bluuebunny/crossref_metadata_2025 huggingface dataset:
Overview radiography of abdominal organs in dogs and cats
Authors:   Doctor of Veterinary Sciences, Professor of Department of the Common Pathology,Moscow State Academy of Veterinary Medicine and Biotechnology – MVA by K. I. Skryabin, Moscow, Russian Federation, Baymatov V. N. , Metelev A. V. ,   Candidate of Veterinary Sciences, Maxima vet Veterinary Clinic, Moscow, Russian Federation
Published: March 2021

Today, less attention has been paid to x-ray studies of animals in connection with the advent of more modern methods. At the same time, the quality of radiopaque substances decreased, which is reflected in this article. The Omnipack, Urografin, Visipack, Barium sulfate, Iodipamide, Bilignost and Biligrafin used in radiopaque contrast studies in most cases did not reveal the hepatobiliary system. Only the last two drugs gave satisfactory results. The complexity of the X-ray examination of the biliary system in cats and dogs lies in the features of the location of the extrahepatic bile ducts and the condition of the intestine, the size of the liver, its structure, echogenicity, the state of the vessels and surrounding organs. In this work, we took into account the anatomical variants of the development and location of organs in animals, which may complicate the study or evaluation of the data obtained. These included changes in the contours, shape, location of the gallbladder and its filling. The results obtained were compared with the available general species standards, taking into account the possible individual characteristics of each animal. Other studied drugs gave satisfactory results with an overall assessment of the abdominal organs, but we preferred Omnipack. Survey radiography provides a large amount of information with the correct selection of radiopaque substances.
Pharmacological properties of the hemostatic drug based on the incomplete silver salt of polyacrylic acid on the model of parenchymal bleeding
Authors:   Graduate student of the Department of Physiology, Pharmacology and Toxicology named after A. N. Golikova and I. E. Mozgova, Moscow State Academy of Veterinary Medicine and Biotechnology – MVA by K.I. Skryabin, Moscow, Russian Federation, Averin N. A. , Del’tsov A. A. ,   Doctor of Veterinary Sciences, Professor of the Department of Physiology, Pharmacology and Toxicology named after A. N. Golikova and I. E. Mozgova, Moscow State Academy of Veterinary Medicine and Biotechnology – MVA by K. I. Skryabin, Moscow, Russian Federation
Published: March 2021

In this work, hemostatic properties of the Hemoblock preparation were studied using a model of parenchymal bleeding, in comparison with a 3% aqueous solution of hydrogen peroxide. The study was carried out on 30 white rats of both sexes weighing 180–200 g. under general anesthesia. Parenchymal bleeding was modeled by applying a wound on the left lobe of the liver with a scalpel. After the use of drugs, the wound was sutured in layers, tightly. An intradermal suture was applied to the skin. To assess the hemostatic effect, the time to stop bleeding was taken into account, as well as the presence or absence of relapse of parenchymal bleeding. As a result of studies, it was found that in animals after application of the studied drug, a complete stop of bleeding was observed after 120,7±4,3 seconds. Relapses of bleeding were absent. The wound surface was without signs of tissue damage. Stable hemostasis was achieved in 100% of cases.
Efficiency of breeding of multiparous sheep of the new genotype
Authors:   Doctor of Agricultural Sciences, Honored Worker of Science of the Kuban, Chief Researcher, Department of breeding and genetics of agricultural animals, Krasnodar Research Centre for Animal Husbandry and Veterinary Medicine, Krasnodar, Russian Federation, Kravchenko N. I.
Published: March 2021

The paper provides a comparative assessment of the productivity of multiparous sheep of a new genotype with animals of the original breeds in order to show the possibility of converting domestic sheep breeding from a loss-making industry to a profitable one. The superiority of the new genotype over peers of fine-wool breed is: for the production of lamb in live weight of 174–183% to (40–45 kg per initial sheep, 71–74,5 kg per ewe), slaughter weight 20% and profitability 104–117 absolute percentages (up to +165, +178%) due to an increase in multiple pregnancy by 158,1–162,4% (1,85–1,90 lambs from one ewe).
The influence of the duration of gestation of sows on the development and the preservation of piglets in the suckling period
Authors:   Candidate of Agricultural Science, a Leading Scientific Worker, Udmurt Federal Research Center of the Ural Branch of the Academy of Sciences, Izhevsk, Russian Federation, Faizullin R. A. , Saifutdinov M. R. ,   Research Worker, Udmurt Federal Research Center of the Ural Branch of the Academy of Sciences, Izhevsk, Russian Federation
Published: March 2021

