A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

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Identification of Neoceratitis asiatica (Becker) (Diptera: Tephritidae) based on morphological characteristics and DNA barcode

Zootaxa ◽

10.11646/zootaxa.4363.4.7 ◽

2017 ◽

Vol 4363 (4) ◽

pp. 553

Author(s):

SHAOKUN GUO ◽

JIA HE ◽

ZIHUA ZHAO ◽

LIJUN LIU ◽

LIYUAN GAO ◽

...

Keyword(s):

Dna Sequences ◽

Phylogenetic Trees ◽

Gap Analysis ◽

Dna Barcode ◽

Morphological Characteristics ◽

Coi Gene ◽

Economic Losses ◽

Morphological Identification ◽

Lycium Barbarum ◽

Accurate Identification

Neoceratitis asiatica (Becker), which especially infests wolfberry (Lycium barbarum L.), could cause serious economic losses every year in China, especially to organic wolfberry production. In some important wolfberry plantings, it is difficult and time-consuming to rear the larvae or pupae to adults for morphological identification. Molecular identification based on DNA barcode is a solution to the problem. In this study, 15 samples were collected from Ningxia, China. Among them, five adults were identified according to their morphological characteristics. The utility of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) gene sequence as DNA barcode in distinguishing N. asiatica was evaluated by analysing Kimura 2-parameter distances and phylogenetic trees. There were significant differences between intra-specific and inter-specific genetic distances according to the barcoding gap analysis. The uncertain larval and pupal samples were within the same cluster as N. asiatica adults and formed sister cluster to N. cyanescens. A combination of morphological and molecular methods enabled accurate identification of N. asiatica. This is the first study using DNA barcode to identify N. asiatica and the obtained DNA sequences will be added to the DNA barcode database.

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DNA barcodes and new primers for nature’s pest controllers: the social wasps

Genome ◽

10.1139/gen-2019-0193 ◽

2020 ◽

Author(s):

Ikechukwu Eugene Onah ◽

Seirian Sumner

Keyword(s):

Dna Barcode ◽

Pcr Amplification ◽

Social Wasps ◽

Coi Gene ◽

Dna Barcodes ◽

Anthropogenic Pressures ◽

Specific Primers ◽

Prime Concern ◽

The Social ◽

Polistine Wasps

Globally, biodiversity is declining as a result of anthropogenic pressures, and this could lead to extinction of some species before they are discovered. The loss of insect taxa is of prime concern, given recent reports of significant declines in the populations of many taxa across the globe. Efforts to document biodiversity have met with several challenges, amongst which are the difficulties in using morphological features to discriminate species, especially in insects. DNA barcoding is a rapid and reliable method for species identification and discovery, but choosing appropriate primers to amplify the barcode region without coamplifying contaminants remains a key challenge. We developed and tested a set of primers for PCR amplification of the DNA barcode region of the COI gene in polistine wasps. We tested their efficacy in 36 species of vespid wasps, and the solitary wasp Zethus miniatus Saussure. Samples were obtained from Africa, Americas, Asia and Europe. The polistine-specific primers successfully amplified the barcode region for all polistines tested, without amplifying any Wolbachia present; they also worked with many species from the other Vespidae wasp subfamilies. The new primers are valuable for the discovery and accurate documentation of polistine wasps in the four continents.

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A molecular framework for the identification of planthopper vectors (Hemiptera: Delphacidae) of central Argentina

Bulletin of Entomological Research ◽

10.1017/s0007485315000735 ◽

2015 ◽

Vol 105 (6) ◽

pp. 754-762

Author(s):

E.B. Argüello Caro ◽

A.D. Dumón ◽

M.F Mattio ◽

V. Alemandri ◽

G. Truol

Keyword(s):

