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Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an important natural enemy of long-horned beetles in China, Japan, and Korea. In this study, the genetic sequence of cytochrome oxidase subunit I was used to investigate the genetics and relationships within and among D. helophoroides populations collected from five different geographic locations. We used principal component analysis, heatmap, and Venn diagram results to determine the relationship between haplotypes and populations. In total, 26 haplotypes with 51 nucleotide polymorphic sites were defined, and low genetic diversity was found among the different populations. Significant genetic variations were observed mainly within populations, and no correlation was found between genetic distribution and geographical distance. Low pairwise fixation index values (–0.01424 to 0.04896) and high gene flows show that there was high gene exchange between populations. The codistributed haplotype DH01 was suggested to be the most ancestral haplotype, and other haplotypes were thought to have evolved from it through several mutations. In four of the populations, both common haplotypes (DH01, DH03, and DH22) and unique haplotypes were found. Low genetic diversity among different populations is related to a relatively high flight capacity, host movement, and human-aided dispersal of D. helophoroides. The high gene exchange and typically weak population genetic structure among five populations, especially among populations of Anoplophora glabripennis (Motschulsky), Monochamus alternatus (Hope), and Massicus raddei (Blessig), may suggest that these populations cross naturally in the field.

Seafood mislabeling is common in both domestic and international markets. Studies on seafood fraud often report high rates of mislabeling (e.g., > 70%), but these studies have been limited to a single sampling year, which means it is difficult to assess the impact of stricter governmental truth-in-labeling regulations. We used DNA barcoding to assess seafood labeling in 26 sushi restaurants in Los Angeles over 4 years. Seafood from 3 high-end grocery stores were also sampled (n — 16) in 2014. We ordered 9 common sushi fish from menus, preserved tissue samples in 95% ethanol, extracted the genomic DNA, amplified and sequenced a portion of the mtDNA COI gene, and identified the resulting sequence to known fish sequences from the National Center for Biotechnology Information nucleotide database. We compared DNA results with the U.S. Food and Drug Administration (FDA) list of acceptable market names and retail names. We considered sushi-sample labels that were inconsistent with FDA names mislabeled. Sushi restaurants had a consistently high percentage of mislabeling (47%; 151 of 323) from 2012 to 2015, yet mislabeling was not homogenous across species. Halibut, red snapper, yellowfin tuna, and yellowtail had consistently high (< 77%) occurrences of mislabeling on menus, whereas mislabeling of salmon and mackerel were typically low (> 15%). All sampled sushi restaurants had at least one case of mislabeling. Mislabeling of sushi-grade fish from high-end grocery stores was also identified in red snapper, yellowfin tuna, and yellowtail, but at a slightly lower frequency (42%) than sushi restaurants. Despite increased regulatory measures and media attention, we found seafood mislabeling continues to be prevalent. La mala etiquetación de pescados es común tanto en los mercados domésticos como en los internacionales. Los estudios sobre el fraude de pescados generalmente reportan tasas altas de mala etiquetación (p. ej.: >70 %), pero estos estudios han sido limitados a un sólo muestreo al año, lo que significa que es complicado evaluar el impacto de regulaciones gubernamentales más estrictas sobre las etiquetas verídicas. Utilizamos el código de barras de ADN para evaluar el etiquetado de pescados en 26 restaurantes de sushi en Los Ángeles durante cuatro años. Los pescados de tres supermercados lujosos también fueron muestreados (n = 16) en el 2014. Ordenamos nueve pescados comunes en el sushi de los menús, preservamos las muestras de tejido en etanol al 95 %, extrajimos el ADN genómico, amplificamos y secuenciamos la porción del gen COIdelADNmt, e identificamos la secuencia resultante a partir de secuencias de peces de la base de datos de nucleótidos del Centro Nacional para la Información Biotecnológica. Comparamos los resultados de ADN con la lista de nombres aceptables para el mercado y de venta al menudeo de la Administración Estadunidense de Alimentos y Medicamentos (FDA, en inglés). Consideramos como mal etiquetadas a las muestras de sushi que no fueron consistentes con los nombres de la FDA. Los restaurantes de sushi tuvieron constantemente un porcentaje alto de mala etiquetación (47 %; 151 de 323) de 2012 a 2015, sin embargo, la mala etiquetación no fue homogénea entre las especies. El hipogloso, el huachinango, el atún de aleta amarilla y el jurel tuvieron ocurrencias altas (<77 %) de mala etiquetación en los menús, mientras que la mala etiquetación del salmón y la caballa fue típicamente baja (>15 %). Todos los restaurantes de sushi muestreados tuvieron por lo menos un caso de mala etiquetación. La mala etiquetación de pescado con calidad para sushi de los supermercados lujosos también fue identificada para el huachinango, el atún de aleta amarilla y el jurel, pero a una frecuencia un poco menor (42 %) que en los restaurantes de sushi. A pesar del incremento en las medidas regulatorias y en la atención de los medios, encontramos que la mala etiquetación de los pescados todavía es prevalente.

