Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.     

DNA barcodes of the native ray‐finned fishes in Taiwan

Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray‐finned fishes in Taiwan. A total of 2993 individuals, belonging to 1245 species within 637 genera, 184 families and 29 orders of ray‐finned fishes and representing approximately 40% of the recorded ray‐finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10‐fold higher than the mean conspecific one (1.51%), but approximately 1.4‐fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2993 specimens represent 1275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity.     

New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.     

Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles

DNA barcoding facilitates the identification of species and the estimation of biodiversity by using nucleotide sequences, usually from the mitochondrial genome. Most studies accomplish this task by using the gene encoding cytochrome oxidase subunit I (COI; Entrez COX1). Within this barcoding framework, many taxonomic initiatives exist, such as those specializing in fishes, birds, mammals, and fungi. Other efforts center on regions, such as the Arctic, or on other topics, such as health. DNA barcoding initiatives exist for all groups of vertebrates except for amphibians and nonavian reptiles. We announce the formation of Cold Code, the international initiative to DNA barcode all species of these ‘cold‐blooded’ vertebrates. The project has a Steering Committee, Coordinators, and a home page. To facilitate Cold Code, the Kunming Institute of Zoology, Chinese Academy of Sciences will sequence COI for the first 10 specimens of a species at no cost to the steward of the tissues.     

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

Molecular taxonomic identification in the absence of a ‘barcoding gap’: a test with the endemic flora of the Canarian oceanic hotspot

We use a comprehensive subset of Canarian angiosperms corresponding to 23 families, 35 genera and 60 Canarian endemic taxa to test whether this flora is suitable to taxonomic identification with the two proposed plant DNA barcode sequences and whether these sequences may reveal the existence of cryptic species overlooked by morphology. The rate of discrimination success between the insular congeneric samples using the rbcL+matK combination and a ‘character‐based’ approach (where we use only the combination of nucleotide positions in an alignment that allows unambiguous species identification) is higher (82.29%) than that obtained with the ‘distance‐based’ approach (80.20%) used by the CBOL Plant Working Group in 2009 and also when compared with tests conducted in other floras. This suggests that the molecular identification of the Canarian endemic flora can be achieved as successfully as in other floras where the incidence of radiation is not as relevant. The facts that (i) a distance‐based criterion was unable to discriminate between congeneric and conspecific comparisons and (ii) only the character‐based discrimination criterion resolved cases that the distance‐based criterion did not, further support the use of a character discrimination approach for a more efficient DNA barcoding of floras from oceanic islands like the Canaries. Thus, a barcoding gap seems not to be necessary for the correct molecular characterization of the Canarian flora. DNA barcodes also suggest the possible existence of cryptic taxa to be further investigated by morphology and that the current taxonomic status of some of the taxa analysed may need revision.     

Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Mouthpart evolution in adults of the basal, 'symphytan', hymenopteran lineages

We review feeding biology and mouthpart structure generally among adults of the basal hymenopteran, or ‘symphytan’, lineages (sawflies, woodwasps, horntails and their relatives). These insects feed on a wide range of materials: floral and extrafloral nectar, pollen, plant (floral and leaf) tissues, plant (angiosperm) sap, the juice of ripe fruit, die spermatial fluid of rust fungi, sternorrhynchan bug honeydew, and insect tissues. Adults show feeding‐related mouthpart specialization either for consuming pollen (the Xyelidae only) or for consuming ‘concealed’ floral nectar (several families). Seven functional types of elongated proboscis or ‘concealed‐nectar extraction apparatus (GNEA)’ have previously been recognized among Hymenoptera. We identify an additional type, which appears to be unique among Hy‐menoptera and has probably evolved direcdy from unspecialized mouthparts (labiomaxillary complex). In total, three types of CNEA are known to occur in ‘Symphyta’. Type 1 occurs in Pamphiliidae, Megalodontesidae, Argidae, Pergidae, Tenthredinidae, Cimbicidae and Cephidae. Type 5 occurs in Pergidae (in two unidentified species of Euryinae). Type 8 occurs in Tenthredinidae (in the genus Nipponorhynchus Takeuchi). CNEA of some type or other has arisen at least twice within the family Tenthredinidae and at least twice widiin die pergid subfamily Euryinae. Evolutionary parallelism in CNEA structure has occurred between the basal, ‘symphytan’, hymenopteran lineages and die Apocrita, a phenomenon hitherto not mentioned in the literature. Within the ‘Symphyta’, possession of Type 1 CNEA appears to be a ground plan feature of each of the following taxa: the pergid genus Eurys Newman, the megalodontesid genus Megalodontes Latreille (the only extant representative of the Megalodontesidae) and the tenthredinid genus Cuneala Zirngiebl, while possession of Type 8 appears to be a ground plan feature of die tenthredinid genus Mpponorhynchus Takeuchi. However, in general among ‘Symphyta’, possession of CNEA is characteristic of only small and taxonomically subordinate groups, suggesting that CNEA has evolved independendy many times within the basal hymenopteran lineages rather than being inherited from a common ancestor early in the evolutionary history of the Hymenoptera. In other words, ecological expediency radier than phylogenetic history mainly accounts for its distribution pattern within the basal lineages. The results of a morphological survey of ‘Symphyta’ indicate that the habit of exploiting ‘concealed nectar’, by means of CNEA, is fairly     

DNA barcoding for the identification of smoked fish products

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.     

Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades‐old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high‐throughput laboratory workflows. The strategy uses hemi‐nested, degenerate, M13‐tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard‐compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

BOLD’s role in barcode data management and analysis: a response

DNA barcoding is a very effective tool for the identification of specimens when a carefully validated and taxonomically comprehensive library of reference DNA barcodes is available. Libraries meeting this criterion are now available for some taxonomic groups in some geographic regions, provoking their use as a tool for the identification of samples that would otherwise remain as unknowns. In this article, we emphasize the need for caution in the interpretation of identifications based on a reference library with entries that have seen limited validation. We also emphasize the need for the deposition of sequence records for ‘unknowns’ so that presumptive identifications can be tested by other researchers and updated as the barcode reference library gains increased coverage and validation.     

Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys

DNA barcoding is a powerful tool for species detection, identification and discovery. Metazoan DNA barcoding is primarily based upon a specific region of the cytochrome c oxidase subunit I gene that is PCR amplified by primers HCO2198 and LCO1490 (‘Folmer primers’) designed by Folmer et al. (Molecular Marine Biology and Biotechnology, 3 , 1994, 294). Analysis of sequences published since 1994 has revealed mismatches in the Folmer primers to many metazoans. These sequences also show that an extremely high level of degeneracy would be necessary in updated Folmer primers to maintain broad taxonomic utility. In primers jgHCO2198 and jgLCO1490, we replaced most fully degenerated sites with inosine nucleotides that complement all four natural nucleotides and modified other sites to better match major marine invertebrate groups. The modified primers were used to amplify and sequence cytochrome c oxidase subunit I from 9105 specimens from Moorea, French Polynesia and San Francisco Bay, California, USA representing 23 phyla, 42 classes and 121 orders. The new primers, jgHCO2198 and jgLCO1490, are well suited for routine DNA barcoding, all‐taxon surveys and metazoan metagenomics.     

Promise and Challenge of DNA Barcoding in Venus Slipper (Paphiopedilum)

Orchidaceae are one of the largest families of flowering plants, with over 27,000 species described and all orchids are listed in CITES. Moreover, the seedlings of orchid species from the same genus are similar. The objective of DNA barcoding is rapid, accurate, and automated species identification, which may be used to identify illegally traded endangered species from vegetative specimens of Paphiopedilum (Venus slipper), a flagship group for plant conservation with high ornamental and commercial values. Here, we selected eight chloroplast barcodes and nrITS to evaluate their suitability in Venus slippers. The results indicate that all tested barcodes had no barcoding gap and the core plant barcodes showed low resolution for the identification of Venus slippers (18.86%). Of the single-locus barcodes, nrITS is the most efficient for the species identification of the genus (52.27%), whereas matK + atpF-atpH is the most efficient multi-locus combination (28.97%). Therefore, we recommend the combination of matK + atpF-atpH + ITS as a barcode for Venus slippers. Furthermore, there is an upper limit of resolution of the candidate barcodes, and only half of the taxa with multiple samples were identified successfully. The low efficiency of these candidate barcodes in Venus slippers may be caused by relatively recent speciation, the upper limit of the barcodes, and/or the sampling density. Although the discriminatory power is relatively low, DNA barcoding may be a promising tool to identify species involved in illegal trade, which has broad applications and is valuable for orchid conservation.     

The efficacy of DNA barcoding in the classification,genetic differentiation,and biodiversity assessment of benthic macroinvertebrates

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.     

桔小实蝇幼体及成虫残体DNA条形码识别技术的建立与应用

实蝇类害虫多为国内外检疫对象, 其鉴定识别方法主要依据成虫的外部形态特征, 而传统的形态学识别法对口岸经常截获的幼体及残缺的虫体, 则无能为力。本研究以桔小实蝇Bactrocera dorsalis的幼体（卵、 幼虫、 蛹）以及成虫残体（足、 翅、 头部、 胸部、 腹部）为对象, 利用 DNA 条形码技术, 构建实蝇类害虫快速鉴定技术体系, 并以其他4种常见实蝇（包括番石榴实蝇B. correcta、 瓜实蝇B. cucurbitae、 南亚果实蝇B. tau、 柑桔大实蝇B. minax）为对象对该技术体系进行应用验证。结果显示, 桔小实蝇幼体以及成虫残体的碱基序列与数据库中靶标种COⅠ基因碱基序列的一致性为99.51%~99.84%, 其他4种实蝇相应序列与数据库中靶标种COⅠ基因序列的一致性分别为100%, 100%, 99.81%~99.83%和100%; 以邻接法（NJ法）构建系统发育树, 靶标种实蝇均与数据库中对应种实蝇聚为一支, 且置信度均为100%。以K2-P模型计算种内及种间遗传距离得出, 5种实蝇的种间遗传距离为0.0597~0.2363, 平均为0.1693; 种内遗传距离为0.0000~0.0041, 平均为0.0019。这些结果表明, 基于DNA条形码的物种识别技术完全可用于口岸截获的实蝇类害虫幼体及残体的准确鉴定。     

Length–weight relationships of wild fish captured at the mouth of Río Verde,Oaxaca, México and connected lagoons (Miniyua,El Espejo,Chacahua and Pastoría)

Length–weight relationships (LWR) were estimated for 34 fish species belonging to 25 genera and 17 families, specimens were collected near the mouth of the Río Verde, Oaxaca, Mexico. All r² values are statistically significant (P > 0.0001). The values of ‘b’ ranged from 2.55 and 3.3, within the usual range (2.5–3.5). This study provides the first LWR references for six species, and new maximum length records for eight species.