Species Identification ◽

Regional Scale ◽

Dna Barcode ◽

Restriction Enzymes ◽

Common Species ◽

Molecular Techniques ◽

Coi Gene ◽

Morphological Identification ◽

Accurate Identification ◽

Central Argentina

AbstractPlanthoppers are important worldwide crop pests as well as vectors of numerous diseases. Different species transmit Mal de Río Cuarto virus, which causes the most economically important corn disease in central Argentina. Epidemiological studies rely on the accurate identification of the species present in the field. Presently, morphological identification of planthoppers requires taxonomic expertise and there are no taxonomic keys for females and nymphs. Nevertheless, no molecular protocols are available for accurate species identification of most frequent delphacid species from central Argentina. In this context, the aim of this study was to evaluate the utility of the cytochrome oxidase I gene (COI) as a DNA barcode and its digestion with restriction enzymes (Restriction Fragment Length Polymorphism, RFLP) for the identification of the most common species of planthoppers in central Argentina. We amplified and sequenced a 843 bp fragment of the COI gene of taxonomically identified specimens and evaluated its use as a DNA barcode. Restriction enzymes were also selected for digesting the COI fragment via RFLP. The high interspecific variability (20.79%; ± 2.32%) and low intraspecific divergence (0.12%; ± 0.17%) observed in the studied species, demonstrate the effectiveness of the COI gene for species identification of major vector delphacids affecting corn crops in Argentina. Moreover, the digestion of this COI gene fragment with Bfa I and Apo I enzymes allows a fast and cost-effective species identification method when numerous specimens need to be processed. Both molecular techniques developed here, allow the accurate identification of planthopper species at regional scale. These new tools would assist traditional identification of these insects, especially for aiding non-experts in morphological taxonomy.

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DNA barcodes provide evidence for the independent origin of the cultivated silkworms Samia ricini and Samia cynthia

Journal of Insects as Food and Feed ◽

10.3920/jiff2021.0077 ◽

2021 ◽

pp. 1-8

Author(s):

J.-C. Huang ◽

X.-Y. Li ◽

Y.-P. Li ◽

R.-S. Zhang ◽

D.-B. Chen ◽

...

Keyword(s):

Genetic Distance ◽

Phylogenetic Relationship ◽

Dna Barcode ◽

Distinct Species ◽

Coi Gene ◽

Molecular Evidence ◽

Dna Barcodes ◽

Close Relationship ◽

Molecular Phylogenetic ◽

Phylogenetic Framework

Samia ricini (Wm. Jones) and Samia cynthia (Drury) (Lepidoptera: Saturniidae) have been used as traditional sources of food as well as silk-producing insects. However, the phylogenetic relationship between the two silkworms remains to be addressed. In this study, the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences corresponding to DNA barcodes from 13 Samia species were analysed, and a DNA barcode-based phylogenetic framework for these Samia species was provided. Phylogenetic analysis showed that multiple individuals of a species could be clustered together. Our analysis revealed a close relationship among Samia yayukae Paukstadt, Peigler and Paukstadt, Samia abrerai Naumann and Peigler, Samia kohlli Naumann and Peigler, Samia naessigi Naumann and Peigler, Samia naumanni Paukstadt, Peigler and Paukstadt, and Samia kalimantanensis Paukstadt and Paukstadt. The mixed clustering relationship and low Kimura-2-parameter (K2P) genetic distance (0.006) between individuals of S. ricini and Samia canningi (Hutton) indicated that the cultivated silkworm S. ricini was derived from the non-cultivated silkworm S. canningi. The remote phylogenetic relationship and high K2P genetic distance (0.039) indicated that S. ricini and S. cynthia are distinct species, thus providing solid molecular evidence that they had entirely independent origins. The relationships between S. kalimantanensis and S. naumanni and between S. cynthia and Samia wangi Naumann and Peigler, as well as the potential cryptic species within S. abrerai were also discussed. This is the first study to assess the DNA barcodes of the genus Samia, which supplements the knowledge of species identification and provides the first molecular phylogenetic framework for Samia species.

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DNA barcode identification of commercial fish sold in Mexican markets

Genome ◽

10.1139/gen-2017-0222 ◽

2018 ◽

Vol 61 (6) ◽

pp. 457-466 ◽

Cited By ~ 8

Author(s):

Stephanie Sarmiento-Camacho ◽

Martha Valdez-Moreno

Keyword(s):

