DNA barcoding of the vegetable Leafminer Liriomyza sativae Blanchard (Diptera: Agromyzidae) in...

DNA Barcodes 2014; Volume 2: 29–33

The most damaging Liriomyza leafminers are the invasive New World species L. huidobrensis, L. trifolii and L. sativae, all of which are polyphagous and have spread to most regions of the world [1, 5]. Introduced populations are notoriously difficult to control and may cause significant crop damage [6]. In Bangladesh, leafmining flies have been found on 45 vegetable crops, including tomato, potato, long bean, common cowpea, bush bean, french bean, common bean, ground nut, okra, onion, snake gourd, bottle gourd, cucumber, sweet cucumber, watermelon, cauliflower, mustard, cabbage, eggplant, squash, and red spinach [7; this study]. However, the species identity of the leafminer(s) found on these crops has not previously been identified.

DNA barcoding has been developed as a tool for molecular identification of specimens [8-10]. Most typically, a portion of the mitochondrial cytochrome c oxidase subunit one (COI) is sequenced from the unidentified specimen and is compared to COI sequences from identified samples. DNA barcoding has become a common means of identifying species, particularly those that are difficult to identify using morphological characters; in addition, this technique can be performed on any life stage, making reliable identifications of juvenile or even partial specimens are possible [8, 11, 12].

Adult Liriomyza leafminers are difficult to identify using only morphological characters, and juvenile leafminers, the most conspicuous life stage in the field, typically do not possess diagnostic morphological characters beyond those to family. Several studies investigating mitochondrial sequence variation in pest Liriomyza leafminers demonstrate the utility of mitochondrial sequences for species identification [13-15]. Recently, DNA-barcoding was used to document the identities and distributions of invasive leafminers in the Philippines [16].

The goal of this study was to use DNA barcoding to identify the major pest leafminer sampled from crops in Bangladesh.

Abstract: DNA barcoding revealed the presence of the polyphagous leafminer pest Liriomyza sativae Blanchard in Bangladesh. DNA barcode sequences for mitochondrial COI were generated for Agromyzidae larvae, pupae and adults collected from field populations across Bangladesh. BLAST sequence similarity searches of GenBank showed 99-100% sequence similarity with populations of L. sativae Blanchard and 97% sequence similarity with L. trifolii (Burgess). Neighbor-joining distance analysis was used to investigate patterns of clustering of the Bangladeshi sequences with other pest species of Liriomyza. Sequences for the Bangladeshi specimens clustered unambiguously with L. sativae sequences from GenBank. This represents the first molecular confirmation of L. sativae in Bangladesh, and we conclude that this destructive leafminer is continuing its geographic expansion within Asia.

Keywords: Molecular taxonomy, DNA barcodes, Liriomyza sativae, Agromyzidae, Bangladesh

Doi: 10.2478/dna-2014-0005received December 05, 2013 accepted June 14, 2014

1 IntroductionIn recent years, Liriomyza leafminers (Diptera: Agromyzidae) have increasingly become important pests on vegetable and cut flower crops around the world [1-4].

Research Article Open Access

© 2014 Sabrina Amin, et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

Sabrina Amin*, Sonja J. Scheffer, Matthew L. Lewis, Mostafa Kamal Pasha, Badrul Amin Bhuiya

DNA barcoding of the vegetable Leafminer Liriomyza sativae Blanchard (Diptera: Agromyzidae) in Bangladesh

*Corresponding author: Sabrina Amin: Department of Biochemistry & Biotechnology, University of Science & Technology Chittagong, Bangladesh, e-mail: [email protected] J. Scheffer, Matthew L. Lewis: Systematic Entomology Labora-tory, Plant Science Institute, ARS, USDA, Beltsville, USAMostafa Kamal Pasha: Department of Botany, University of Chitta-gong, BangladeshBadrul Amin Bhuiya: Department of Zoology, University of Chitta-gong, Bangladesh

