetic

analysis using Parsimony 4.0b10 (Swofford 2002) and

MrBayes v3.1.2 (Huelsenbeck et al. 2001), respectively,

for the dataset of the braconids. MP analysis was per-

formed to find the most parsimonious tree(s) with a heu-

ristic search (Hillis et al. 1996) of 1,000 replications in

random addition sequences and the tree bisection recon-

nection option for branch swapping. Bootstrap support

value for each clade was calculated by performing boot-

strap analysis with 1,000 replications (Felsenstein 1985). In

this study, Aspilota sp. (Alysiinae species, a sister group of

Opiinae) (GenBank Accession no. JF962946 and Z93667)

was selected as outgroup in the dataset of the COI and Cyt

b genes. For Bayesian analysis, the best-fit model was

selected using Modeltest 3.7 (Posada and Crandall 1998)

based on the Akaike information criterion (AIC). Modeltest

3.7 was also used to derive best-fit estimates of base pair

frequencies, proportion of invariant sites, and the gamma

shape parameter. MrBayes V3.1.2 calculates the posterior

probability of the phylogenetic tree where these probabil-

ities are obtained by running two Monte Carlo Markov

Chains simultaneously (Huelsenbeck and Ronquist 2001).

Each run was carried out with a sample frequency of 100

generations. The first 25 % of these generations were dis-

carded and considered as burn-in. The genetic divergences

between the six braconids species were estimated by

pairwise genetic distance based on a Kimura-two-

parameter algorithm (Kimura 1980). This algorithm dis-

tinguishes between two types of substitutions (transitions

and transversions).

Results

PCR assay

The COI and Cyt b genes on the opiine samples were

successfully amplified in a single reaction and produced

fragments of 710 and 440 bp, respectively (Fig. 1).

Nucleotide data characterization and genetic distance

of opiinae COI and Cytb

Fragments of 707 and 438 bp were obtained from the

multiple alignments of COI and Cyt b genes, respectively.

Sequence analysis indicated that 192 (27.2 %) were vari-

able sites within the COI gene and 173 (24.5 %) were

parsimony informative. The Cyt b fragments showed more

variability and were more parsimony informative with

values of 134 (30.6 %) and 122 (27.9 %), respectively

(Table 4). Additionally, the conserved sites constituted 515

Table 2 List of the primers used for PCR amplifications

Gene Primer name Sequences (50–30) References

COI COI F 5-TAC AAT TTA TCG CCT AAA CTT CAG CC-3 (Forward) Han and Ro (2005)

COI R 5-CAT TTC AAG TTG TGT AAG CAT C-3 (Reverse) Han and Ro (2005)

LCO1490 5-GGT CAA CAA ATC ATA AAG ATA TTG G-3 (Forward) Folmer et al. (1994)

HCO2198 5-TAA ACT TCA GGG TGA CCA AAA AAT CA-3 (Reverse) Folmer et al. (1994)

Cyt b CB-J-10933 5-TCT TTT TGA GGA GCW ACW GTW ATT AC-3 (Forward) Simon et al. (1994)

CB-N-11367 5-AAT TGA ACG TAA AAT WGT RTA AGC AA-3 (Reverse) Simon et al. (1994)

Fig. 1 Result of the PCR amplification by using multiplex PCR; 710bp (COI) and 440 bp (Cyt b)


123


http://www.ncbi.nlm.nih.gov/genbankhttp://www.ncbi.nlm.nih.gov/genbank

(72.8 %), showing that the COI gene is highly conserved

compared with the Cyt b gene. There were 304 (69.4 %)

conserved sites for Cyt b. The genetic divergences between

the six braconid species were estimated by pairwise genetic

distance based on a Kimura-two-parameter algorithm

(Kimura 1980). This algorithm distinguishes between two

types of substitutions (transitions and transversions). The

transition/transversion rates were 0.766 and 4.086 for COI

and Cyt b, respectively (Table 4). The highest nucleotide

distance estimated from the COI sequences was between D.

longicaudata and P. incisi, with a value of 0.192, followed

by 0.184 between D. longicaudata and Psyttalia sp. The

highest nucleotide distance estimated from the Cyt

b sequences was between D. longicaudata and Fopius

vandenboschi (Fullaway), with a value of 0.240, followed

by 0.232 between D. longicaudata and P. incisi (Table 3).