Conservation Status ◽

Dna Barcode ◽

Common Species ◽

Morphological Identification ◽

Dna Barcodes ◽

Commercial Fish ◽

Sphyrna Lewini ◽

Pangasianodon Hypophthalmus ◽

Makaira Nigricans ◽

The Status

The substitution of high-value fish species for those of lower value is common practice. Although numerous studies have addressed this issue, few have been conducted in Mexico. In this study, we sought to identify fresh fillets of fish, sharks, and rays using DNA barcodes. We analyzed material from “La Viga” in Mexico City, and other markets located on the Gulf and Caribbean coasts of Mexico. From 134 samples, we obtained sequences from 129, identified to 9 orders, 28 families, 38 genera, and 44 species. The most common species were Seriola dumerili, Pangasianodon hypophthalmus, Carcharhinus falciformis, Carcharhinus brevipinna, and Hypanus americanus. Pangasianodon hypophthalmus was most commonly used as a substitute for higher-value species. The substitution rate was 18% of the total. A review of the conservation status of the specimens identified against the IUNC list enabled us to establish that some species marketed in Mexico are threatened: Makaira nigricans, Lachnolaimus maximus, Hyporthodus flavolimbatus, and Isurus oxyrinchus are classified as vulnerable; Lopholatilus chamaeleonticeps and Sphyrna lewini are endangered; and the status of Hyporthodus nigritus is critical. These results will demonstrate to the Mexican authorities that DNA barcoding is a reliable tool for species identification, even when morphological identification is difficult or impossible.

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DNA barcoding to resolve the confusion in identifying Tribolium confusum and Tribolium castaneum

Bangladesh Journal of Zoology ◽

10.3329/bjz.v47i2.44344 ◽

2019 ◽

Vol 47 (2) ◽

pp. 333-342

Author(s):

Abu Faiz Md Aslam ◽

Sharmin Sultana ◽

Sumita Rani Das ◽

Abdul Jabber Howlader

Keyword(s):

Dna Barcoding ◽

Tribolium Castaneum ◽

Life Stage ◽

Dna Barcode ◽

Tribolium Confusum ◽

Likelihood Method ◽

Coi Gene ◽

Pest Species ◽

Stored Grain ◽

Accurate Identification

Tribolium confusum and Tribolium castaneum (Coleoptera: Tenebrionidae) are two very confusing pest species while identification is done on the basis of morphology only. Such pests are discovered in stored grain as immature stages, which further complicates the identification process. Accurate identification of these pests is urgently required for integrated pest management. In this research, DNA barcoding was used to identify these pests accurately at any life stage. A 658 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was analyzed. DNA barcode dataset of T. confusum (GeneBank Acc. no. MK120453.1) and T. castaneum (Acc. no. MK411585.1) were constructed. The nucleotide composition reveals that average AT contents (59.9%) were higher than the GC contents (38.6%). Phylogenetic analysis by maximum likelihood method showed that both the species were originated from a common major clade. About 17.13% nucleotide differences were noted between the CO1 sequences by multiple sequence alignment. The interspecies nucleotide genetic distance (0.200) was calculated using Kimura 2 parameter. Haplotype analysis showed high genetic diversity (112 mutaional steps) among them. Bangladesh J. Zool. 47(2): 333-342, 2019

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MOLECULAR IDENTIFICATION OF EXOTIC FRUIT FLY Bactrocera occipitalis (DIPTERA: TEPHRITIDAE) USING MITOCHONDRIAL CYTOCHROME OXIDASE I (COI) GENE

International Journal of Biosciences and Biotechnology ◽

10.24843/ijbb.2018.v06.i01.p03 ◽

2018 ◽

Vol 6 (1) ◽

pp. 34

Author(s):

I Putu Sudiarta ◽

Dwi Martiningsia ◽

I Nyoman Wijaya

Keyword(s):