The duration of gestation is one of the important characteristics of pigs, which affects on the live mass young animal under birth, the him postembryonic development and him future productivity, in particular on the early maturity and the spending of feeds. The aim of the researches was to study the influenceof the duration of gestation of sows on the growth, development and preservation of suckling piglets. The researches were contacted on the basis of the economy of LLC «Russia» of the Mozhginsky district of the Udmurt Republic. For conduct researches the method of the analogs was formed 2 groups of the sucking sows of large white breed. In the first group of sows the average duration of gestation was 110.08 days, and in the second group of sows – 115,16 days. The age of the sows was 36 months, and the number of farrow was 4. The number of the sows in the groups was 12 and 30 heads; respectively. In the results conducted researches was established that young animals obtained from the sows with duration of gestation – 115,16 days authentically exceeded peers born from sows with duration of gestation – 110,08 days on the live mass under birth – by 6,40%, live mass under weaning in the 30 days – by 5,91%, average daily gain – by 5,85% and the preservation of piglets – by 18,84%.
Fourteen Years of Event Management: a Bibliometric Analysis
Authors: Srivastava Praveen Ranjan , Eachempati Prajwal , Sharma Dheeraj
Published: March 2021

Event Management is a well-established international journal in event, festival, and culture management, which has published high-quality peer-reviewed research from the last 20 years. The current study represents a retrospective overview of the journal's publication journey
over the previous 14 years from 2006 to 2020. This period was chosen due to the availability of data in Scopus. The number of publications and citations received by these publications globally has exhibited continuous growth, which signifies the journal's widespread presence. This article
also analyzes the journal's impact, its prominent topics, and its most prolific authors, including their affiliated institutions and countries. The multiple dimensionality scaling analysis initially classified the publications of the journal into five broad clusters: Cluster 1 ("Event Management"),
Cluster 2 ("Customer Satisfaction and Service Quality"), Cluster 3 ("Mega-Events"), Cluster 4 ("Legacy Sports"), and Cluster 5 ("Economic Feasibility"). Clusters 4 and 5 are combined into a single cluster to improve the journal coverage. The coauthorship networks among authors, institutions,
countries, and keywords unveil the collaboration structure among this journal's contributors. The results of the current study provide useful insights for the potential contributors and global audience of the journal and the editorial board of the journal in terms of emerging topics for research.
Searching for the missing genetic determinants of hereditary breast cancer risk by whole-exome sequencing of BRCA-negative patients: new candidate genes USP39, SLIT3, CREB3
Authors: Zagorodnev Kirill , Romanko Aleksandr , Gorgul Uliy , Ivantsov Aleksandr , Sokolenko Anna , Bizin Ilya , Kuligina Ekaterina
Published: March 2021

The search for the new hereditary mutations and a precise molecular genetic diagnosis that determines the causative mutation in each specific case of hereditary breast cancer (BC) is a clinically important task since it helps to define the personal therapeutic approach and increase the effectiveness of preventive measures. Using whole-exome sequencing (WES) we analyzed the full spectrum of hereditary variations in 49 Russian patients with clinical signs of a hereditary disease which allowed us to compile a list of 229 candidate probably pathogenic germ-line variants. Then, the selected candidate mutations were validated by Sanger sequencing and molecular-epidemiological studies, the predisposing roles of three oncologically relevant mutations (USP39 c.*208G>C, SLIT3 p.Arg154Cys, and CREB3 p.Lys157Glu) were confirmed. Our candidate genes are first mentioned in connection with the hereditary risk of BC. The final proofs of the causative roles of these variants could be obtained through functional tests as well as via the analysis of the mutations segregation in BC families.
Mirna expression profiling of anaplastic thyroid cancer
Authors: Knyazeva Margarita , Zabegina Lidia , Sidina Elena , Karizky Aleksey , Tsyrlina Evgeniya , Atrem’eva Anna , Kuznetsova Irina , Vorobyev Sergei , Malek Anastasia
Published: March 2021

Introduction: Malignant transformation of follicular epithelium of the thyroid gland is associated with specific alterations of miRNA profile. Evaluation of miRNAs expression changes is being applied for primary or differential diagnostic of thyroid nodes. Anaplastic thyroid cancer (ATC) is relatively rare form of thyroid cancer with high malignant potency and rate of lethality. Investigation of miRNAs role in ATC might provide with soul for development of new diagnostic and therapeutic approaches. Goal: To analyze expression profile of miRNA in ATC and to identify miRNAs involved in pathogenesis of ATC. Material and Methods: Samples of ATC (n.20) and normal thyroid tissue (n. 22) were included in the study, expression levels of 85 cancer-associated miRNAs were analyzed by RT-PCR. Results: Expression of miR-375, miR-1246 and miR-21 is activated while expression of miR-Let7b, miR-125b and miR-181a is suppressed in cells of ATC. Conclusions: Further investigation of miRNA involvement into carcinogenesis of ATC is needed for development of new diagnostic and therapeutic approaches.
Experience of performing radical trachelectomy with uterine transposition in organ-preserving treatment of stage IB-II invasive cervical cancer
Authors: Chernyshova Alyena , Аntipov Vitaly , Коlomiets Larisa , Chernov Vladimir , Gyunter Victor , Маrchenko Ekaterina , Chekalkin Timofei
Published: March 2021