Tree reconstructions of braconids

Maximum parsimony (MP)

For the dataset of the COI gene, MP analysis is based on

equally weighted values produced of four parsimonious

trees (Fig. 2). The best tree had a minimum evolution of

325 steps, with a consistency index of 0.7262, a homoplasy

index of 0.2738, and a retention index of 0.9311. MP

analysis for the Cyt b gene produced three parsimonious

trees with a tree length of 228 (Fig. 3).The best tree had a

consistency index of 0.7325, a homoplasy index of 0.2675,

and a retention index of 0.9363. The MP bootstrap trees

produced for both genes showed high congruence after

bootstrap analysis with 1,000 replications. In the MP ana-

lysis of the COI gene, D. longicaudata was highly dis-

tanced from the other species. The highest genetic distance

shown between D. longicaudata and P. incisi can also be

demonstrated in the topology of the MP tree. In the MP

analysis of the Cyt b gene, a species from the genus

Psyttalia is shown to be highly distanced from the other

species; however, D. longicaudata and F. vandenboschi

showed the highest genetic distance, which was not con-

gruent in the MP tree and had a low support (53 %) of the

bootstrap value. Overall, the same species were grouped

together to form a monophyletic clade according to their

infested crops species; this was supported by the higher

bootstrap value (Table 4).

Bayesian inference

Modeltest 3.7 was utilized to obtain the best model with

best-fit estimates of base pair frequencies, proportion of

invariant sites, and the gamma shape parameter. In the

Bayesian analysis of the COI dataset, the most appropriate

substitution model selected by AIC was general time

reversible with a modified proportion of invariable sites

(GTR ? I). The proportion of invariable sites was 0.6449,

and these were equal. Meanwhile, for Cyt b, the best model

computed for Bayesian analysis was general time reversible

with a modified proportion of invariable sites and gamma

distribution (GTR ? I ? G). The proportion of invariable

sites and gamma distribution shape parameters were 0.5551

and 2.1458, respectively. BI for COI was generated by

running 170,000 generations with 0.008048 split frequen-

cies probability. A tree was sampled for every 100 genera-

tions, and 25 % of generations (425 trees) were discarded as

burn-in. BI for Cyt b was generated by running 230,000

generations for every 100 sample frequencies. As a result,

25 % of the generations produced 575 trees as burn-in. The

split frequencies probability produced was 0.009037. In

both genes, Bayesian analysis produced essentially the same

topology as presented in the MP tree. However, species of

the genus Psyttalia and Fopius were clustered and grouped

together, supported with relatively high values of posterior

probability (1.00) from the COI dataset (Fig. 2). Likewise,

in the Cyt b dataset, D. longicaudata and species from the

genus Fopius were clustered and supported with a very low

posterior probability value (0.79) (Fig. 3).

Discussion

An accurate determination of parasitoid species by using a

molecular approach is highly desirable for an effective

Table 3 The pairwise genetic distance between braconids species of COI gene (below diagonal) and Cyt b gene (above diagonal)

Diachasmimorpha

longicaudata

Fopius

arisanus

Fopius

vandenboschi

Psyttalia

incisi

Psyttalia

sp.

Psyttalia

fletcheri

Diachasmimorpha longicaudata – 0.188 0.24 0.232 0.202 0.193

Fopius arisanus 0.169 – 0.142 0.198 0.201 0.209

Fopius vandenboschi 0.155 0.111 – 0.229 0.22 0.231

Psyttalia incisi 0.192 0.142 0.164 – 0.11 0.117

Psyttalia sp. 0.184 0.148 0.145 0.088 – 0.043

Psyttalia fletcheri 0.171 0.144 0.153 0.077 0.038 –

S. Yaakop et al.

123


management program against Bactrocera species. Multi-

plex PCR should be able to give additional genetic infor-

mation of the species by simultaneously amplifying

different fragments of COI and Cyt b genes in a single PCR

reaction. Therefore, multiplex PCR directly provides pre-

cise and important information in identifying the species

under study. Furthermore, with two different primers being

used in the same reaction, the resulting sensitivity of the

primers was increased (Gariepy and Messing 2012; Trau-

gott et al. 2006, 2012; Traugott and Symondson 2008).

This procedure is also more cost-effective as it reduces the

lengthy process due to the multiple targets of DNA that

could be generated in a single reaction (Gariepy et al. 2007;

Greenstone 2006).

Multiplex PCR has been utilized in host–parasitoid

studies to identify multiple parasitoid species (Traugott

et al. 2006; Traugott and Symondson 2008). Due to the

high degree of nucleotide variations, COI has been used as

DNA barcoding for rapid identification of the species

(Boehme et al. 2011; Foottit et al. 2009; Hebert et al. 2004)

and because DNA barcodes have low levels of intraspecific

diversity and higher interspecific divergence (Boehme et al.