Cytochrome Oxidase ◽

Molecular Identification ◽

Fruit Fly ◽

Fruit Flies ◽

Cytochrome Oxidase I ◽

Coi Gene ◽

Morphological Identification ◽

Mitochondrial Cytochrome ◽

Accurate Identification ◽

Mitochondrial Cytochrome Oxidase

Some of fruit flies have been reported as the important pest on fruits and vegetables in the world. Agricultural Quarantine Agency Denpasar reported that there was new coming species (exotic) of fruit flies in Bali in 2014 based on the morphological identification, namely Bactrocera occipitalis. However Bactrocera dorsalis complex have similar morphological characters and have a less distinctive character for taxonomic identification, therefore it is difficult to identify fruit flies accurately. Based on that phenomena, the accurate identification is needed. One of the more accurate identification techniques is based on molecular identification using DNA-based barcode. To identify fruit flies, DNA-based barcode using mitochondrial cytochrome oxidase I (COI) gene has been conducted. PCR analysis using Fruit Fly MT-CO1-F (FFMT-CO1-F) 5’-GGAGCATTAATYGGRGAYG-3’ as forward primer and HCO 5’-TAAACTTCAGGGTGACCAAAAATCA-3’ as reverse primer was successfully amplified around 600 bp of COI gene of fruit flies. Based on similarity of sequence product, the species was identifiedas Bactrocera occipitalis and same result was revealed using morphological identification. Phylogenetic analysis of B. occipitalis based on COI genes showed that B. occipitalis from Bali were in the same groups with Bactrocera species from Tarakan and Philippines. In addition, Bactrocera occipitalis as exotic fruit fly is a new report in Bali, Indonesia.

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Revision of the subgenus Phortica (sensu stricto) (Diptera, Drosophilidae) from East Asia, with assessment of species delimitation using DNA barcodes

Zootaxa ◽

10.11646/zootaxa.4678.1.1 ◽

2019 ◽

Vol 4678 (1) ◽

pp. 1-75

Author(s):

JIA HUANG ◽

LU GONG ◽

SHUN-CHERN TSAUR ◽

LIN ZHU ◽

KEYING AN ◽

...

Keyword(s):

New Species ◽

Cytochrome C Oxidase ◽

Species Delimitation ◽

Species Complex ◽

Dna Barcode ◽

Sensu Stricto ◽

Coi Gene ◽

Mitochondrial Cytochrome ◽

Dna Barcodes ◽

New Synonyms

A total of 50 (43 known and seven new) species in the subgenus Phortica (sensu stricto) were surveyed and (re)described from China: P. bicornuta (Chen & Toda, 1997); P. bipartita (Toda & Peng, 1992); P. biprotrusa (Chen & Toda, 1998); P. cardua (Okada, 1977); P. chi (Toda & Sidorenko, 1996); P. conifera (Okada, 1977); P. eparmata (Okada, 1977); P. eugamma (Toda & Peng, 1990); P. excrescentiosa (Toda & Peng, 1990); P. fangae (Máca, 1993); P. flexuosa (Zhang & Gan, 1986); P. foliata (Chen & Toda, 1997); P. gamma (Toda & Peng, 1990); P. gigas (Okada, 1977); P. glabtabula Chen & Gao, 2005; P. hainanensis (Chen & Toda, 1998); P. hongae (Máca, 1993); P. huazhii Cheng & Chen, 2008; P. iota (Toda & Sidorenko, 1996); P. jadete Zhu, Cao & Chen, 2018; P. kappa (Máca, 1977); P. lambda (Toda & Peng, 1990); P. latifoliacea Chen & Watabe, 2008; P. magna (Okada, 1960); P. okadai (Máca, 1977); P. omega (Okada, 1977); P. orientalis (Hendel, 1914); P. pangi Chen & Wen, 2005; P. paramagna (Okada, 1971); P. perforcipata (Máca & Lin, 1993); P. pi (Toda & Peng, 1990); P. protrusa (Zhang & Shi, 1997); P. pseudopi (Toda & Peng, 1990); P. pseudotau (Toda & Peng, 1990); P. psi (Zhang & Gan, 1986); P. rhagolobos Chen & Gao, 2008; P. saeta (Zhang & Gan, 1986); P. setitabula Chen & Gao, 2005; P. subradiata (Okada, 1977); P. tau (Toda & Peng, 1990); P. uncinata Chen & Gao, 2005; P. unipetala Chen & Wen, 2005; P. allomega Gong & Chen, sp. nov.; P. archikappa Gong & Chen, sp. nov.; P. dianzangensis Gong & Chen, sp. nov.; P. imbacilia Gong & Chen, sp. nov.; P. liukuni Gong & Chen, sp. nov.; P. tibeta Gong & Chen, sp. nov.; and P. xianfui Gong & Chen, sp. nov. In addition, seven new synonyms were recognized: P. acongruens (Zhang & Shi, 1997), syn. nov.; P. antillaria (Chen & Toda, 1997), syn. nov.; P. kukuanensis Máca, 2003, syn. nov.; P. linae (Máca & Chen, 1993), syn. nov.; P. shillongensis (Singh & Gupta, 1979), syn. nov.; P. takadai (Okada, 1977), syn. nov.; and P. watanabei (Máca & Lin, 1993), syn. nov. A key to all Asian species (except for the eparmata species complex) of this subgenus was provided. All currently available DNA barcode (partial mitochondrial cytochrome c oxidase subunit I (COI) gene) sequences of this subgenus (217 sequences of 54 species) are employed in a molecular analysis using different species delimitation methods. The results indicate that approximately 68.5% (37 of 54 spp.) of Phortica (s. str.) species could be clearly distinguished from closely related morphospecies or cryptic species.