The development and implementation of organ-preserving treatment modalities is the priority trend in the current cancer treatment of patients of the reproductive age. Methods of expanding indications for organ-preserving treatment in cancer patients are becoming relevant. We present our experience in performing radical trachelectomy with uterine transposition in patients with stage IB-II cervical cancer. Our surgical technique allows the preservation of the uterus and adnexa in patients with invasive cervical cancer, who need to receive combined modality treatment including extended surgery followed by chemotherapy or radiation therapy to the pelvis. Transposition of the uterus and adnexa after radical abdominal or laparoscopic trachelectomy allows the movement of the uterus and ovaries out of the radiation field. After completion of radiation therapy, the uterus with adnexa is repositioned into the pelvis and uterine-vaginal anastomosis is formed using an implant made of titanium nickelide. Independent pregnancy and the use of assisted reproductive technologies are permitted after passing the time when distant metastases are most likely to develop.
Arrheniusite-(Ce), CaMg[(Ce7Y3)Ca5](SiO4)3(Si3B3O18)(AsO4)(BO3)F11, a New Member of the Vicanite Group, from the Östanmossa Mine, Norberg, Sweden
Authors: Holtstam Dan , Bindi Luca , Bonazzi Paola , Förster Hans-Jürgen , Andersson Ulf B.
Published: March 2021

Arrheniusite-(Ce) is a new mineral (IMA 2019-086) from the Östanmossa mine, one of the Bastnäs-type deposits in the Bergslagen ore region, Sweden. It occurs in a metasomatic F-rich skarn, associated with dolomite, tremolite, talc, magnetite, calcite, pyrite, dollaseite-(Ce), parisite-(Ce), bastnäsite-(Ce), fluorbritholite-(Ce), and gadolinite-(Nd). Arrheniusite-(Ce) forms anhedral, greenish-yellow translucent grains, exceptionally up to 0.8 mm in diameter. It is optically uniaxial (–), with ω = 1.750(5), ε = 1.725(5), and non-pleochroic in thin section. The calculated density is 4.78(1) g/cm3. Arrheniusite-(Ce) is trigonal, space group R3m, with unit-cell parameters a = 10.8082(3) Å, c = 27.5196(9) Å, and V = 2784.07(14) Å3 for Z = 3. The crystal structure was refined from X-ray diffraction data to R1 = 3.85% for 2286 observed reflections [Fo > 4σ(Fo)]. The empirical formula for the fragment used for the structural study, based on EPMA data and results from the structure refinement, is: (Ca0.65As3+0.35)Σ1(Mg0.57Fe2+0.30As5+0.10Al0.03)Σ1[(Ce2.24Nd2.13La0.86Gd0.74Sm0.71Pr0.37)Σ7.05(Y2.76Dy0.26Er0.11Tb0.08Tm0.01Ho0.04Yb0.01)Σ3.27Ca4.14]Σ14.46(SiO4)3[(Si3.26B2.74)Σ6O17.31F0.69][(As5+0.65Si0.22P0.13)Σ1O4](B0.77O3)F11; the ideal formula obtained is CaMg[(Ce7Y3)Ca5](SiO4)3(Si3B3O18)(AsO4)(BO3)F11. Arrheniusite-(Ce) belongs to the vicanite group of minerals and is distinct from other isostructural members mainly by having a Mg-dominant, octahedrally coordinated site (M6); it can be considered a Mg-As analog to hundholmenite-(Y). The threefold coordinated T5 site is partly occupied by B, like in laptevite-(Ce) and vicanite-(Ce). The mineral name honors C.A. Arrhenius (1757–1824), a Swedish officer and chemist, who first discovered gadolinite-(Y) from the famous Ytterby pegmatite quarry.
The Role of Intern Pharmacists in Implementing Innovative and Sustainable Practices to Enhance Rates of Non-influenza Immunizations
Authors: Nguyen Kevin , Ignatyeva Natalya , Jorgensen-Ponce Karla , Nijum Anamika , Nguyen Analia , Patel Akash , Pham Yvonne , Hollenbach Kathryn , Mnatzaganian Christina
Published: March 2021