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30 Sabrina Amin, Sonja J. Scheffer, Matthew L. Lewis, Mostafa Kamal Pasha, Badrul Amin Bhuiya

L. trifolii, L. huidobrensis, L. bryoniae, and Chromatomyia horticola as the outgroup taxon. All COI sequences have been deposited in GenBank under accession numbers KF962572 - KF962596. Sequences were also deposited in BOLD under AGBD001-14 - AGBD023-14 (GenBank and BOLD process ID’s are given in Table 1). The remains of the adult L. sativae specimens associated with this project have been deposited in the Insect Museum of Zoology Department at Chittagong University (accession numbers IMZCU001 – IMZCU011).

3 Results and discussionWe sequenced approximately 700bp of COI from leafminer specimens from seven crop species and malaise trap samples from Bangladesh. BLAST searching of GenBank returned the same result, i.e., L. sativae sequences having 99-100% sequence similarity with 25 of our samples from Bangladesh. Similarity of our sequences to GenBank sequences from the closely related L. trifolii hovered around 97%. In the Neighbor-joining analysis, all of the Bangladeshi L. sativae sequences clustered unambiguously with the L. sativae sequences obtained from GenBank with 100% bootstrap support (Figure 1). L. trifolii obtained from GenBank formed a cluster distinct from and sister to the L. sativae group (Figure 1), as expected based on morphology and previous molecular results [14, 15]. BOLD result generated BIN number as BOLD:AAF6797.

Recently, as part of this study, the first report of L. sativae from Bangladesh has been published [25]. We used DNA barcoding to provide molecular corroboration that L. sativae has indeed reached Bangladesh and is the widespread and polyphagous leafminer currently attacking vegetable crops there. Liriomyza sativae is New World in origin, but over decades it has spread worldwide as a result of global trade of vegetables [2]. Although present in India for a considerable length of time [26], it has been recorded only recently from several Asian countries including Japan [27], Turkey [28], China [29], Malaysia and Sri Lanka [14], The Philippines [16], and Vietnam [30]. As this species continues to invade new vegetable-growing regions, additional crop losses can be expected.

3.1 Diagnostic Notes

Small, greyish-black, compact bodied fly, 1.3-2.3mm in body length, 1.3-2.3mm in wing length.

Head with frons and orbits yellow, third antennal segment yellow, femora bright yellow, mesonotum black,

2 Materials and methods

2.1 Sample collection and preservation

Specimens were collected using sweep nets and malaise traps at 44 sites in Bangladesh during the period of October 2008 to September 2011. In addition, some specimens were reared from leafminers collected in the field and brought to the laboratory. Crops from which our samples were obtained include Cucumis sativus (cucumber), Cucurbita máxima (squash), Lablab purpureus (hyacinth bean), Lagenaria siceraria (bottle gourd), Solanum lycopersicum (tomato), Vicia faba (fava bean), and Vigna unguiculata (cowpea). Collection information for specimens is given in Table 1. A number of adult leafminer specimens were identified using morphological characters [15, 17-19] by the fifth author at the NMNH in Washington, D.C.

Larval, pupal and adult specimens were preserved in 95% ethanol and stored at -20°C when available. Genomic DNA from each specimen was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA) following the manufacturer’s protocol for animal tissue.

Reactions for PCR amplification of mitochondrial COI were carried out using a Tetrad 2 DNA Engine thermal cycler (BioRad, Hercules, CA) with a touchdown amplification program: initial denaturation at 92°C for 2 min, followed by touchdown cycles from 58 to 46°C (10s at 92°C, 10s at 58-46°C, 45s at 72°C), 29 cycles of 10s at 92°C, 10s at 45°C, 2min at 72°C, and a final extension step for 7 min at 72°C.

