DNA metabarcoding reveals the importance of gelatinous zooplankton in the diet of Pandalus borealis, a keystone species in the Arctic.

Information about the dietary composition of a species is crucial to understanding their position and role in the food web. Increasingly, molecular approaches such as DNA metabarcoding are used in studying trophic relationships, not least because they may alleviate problems such as low taxonomic resolution or underestimation of digestible taxa in the diet. Here, we used DNA metabarcoding with universal primers for cytochrome c oxidase I (COI) to study the diet composition of the northern shrimp (Pandalus borealis), an Arctic keystone species with large socio-economic importance. Across locations, jellyfish and chaetognaths were the most important components in the diet of P. borealis, jointly accounting for 40%-60% of the total read abundance. This dietary importance of gelatinous zooplankton contrasts sharply with published results based on stomach content analysis. At the same time, diet composition differed between fjord and shelf locations, pointing to different food webs supporting P. borealis in these two systems. Our study underlines the potential of molecular approaches to provide new insights into the diet of marine invertebrates that are difficult to obtain with traditional methods, and calls for a revision of the role of gelatinous zooplankton in the diet of the key Arctic species P. borealis, and in extension, Arctic food webs.

Cryptic genetic diversity and complex phylogeography of the boreal North American scorpion, Paruroctonus boreus (Vaejovidae).

Diverse studies in western North America have revealed the role of topography for dynamically shaping genetic diversity within species though vicariance, dispersal and range expansion. We examined patterns of phylogeographical diversity in the widespread but poorly studied North American vaejovid scorpion, Paruroctonus boreus Girard 1854. We used mitochondrial sequence data and parsimony, likelihood, and Bayesian inference to reconstruct phylogenetic relationships across the distributional range of P. boreus, focusing on intermontane western North America. Additionally, we developed a species distribution model to predict its present and historical distributions during the Last Glacial Maximum and the Last Interglacial Maximum. Our results documented complex phylogeographic relationships within P. boreus, with multiple, well-supported crown clades that are either geographically-circumscribed or widespread and separated by short, poorly supported internodes. We also observed subtle variation in predicted habitat suitability, especially at the northern, eastern and southern edges of the predicted distributional range under past climatic conditions. The complex phylogenetic relationships of P. boreus suggests that historical isolation and expansion of populations may have occurred. Variation in the predicted distributional range over time may implicate past climatic fluctuations in generating the patterns of genetic diversity observed in P. boreus. These findings highlight both the potential for cryptic biodiversity in widespread North American scorpion species and the importance of phylogeographical studies for understanding the factors responsible for generating the biodiversity of western North America.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

With DNA barcoding revealing sexual dimorphism in a water mite: the first description of male Sperchon fuxiensis (Acari: Hydrachnidia: Sperchontidae).

Sperchon fuxiensis Zhang, 2017 was published as a new species based on females alone. Two males of Sperchon were found in the same locality during our recent collection. The males resemble S. fuxiensis female in the integument pattern, excretory pore and the palps shape, but the chitinous plates of both dorsum and venter differ greatly. The males were paired with the female of S. fuxiensis using DNA barcoding, revealing unusual sexual dimorphism in the species. Descriptions and illustrations of the male of S. fuxiensis are given in the present study. Species identification based on the full-length DNA barcoding (658bp) of COI in water mites is also discussed.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification.

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/µl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

A taxonomic revision of the Western Palaearctic genus Cacochroa Heinemann, 1870 (Lepidoptera, Depressariidae, Cryptolechiinae) with description of a new genus and a new species.

Following the description of Cacochroa rosetella Corley, 2018 it soon became clear that there was considerable confusion regarding the identity of Cacochroa permixtella (Herrich-Schäffer, 1854). In this paper the genus Cacochroa is revised and this confusion is resolved, a neotype is chosen for C. permixtella and nearly all records verified. Male and female genitalia of C. permixtella are remarkably different from those of the remaining species, which are here placed in Rosetea Corley Ferreira, gen. nov. The distributions of the three species previously described in Cacochroa are clarified. Cacochroa permixtella has a distribution limited to Macedonia, Bulgaria, Greece and Turkey. Rosetea corfuella (Lvovsky, 2000), comb. nov., is recorded for the first time from Crete, Croatia, Macedonia, Turkey and Israel; the male of R. rosetella (Corley, 2018), comb. nov., is described for the first time and the species is recorded for the first time from Spain, France (mainland and Corsica), Italy (mainland and Sardinia), Greece (mainland and Crete), Croatia and Algeria. Rosetea sara sp. nov. is described from North Africa (Morocco and Tunisia). Male and female genitalia and DNA barcode data are presented for all four species.

DNA Barcoding reveals sexual dimorphism in Isotrias penedana Trematerra, 2013 (Lepidoptera: Tortricidae, Chlidanotinae).

Isotrias penedana Trematerra, 2013 was described from north Portugal based on males alone. Unidentified females were associated with the males using DNA barcoding, revealing sexual dimorphism in the species. Males and females differ in forewing shape, markings, and size, with females significantly smaller than males. The female is described and illustrated for the first time. We also document the species' occurrence in northern Spain.

First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea.

The Mediterranean Sea is increasingly under threat from invasive species that may negatively affect biodiversity and/or modify ecosystem structure and function. The bivalve mollusc Pinctada imbricata radiata is listed among the 100 most invasive species in the Mediterranean. A first finding of an established population of P. imbricata radiata in the coastal waters of the eastern Adriatic Sea, is presented in this paper. Six and then 30 live specimens were collected in 2015 and in 2017, respectively, at depths of 5 to 15m from the island of Mljet, Croatia. DNA sequencing of the mitochondrial cytochrome c oxidase I gene (COI) revealed three different haplotypes. All samples showed greatest similarity (98 to >99%) to P. radiata COI sequence records in GenBank (=P. imbricata radiata as used in this paper). A Neighbour Joining tree placed all Croatian samples within the 100% bootstrap supported clade for P. imbricata radiata.