2011). Furthermore, COI also has the capability to resolve

relationships at various taxonomic levels because of its

higher substitution rate within the Hymenoptera (Lin and

Danforth 2004; Mardulyn and Whitfield 1999). For

instance, B. carambolae and B. papayae are closely related

species (Liu et al. 2011; Chua et al. 2010); therefore, COI

is useful in detecting and determining the immature

developmental stages of these Bactrocera species accu-

rately. Although species identification using the Cyt b gene

is still limited, the Cyt b gene has slowly and rapidly been

evolving codon positions (Farias et al. 2001), and these

nucleotide variations can be ascertained to discriminate the

species (Irwin et al. 1991). However, there is limited

information in GenBank on Cyt b sequences pertaining to

the Opiinae parasitoid species (Gimeno et al. 1997; Shariff

et al. 2014). Therefore, the Cyt b sequences of Opiinae

parasitoids obtained from this study will constitute new

submissions to the NCBI database.

Fig. 2 Fifty percent majorityrules consensus tree resulting

from Maximum Parsimony

(MP) and Bayesian Inference

(BI) analyses by using COI

dataset. Numbers above

branches are the bootstrap

values and those below

branches are the posterior

probabilities


123


Comparative information between the two genes (COI

and Cyt b) can be clarified and facilitated by constructing a

phylogenetic tree with MP and BI to reconfirm the related

species. Based on MP and BI of the COI dataset, D. lon-

gicaudata indicated the highest genetic distance with

Psyttalia fletcheri, a value of 0.193 and congruent with the

tree topology. The monophyletic group of D. longicaudata

showed the most divergent lineage from the other species,

supported with 100 % bootstrap value and a value of 1.00

posterior probability. However, within the Cyt b gene, D.

longicaudata and F. vandenboschi showed the highest

genetic distance with a value of 0.240. Even though these

species were claded together, they were supported with a

lower bootstrap value and posterior probability (53 % and

0.79, respectively). However, the genetic distance was not

congruent with the tree topology of MP and BI. Otherwise,

individuals from the genus Psyttalia showed the most

distance from other species. According to Leache and

Reeder (2002), only a posterior probability of 95 % (0.95)

or greater should be considered as significant and resolved.

Hence, COI has been used widely to solve various taxo-

nomic problems in the phylogenetic analyses of braconid

wasps (Zaldivar-Riveron et al. 2010). However, the

grouping of each species in this study was similar for both

genes in all tree topologies.

This study showed that D. longicaudata, F. arisanus,

and Psyttalia incisi are parasitoids associated with B.

carambolae and B. papayae. (Figs. 2, 3). However, F.

Fig. 3 Fifty percent majorityrules consensus tree resulting

from Maximum Parsimony

(MP) and Bayesian Inference

(BI) analyses by using Cyt b

dataset. Numbers above

branches are the bootstrap

values and those below

branches are the posterior

probabilities

Table 4 Nucleotide composition in COI and Cyt b genes of Opiinaeparasitoids

Region COI Cyt b

Total fragments 707 438

Constant sites 515 (72.8 %) 304 (69.4 %)

Informative sites 173 (24.5 %) 122 (27.9 %)

Variable sites 192 (27.2 %) 134 (30.6 %)

Transition (Ti) 43.38 % 80.38 %

Transversion (Tv) 56.62 % 19.67 %

Ti/Tv 0.766 4.086

S. Yaakop et al.

123


vandenboschi has been recorded parasitizing B. papayae in

guavas, and damage to wax apple was caused by B. pa-

payae. The parasitoids that have been determined in this

study were D. longicaudata and F. vandenboschi. We also

documented a new record of P. fletcheri, which is associ-

ated with B. cucurbitae infesting the ridge gourd in

Malaysia (Figs. 2, 3). All our findings are supported by

Chinajariyawong et al. (2000), who reported that D. lon-

gicaudata, F. arisanus, F. vandenboschi, and P. incisi

parasitize B. carambolae and B. papayae are associated

with star fruits and guavas. Several researchers studying

star fruit orchards have also recorded D. longicaudata (I-

brahim et al. 1995) and F. arisanus (Chua and Khoo 1995)

as parasitoids associated with B. carambolae. Recently,

species identification using both morphological and

molecular characters was recorded for F. arisanus para-

sitizing B. carambolae in wax apple (Yaakop and Aman

2013). Furthermore, other studies based on molecular work

have determined that D. longicaudata, F. arisanus, and P.