A single fragment of 731 bp encompassing the DNA barcode region resulted from PCR with primers TY-J-1461 (5’-TTTACRTTTACCGCCTATTRTCAGCCA-3’) [20] and C1-N-2191 (5’-CCCGGTAAAATTAAAATATAAACTTC-3’) [21]. PCR products were purified using ExoSAPIT (USB Corporation, Cleveland, OH). Sequencing reactions were carried out using the ABI Big Dye v3.1 sequencing kit (Applied Biosystems, Foster City, CA) and fractionated on an ABI 3130 automated DNA sequencer (Applied Biosystems). Standard barcoding primers LCO (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) [22] were used for sequencing.

Sequences were assembled into contigs, edited, and aligned using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI). Edited sequences were submitted to BLAST for species identification using the standard BLASTn algorithm [23]. Neighbor-joining trees and bootstrap values were generated using the program PAUP* [24] with other pest agromyzids including


DNA barcoding of agromyzid leaf miner in Bangladesh 31

Table 1: Collection information for specimens from Bangladesh. Host plants listed in “Collection Source” indicate that the specimen origina-ted as a leafminer on that host and thus represent a true rearing record.

Specimen Code

LifeStage

Collection Date

Collection Source

Location Lat./Long. GenBank Accessions BOLD Process ID Specimen

voucher ID

SAB0005 Larva 18-12-2010 Cucumis sativus L. Banskhali, Chittagong

21⁰59’28.48’’N91⁰57’03.43’’E

KF962586 AGBD001-14 --


21⁰59’28.48’’N91⁰57’03.43’’E

KF962574 AGBD002-14 --


21⁰59’28.48’’N91⁰57’03.43’’E

KF962581 AGBD003-14 --

SAB0010 Pupa 13-10-2010 Vicia faba L. Anwara, Chittagong

22⁰14’27.17’’N91⁰52’48.53’’E

KF962578 AGBD004-14 --

SAB0013 Larva 27-02-2010 Lablab purpureus (L.) Sweet

St. Martin’s Island

20⁰37’47.68’’N92⁰19’15.98’’E

KF962580 AGBD005-14 --

SAB0014 Larva 27-02-2010 Lablab purpureus (L.) Sweet

St. Martin’s Island

20⁰37’47.68’’N92⁰19’15.98’’E

KF962585 AGBD006-14 --

SAB0018 Larva 13-10-2010 Solanum lycopersicum L.

Anwara, Chittagong

22⁰14’27.17’’N91⁰52’48.53’’E

KF962575 AGBD007-14 --


Anwara, Chittagong

22⁰14’27.17’’N91⁰52’48.53’’E

KF962577 AGBD008-14 --


Anwara, Chittagong

22⁰14’27.17’’N91⁰52’48.53’’E

KF962587 AGBD009-14 --

SAB0036 Adult 04-02-2009 Cucurbita maxima Duchesne

Expt. Field, Ctg Univ.

22⁰28’02.85’’N91⁰46’47.45’’E

KF962593 AGBD010-14 SAB-36IMZCU-001

SAB0038 Adult 19-10-2010 Solanum lycopersicum L.

Mirswarai, Chittagong

22⁰46’45.20’’N91⁰34’27.16’’E

KF962589 AGBD011-14 --

SAB0040 Larva 19-10-2010 Cucurbita maxima Duchesne

Mirswarai, Chittagong

22⁰46’45.20’’N91⁰34’27.16’’E

KF962576 AGBD012-14COI-5P

--


Patenga, Chittagong

22⁰14’52.12’’N91⁰47’59.00’’E

KF962591 AGBD013-14 --

SAB0112 Adult 18-4-2010 Malaise trap Experimental Field, Ctg Univ.