DNA barcoding of two solitary ascidians, Herdmania momus Savigny, 1816 and Microcosmus squamiger Michaelsen, 1927 from Thoothukudi coast, India.

Morphology-based taxonomical studies of ascidians in India are meagre due to lack of ascidian taxonomist and limitations inherent in conventional system-based identification. The use of short fragment of mitochondrial DNA sequence is proving highly useful in identifying species in a situation where, the traditional morphology-based identification is difficult. In the present study, two adult solitary ascidians collected from the Thoothukudi coast were morphologically identified as Herdmania momus Savigny, 1816 and Microcosmus squamiger Michaelsen, 1927. The genomic DNA of these ascidians was isolated, COI gene was amplified, sequenced and submitted to the GenBank under the accession numbers KM058116, KM411616 and KJ944390. Homology search result using BLAST showed that H. momus showed 100% matched with other H. momus, while M. squamiger showed similarity with Pyura herdmani, a member of the same family Pyuridae. The phylogenetic and genetic distance was maximum in interspecies than in intraspecies. These COI sequences will allow the identification of the species through DNA barcoding technique. Here, we report for the first time the COI gene of H. momus, Savigny 1816 from the Indian coast.

AMDAR y PCR-extra-rápida para la identificación de la tottuga cabezona Caretta caretta (Testudines: Cheloniidae) utilizando el gen mitocondrial citocromo c oxidasa I (COI) / AMDAR and PCR-Extra-fast for Molecular identification of the loggerhead sea turtle Caretta caretta (Testudines: Cheloniidae) using the mitochondrial gene cytochrome c oxidase I (COI) / AMDAR e PCR extra-rápida para a identificação da Tartaruga-Comum Caretta caretta (Testudines: Cheloniidae), utilizando o gene mitocondrial citocromo c oxidase I (COI)

Para identificación molecular de tortuga cabezona Caretta caretta, se utilizó Amplificación Mitocondrial DNA y Análisis de Restricción y PCR Extra-rápida. Se obtuvo muestras de sangre de juveniles de C. caretta de playa Don Diego (Magdalena; n=4) e Islas Del Rosario (Bolívar; n=2) en el Caribe colombiano. Se aisló DNA, se estandarizó la amplificación del gen mitocondrial citocromo c oxidasa I (COI) por PCR extra-rápida, obteniendo fragmentos de 650 pares de bases, disminuyendo en un tercio el tiempo de reacción. El producto amplificado de COI se analizó con enzimas HindIII, HyCH4III y MseI generando perfil electroforético que al compararlo in silico con secuencias para otras especies de tortugas marinas; permitió identificar patrón específico para la tortuga cabezona. Las secuencias nucleótidicas se analizaron con BLAST y similaridad entre 97-99 % con C. caretta en cinco secuencias y 92 % en otra. La información de tortugas muestreadas fue integrada en base datos BOLD y se generó el código de barras. La metodología descrita para identificación de C. caretta es procedimiento rápido y bajo costo que minimiza el tiempo de PCR mejorando su especificidad.

We molecularly identified the loggerhead turtle Caretta caretta using high-speed PCR amplification and restriction analysis of mitochondrial DNA. We isolated the DNA from blood from juvenile C. caretta from Don Diego beach (Magdalena; n=4), in Islas Del Rosario (Bolívar; n=2) in the Colombian Caribbean. By using high-speed PCR amplification of mitochondrial cytochrome c oxidase I (COI), we reduced reaction by one third and obtained fragments of 650 base pairs. We analyzed the amplified IOC product using enzymes HindIII, HpyCH4III and MseI and generated an electrophoretic profile, which compared in silico to other sea turtle species sequences, revealed the loggerhead's specific pattern. We found similarity between 97-99% with C. caretta in five of the BLAST analyzed nucleotide sequences and 92% in another. We generated a bar code for the sampled turtle information and sequences and stored them in the BOLD database. The methodology described for the identification of C. caretta is a fast and inexpensive procedure that minimizes time and improves PCR specificity.

Identificámos molecularmente a tartaruga-comum Caretta caretta usando PCR amplificado de alta-velocidade e análise de ADN mitocondrial restrito. Isolámos o ADN do sangue de juvenis C. carreta das praias Don Diego (Magdalena ; n=4), das Ilhas del Rosario (Bolívas; n=2) no Caribe Colombiano. Ao utilizar a amplificação por PCR de alta velocidade da citocromo c oxidase I (COI ), reduzimos a reacção a um terço, e obtivemos fragmentos de 650 pares de bases. Analisámos o produto IOC amplificado com as enzimas de restrição HindIII , HyCH4III e MseI e gerámos um perfil eletroforético, que comparado em silico com outras seqüéncias de espécies de tartarugas marinhas, revelou padrão específico à tartaruga-comum. Encontramos semelhanças entre 97-99 %, com C. caretta em cinco das seqüéncias de nucleotídeos BLAST analisados e 92% com outro. Gerámos um código de barras para a informação e seqüéncias das tartaruga amostradas e foram armazenados na base de dados BOLD. A metodologia descrita para a identificação de C. caretta é um procedimento rápido e barato, que minimiza o tempo e melhora a especificidade da PCR.