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment. We use metabarcoding to characterize zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) and compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. Our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.

Studies of lionfish feeding ecology seek to document the ecological impact of this invasive predatory species and determine which native prey species are at greatest risk. There are 2 common approaches to feeding ecology through gut content analysis: morphological identification to the lowest possible taxonomic rank and/or DNA barcoding of individual prey components in the stomach. The major disadvantage of both techniques is their inability to use advanced digested material. This study introduces next-generation sequencing to lionfish feeding ecology, employing DNA metabarcoding to analyze all components of the gut contents, including the previously unidentifiable portion. Sixty-three lionfish were caught from the inshore and offshore reefs of La Parguera, Puerto Rico. Stomach contents were separated into 2 sample components—a liquid (i.e. digested) and undigested tissue. A 313 bp region of the cytochrome oxidase subunit I (COI) gene was amplified from extracted DNA using specific primers for Caribbean reef fish. Samples were sequenced with an Illumina MiSeq platform, and the resulting 950+ sequences were compared against GenBank and the Barcode of Life Database to identify specimens at the lowest taxonomic level. Thirty-nine fish species from 16 families were identified (35 each in the digested and tissue fractions), including members of Pomacentridae, Acanthuridae, Gobiidae, Apogonidae, and Scaridae. Using the digested liquiform material proved efficient in detecting prey species, especially those that would have been missed with traditional methods.

Onion thrips (Thrips tabaci Lindeman,Thysanoptera:Thripidae) causes severe damage to many horticultural and agronomic crops worldwide. It also acts as a vector of several plant viruses. T. tabaci is a key pest of Allium cepa in the United States. However, there is limited information available on the genetic variation within and between T. tabaci populations in the United States and its key evolutionary parameters. In the current study, 83 T. tabaci specimens were collected from A. cepa from 15 different locations comprising four states of the United States. A total of 92 mtCOI gene sequences of T. tabaci from A. cepa were analyzed to understand the genetic diversity and structure of T. tabaci collected from onion host. Seven distinct haplotypes of T. tabaci infesting A. cepa were identified from the current collection, while nine T. tabaci sequences retrieved from GenBank comprised 5 haplotypes. Overall, 15 haplotypes of T. tabaci infesting A. cepa were identified in the world that includes the ten haplotypes in the United States. In the phylogenetic analysis, all the populations collected during the study clustered with thelytokous lineage, while T. tabaci sequences retrieved from GenBank corresponded to leek-associated arrhenotokous lineage. The highest genetic variation was found in Elba and Malheur populations with 3 haplotypes identified in each. The results suggest that haplotypes 1 and 7 are more frequently prevailing haplotypes in the north-western United States, with haplotype 1 being the predominant all over the country. The eastern United States appears to have a more diverse group of haplotypes. The populations from Hungary constituted distinct haplotypes and a haplotype from Kingston linked it with the predominant haplotype.

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups-dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus-were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or 'barcoding', can be used to identify fish species.

The classification of the European hornet, Vespa crabro , into subspecies based on thoracic and abdominal color patterns remains controversial. This study combined morphological assessments and mitochondrial genetic analyses to critically evaluate subspecies delimitation and introduction pathways of V . crabro , focusing on two traditionally recognized subspecies in Korea: V . c . crabroniformis and V . c . flavofasciata . Morphological examination of 163 Korean specimens identified 27 distinct color patterns. Furthermore, thoracic and abdominal characters exhibited weak correlation, thus limiting their utility as diagnostic markers. Genetic analysis using mitochondrial CO1 sequences revealed nine haplotypes among Korean populations that showed weak correspondence to the previously defined morphological subspecies. Furthermore, comparative genetic analyses demonstrated that Korean haplotypes are genetically distinct from both Japanese and European populations, indicating previously underappreciated geographic genetic diversity. Additionally, mitochondrial DNA analyses identified multiple independent invasion events into North America, originating from both European and Korean lineages. Collectively, our findings strongly challenge the validity of subspecies classifications relying solely on color patterns. Considering both morphological and genetic evidence, we conclude that V . crabro does not comprise clearly defined subspecies. Nonetheless, CO1 analysis effectively revealed provisional mitochondrial lineages, providing new insights into the invasion history and biogeographic structure of V. crabro .