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Identification of Nepeta olgae Regel and phylogenetic status of some genera in subtribe Nepetinae (Lamiaceae) using DNA markers

BIO Web of Conferences ◽

10.1051/bioconf/20213800087 ◽

2021 ◽

Vol 38 ◽

pp. 00087

Author(s):

Elena Nikitina ◽

Abdurashid Rakhmatov

Keyword(s):

Dna Barcoding ◽

Large Scale ◽

Dna Barcode ◽

Dna Barcodes ◽

Biodiversity Monitoring ◽

Reference Library ◽

Accurate Identification ◽

Unknown Species ◽

Phylogenetic Status ◽

Nuclear Its

The species level diversity is the reference unit for biodiversity accounting, should be systematized and include full information about the species. Reliable identification of any species is critical for a large-scale biodiversity monitoring and conservation. A DNA barcode is a DNA sequence that identifies a species by comparing the sequence of an unknown species with barcodes of a known species sequence database. Accurate identification of important plants is essential for their conservation, inventory. The species diversity assessing exampled on the subtribe Nepetinae (Lamiaceae) representatives, growing in Uzbekistan is given, using DNA barcoding method. The study was aimed to identify indigenous important plants with the nuclear (ITS) and plastid (matK, rbcL, trnL-F) genomes. This work demonstrates the phylogenetic relationships of some genera within the subtribe Nepetinae Coss. & Germ. (Lamiaceae), based on ITS locus gene. All results indicate that the DNA barcoding tool can be successfully used to reliably identify important plants, to inventory the botanical resources of Uzbekistan and to create a reference library of DNA barcodes. So, the combination of three-four locus gene is a good candidate for this approach.

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Molecular identification and characterization of medically and veterinary important flies of Bangladesh based on mitochondrial COI gene sequences

Asia Pacific Journal of Molecular Biology and Biotechnology ◽

10.35118/apjmbb.2019.027.4.08 ◽

2019 ◽

pp. 69-79

Author(s):

Faria Farhana Rain ◽

Abdul Jabber Howlader ◽

Abu Faiz Md. Aslam

Keyword(s):

Research Work ◽

Dna Barcode ◽

Haplotype Network ◽

First Record ◽

Coi Gene ◽

Gene Sequences ◽

Accurate Identification ◽

Haematobia Irritans ◽

Mitochondrial Coi ◽

Mitochondrial Coi Gene

Flies are considered serious pests which cause health problems of human and animal, transmitting many pathogenic microbes. Pest management programs depend on proper identification of pests. The present research work is an initiative to identify the medically and veterinary important flies based on mitochondrial COI gene sequences. Eleven species of the fly pests were identified. Among them, four fly species were the first record from Bangladesh. The phylogenetic analysis of retrieved sequences confirmed that the evolution of these species occurred from a common ancestor. Highest AT percentage (69.9%) was found in Haematobia irritans exigua and lowest GC percentage (30.4%) was found in Haematobia irritans exigua. The substitution rate of codon was found 1.88 in 1st position, 0.73 in 2nd position and 1.22 in 3rd position, respectively. Interspecific genetic divergence range of flies sequences was 5-20%. Haplotype network showed that Atylotus agrestis was mostly diverged from its common ancestors by 37 mutational steps. This research is the first molecular approach to identify the medically and veterinary important flies based on MT-COI gene sequences along with the establishment of first DNA barcode dataset for accurate identification in Bangladesh.

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