incisi are parasitoids associated with B. carambolae

infesting star fruits, while Fopius sp. is a parasitoid of B.

papayae in guavas (Ibrahim et al. 2013; Shariff et al. 2014)

Several Opiinae parasitoids showed a pattern of para-

sitization of Bactrocera species that were infesting crops

(Shariff et al. 2013) This study also revealed that P. fletc-

heri is the main parasitoid associated with B. cucurbitae in

ridge gourd. Based on previous studies, P. fletcheri is noted

to be the main parasitoid of B. cucurbitae, which is the

most serious pest of a member of the cucurbit crops

(Jackson et al. 2003; Uchida et al. 1990; Vargas et al.

2004). Although other Opiinae parasitoids have been

introduced to suppress B. cucurbitae, P. fletcheri is known

to be the most significant parasitoid for the successful

management of this fruit fly species (Nishida 1955). Bio-

logical factors, such as the odor from stem cells, volatiles

from rotting fruit, and green leaves, directly influence P.

fletcheri to be parasitized more frequently with fruit fly in

cucurbit crops (Messing et al. 1996; Nishida 1955).

Therefore, updated information on parasitoid–pest–host

plant relationships are the most important factors in

determining the continual success and effective manage-

ment of fruit fly control programs in heavily infested

regions.

Conclusions

Dual-target detection using simultaneous amplification of

PCR on the COI and Cyt b genes has been successfully

conducted to identify Opiinae parasitoids species. Thus,

multiple targets of genes can be generated simultaneously

within a single reaction. The clustering of the species

through phylogenetic analyses was also performed in order

to facilitate differentiating closely related species. Both MP

and BI topologies showed that each species forms its

respective monophyletic group specifically related to the

infested crops. Based on our results, the interaction among

parasitoids, hosts, and infested crops has been seen. Dia-

chasmimorpha longicaudata, F. arisanus, and P. incisi are

parasitoids associated with B. carambolae and B. papayae

infesting star fruits. However, only B. papayae has been

confirmed to infest guava and wax apple fruits. The Opii-

nae parasitoid was detected in guava is F. vandenboschi,

while in the wax apple, D. longicaudata, F. vandenboschi,

and Psyttalia sp. were detected. Only P. fletcheri, a new

record from Malaysia, is indicated as the most important

parasitoid of B. cucurbitae, which infests the ridge gourd

fruit. Our results provide information that is highly useful

for an effective biological control programme of the tar-

geted fruit fly.

Acknowledgments The authors would like to express our specialgratitude to the Hymenoptera curator of the British Museum Natural

History, London (BMNH), Dr. Gavin Broad, for his hospitality and

assistance during the short visit by the corresponding author for

reconfirming the identity of the Opiinae species. We would also like

to express our sincere gratitude to Prof. Dr. Maimon Abdullah for her

critical comments and editing of the manuscript. We are grateful to

technical staff of the Horticulture Research Center, MARDI Head-

quarters, 43,400 Serdang, Selangor for helping us in collecting the

fruit flies and rearing the braconid samples. This project was fully

supported by ERGS grant 320/1/2011/STWN/UKM/03/9, GUP-2014-

029, FRGS/1/2014/SG03/UKM/02/1 and DPP-2014-086.

Conflict of interest The authors declare that no competing financialinterests exist.

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http://dx.doi.org/10.1006/mpev.2001.0958http://dx.doi.org/10.1006/mpev.2001.0958http://dx.doi.org/10.1093/nar/22.22.4673http://dx.doi.org/10.1603/0022-0493-97.5.1531http://dx.doi.org/10.3954/JAUE12-22.1http://dx.doi.org/10.3109/19401736.2010.523701http://dx.doi.org/10.3109/19401736.2010.523701https://www.researchgate.net/publication/273286124

Dual-target detection using simultaneous amplification of PCR in clarifying interaction between Opiinae species (Hymenoptera: Braconidae) associated with Bactrocera spp. (Diptera: Tephritidae) infesting several cropsAbstractIntroductionMaterials and methodsCollection of samples and insect rearingDNA isolationPCR amplification, DNA purification, and DNA sequencing.Pairwise alignment and basic local alignment search tool analysesPhylogenetic analyses

ResultsPCR assayNucleotide data characterization and genetic distance of opiinae COI and CytbTree reconstructions of braconidsMaximum parsimony (MP)Bayesian inference

DiscussionConclusionsAcknowledgmentsReferences

spp. (diptera · braconidae) associated with bactrocera spp. (diptera: tephritidae) infesting...

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