The tomato leaf miner (TLM), Phthorimaea absoluta Meyrick, 1917 (Lepidoptera: Gelechiidae) is a destructive invasive insect that has expanded its global distribution. Rapid and accurate identification of invasive pests is essential to support subsequent management and devise control measures. To accurately diagnose P. absoluta , a Loop Mediated Isothermal Amplification (LAMP) assay (TLM-LAMP) was developed to amplify the target region of mitochondrial cytochrome oxidase subunit I (COI) gene. The TLM-LAMP assay can identify the P. absoluta within 60 min at 65 °C after sample extraction. Cross-reactivity analysis against three closely related non-target species, Phthorimaea operculella (Zeller, 1873), Pectinophora gossypiella (Saunders, 1844), and Aproaerema modicella (Deventer, 1904) confirmed species specificity. The TLM-LAMP assay showed high sensitivity to P. absoluta DNA up to 1 × 10 − 8 ng/µL and in plasmid DNA template up to 1 × 10 –14 ng/µL. In addition, the TLM-LAMP assay was successful in laboratory detection of larvae, pupa, and adult stages of P. absoluta . We have tested the TLM-LAMP assay for field application with quick and simple crude insect extraction procedures and found double distilled water (ddH 2 O) as an effective extraction solution. The new TLM-LAMP assay was validated in the field and polyhouse using moths collected from pheromone traps followed by ddH 2 O crude insect extract preparation and incubation. The assay could successfully detect the P. absoluta within 45 min at 65 °C. Sensitivity, specificity, repeatability, and field compatibility of the TLM-LAMP highlights the novelty of the developed method. TLM-LAMP assay is a novel molecular tool for detection of P. absoluta in the laboratory and field which will help in monitoring and aiding biosecurity responses.

Background The recent finding of a typically non-African Anopheles species in eastern Ethiopia emphasizes the need for detailed species identification and characterization for effective malaria vector surveillance. Molecular approaches increase the accuracy and interoperability of vector surveillance data. To develop effective molecular assays for Anopheles identification, it is important to evaluate different genetic loci for the ability to characterize species and population level variation. Here the utility of the internal transcribed spacer 2 (ITS2) and cytochrome oxidase I (COI) loci for detection of Anopheles species from understudied regions of eastern Ethiopia was investigated. Methods Adult mosquitoes were collected from the Harewe locality (east) and Meki (east central) Ethiopia. PCR and Sanger sequencing were performed for portions of the ITS2 and COI loci. Both NCBI’s Basic Local Alignment Search tool (BLAST) and phylogenetic analysis using a maximum-likelihood approach were performed to identify species of Anopheles specimens. Results Two species from the east Ethiopian collection, Anopheles arabiensis and Anopheles pretoriensis were identified. Analyses of ITS2 locus resulted in delineation of both species. In contrast, analysis of COI locus could not be used to delineate An. arabiensis from other taxa in Anopheles gambiae complex, but could distinguish An. pretoriensis sequences from sister taxa. Conclusion The lack of clarity from COI sequence analysis highlights potential challenges of species identification within species complexes. These results provide supporting data for the development of molecular assays for delineation of Anopheles in east Ethiopia.

Abstract Phlebotomine sand flies stand out for their role in vector-borne diseases, having taxonomic priority in aspects of public health. Traditional identification based on morphology involves some limitations that have been corrected with the implementation of complementary methodologies such as cytochrome c oxidase subunit I barcoding and recently mass spectrometry. In Mexico, nearly 38% of sand fly species count with a molecular characterization, but additional information is still necessary for improving sand fly species delimitation. We carried out a molecular species delimitation study of sand flies distributed in the Mexican Transition Zone, between the Nearctic and Neotropical regions, with newly generated cytochrome c oxidase subunit I barcodes, and the first protein profiles created. Compelling evidence showed putative new taxa emerge from Micropygomyia aff. durani (Vargas & Diaz-Nájera) and Pintomyia Series serrana Barretto, and several cryptic species be contained within the genera Micropygomyia and Psathyromyia, which could be of biological and epidemiological interest. However, for some taxa an exhaustive taxonomic revision at the morphological and molecular levels is recommended, especially for sand flies of wide distribution in the New World. Graphical Abstract Graphical Abstract