22⁰28’02.85’’N91⁰46’47.45’’E

KF962594 AGBD014-14 SAB-112IMZCU-002

SAB0113 Adult 5-12-10 Malaise trap Godagari, Rajshahi

24⁰28’06.41’’N88⁰19’38.58’’E

KF962595 AGBD015-14 SAB-113IMZCU-003

SAB0114 Adult 5-7-2010 Malaise trap Dinajpur 25⁰45’09.68’’N88⁰42’07.05’’E

KF962572 AGBD016-14 SAB-114IMZCU-004


24⁰28’06.41’’N88⁰19’38.58’’E

KF962573 AGBD017-14 SAB-115IMZCU-005


KF962579 AGBD018-14 SAB-117IMZCU-006


KF962590 AGBD019-14 SAB-118IMZCU-007


24⁰28’06.41’’N88⁰19’38.58’’E

KF962588 AGBD020-14 SAB-119IMZCU-008


24⁰28’06.41’’N88⁰19’38.58’’E

KF962583 AGBD021-14 SAB-120IMZCU-009


24⁰28’06.41’’N88⁰19’38.58’’E

KF962596 AGBD022-14 SAB-121IMZCU-010

SAB0127 Larva 30-1-2010 Lagenaria siceraria (Molina) Standl.

Experimental Field, Ctg Univ.

22⁰28’02.85’’N91⁰46’47.45’’E

KF962582 AGBD023-14 --

SAB0137 Adult 25-3-2011 Malaise trap Experimental Field, Ctg Univ.

22⁰28’02.85’’N91⁰46’47.45’’E

KF962592 AGBD024-14 SAB-137IMZCU-011

SAB0180 Adult 10-3-2011 Vigna unguiculata (L.) Walp.

Experimental Field, Ctg Univ.

22⁰28’02.85’’N91⁰46’47.45’’E

KF962584 AGBD025-14 --


32 Sabrina Amin, Sonja J. Scheffer, Matthew L. Lewis, Mostafa Kamal Pasha, Badrul Amin Bhuiya

Acknowledgements: The authors gratefully acknowledge the generous support of Dr. Michael W. Gates of Systematic Entomology Laboratory, USDA, National Museum of Natural History, Washington D. C., USA, for facilitating this project. The services of Mahmuda Begum, PhD Fellow and Santosh Mazumdar, MPhil Fellow of the Department of Zoology, University of Chittagong, are also acknowledged for collecting and preserving samples for DNA study. USDA is an equal opportunity provider and employer.

shining, outer vertical bristle on black ground and inner vertical bristle on yellow. Wing with last section of CuA1 3-4 times length of penultimate section, wing length 1.3-1.7 mm. Aedeagus: distiphallus with one distal bulb with a slight constriction between upper and lower halves.

Females slightly larger than males. Scutellum bright yellow, outer vertical setae standing on black, inner vertical setae usually standing on yellow ground. Formal identification needs examination of male genitalia.

Figure 1: Neighboring-joining (NJ) phenogram of mitochondrial COI sequences from several invasive Liriomyza species. Sequences from specimens collected in Bangladesh are presented in bold type.

Chromatomyia horticola GenBank EU443642

Liriomyza bryoniae GenBank JN570504 99

100 Liriomyza huidobrensis GenBank EU219615 100

100

100

Liriomyza huidobrensis GenBank JN570505

Liriomyza trifolii GenBank GU327644

Liriomyza trifolii GenBank JN570506 Liriomyza trifolii GenBank EU219614 Liriomyza trifolii GenBank DQ516575

SAB0018 Solanum lycopersicum

SAB0117 malaise trap SAB0120 malaise trap SAB0180 Vigna unguiculata Liriomyza sativae GenBank HQ333260

SAB0137 malaise trap


SAB0005 Cucumis sativus SAB0040Cucurbita maxima SAB0042 Solanum lycopersicum SAB0119 malaise trap SAB0113 malaise trap SAB0127 Lagenaria siceraria SAB0118 malaise trap SAB0112 malaise trap SAB0115 malaise trap SAB0007 Cucumis sativus


SAB0036 Cucurbita maxima

Liriomyza sativae GenBank EU219613

SAB0010 Vicia faba

SAB0038 Solanum lycopersicum SAB0013 Lablab purpureus SAB0014 Lablab purpureus SAB0006 Cucumis sativus SAB0114 malaise trap SAB0121 malaise trap


DNA barcoding of agromyzid leaf miner in Bangladesh 33

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