Background
Intern pharmacists are likely an underutilized resource for addressing immunization barriers and improving immunization rates. Studies have addressed methods that pharmacists use to improve immunization rates, but few have focused on the role of intern pharmacists, with particular emphasis on non-influenza immunizations.
Methods
An online survey was distributed through email listservs associated with California pharmacy schools and organizations. Data collected included title (“pharmacist” or “intern pharmacist”), practice setting, perceptions of the intern pharmacists’ role in improving immunization rates (strongly agree to strongly disagree using a 5-point Likert scale), and opinions regarding potential implementations. Immunization barriers were assessed using a 9-point scale, from least to most feasible to be addressed. Statistical comparisons between intern pharmacist and pharmacist responses were made using chi-square or Fisher’s exact tests.
Results
A total of 133 respondents completed the survey. The immunization barrier most likely to be successfully overcome by intern pharmacists was lack of patient awareness. There was strong support in favor of intern pharmacists having greater involvement in programs that increase patient non-influenza immunization rates. The two most feasible implementations by intern pharmacists were determined to be intern-led college/university campus clinics providing free non-influenza immunizations and educational booths/outreaches that raise awareness about immunizations.
Conclusion
Identification of immunization barriers capable of being addressed by intern pharmacists and effective intern pharmacist-led initiatives may contribute to enhanced immunization outcomes. Future steps include the implementation of intern pharmacist-led activities and further evaluation of whether these intern pharmacist-specific initiatives contribute to increasing non-influenza immunizations.
Properties of waste of chemical processing of germanium concentrates
Authors:   Institute of Metallurgy of Ural Branch of Russian Academy of Sciences, Tanutrov Igor N. , Sviridova Marina N. ,   Institute of Metallurgy of Ural Branch of Russian Academy of Sciences
Published: March 2021

In order to increase the through extraction of germanium in the technology of production of germanium concentrates, as well as finding ways to eliminate the accumulation of toxic waste, using modern techniques and equipment, the physical and chemical properties of waste chemical processing of germanium concentrates (OHGC) of two domestic enterprises were experimentally studied. The main components of OHGC are: sulphate hemihydrate CaSO4·0.5H2O and hypochlorite Ca(OCl)2 calcium. The moisture content of the sludge amounted to 30-50%. The content of germanium in the cakes of both companies is in the range of 0.20 and 0.27%, respectively, indicating the feasibility of recovery in the Ge. At the same time, the samples of cakes differ significantly in the content of impurities, which depends on the types of raw materials in the preparation of concentrates. Granulometric composition of cakes is characterized by high dispersion. With an average diameter of 12 µm, all particle sizes are in the range of 0.5-15 µm. The distribution of particle sizes is shifted in interval of 0-15 µm, and the area of the particles less than 3 µm is not more than 10%. The high dispersion of the cake is reflected in the specific surface area, which is 23.7 m2/g. Thermographic study found that the heating of the sample cake is accompanied by two endothermic effects of dehydration at 110 and 145-168 oC calcium sulfate and hypochloride semihydrate with correspond-ding weight loss of 13.1 and 12.9%. The presence of toxic impurities (arsenic, zinc and lead), as well as chlorine, presents significant challenges for the development of disposal technology with the extraction of germanium. Assuming that the undiscovered part of the germanium in the concentrate is compounds or solid solutions with silicon dioxide, an effective technology should include their reagent high temperature treatment.
Fabrication of composites based on modified vermiculite and study of their surface
Authors:   Far Eastern Federal University, Shapkin Nikolay P. , Khalchenko Irina G. ,   Far Eastern Federal University, Sokolova Larisa I. ,   Far Eastern Federal University, Shkuratov Anton L. ,   Far Eastern Federal University, Razov Valery I. ,   Far Eastern Federal University, Galchenko Daria S. ,   Far Eastern Federal University, Smirnova Maria G. ,   Far Eastern Federal University, Apanasenko Olga A. ,   Far Eastern Federal University, Katkova Svetlana A. ,   Far Eastern State Technical Fisheries University
Published: March 2021

Composites based on vermiculite with different modifiers, including hydroxides on the surface of vermiculite particles, have been fabricated. The composition and structure of the fabricated composites have been investigated by means of the methods of energy-dispersive analysis, X-ray diffraction analysis, and positron annihilation spectroscopy. The adsorption characteristics of the fabricated materials with respect to dye of different nature have been determined, and isotherms of low temperature nitrogen adsorption/ desorption have been obtained. It has been demonstrated that for different modifications the composites sorption properties change significantly in dependence on the modifier nature. At modification of layered natural aluminosilicates by cellulose and polyethylene, the surface nature depends largely on the modifier nature. At treatment by polyethylene, a dramatic decrease of the aluminosilicate surface area due to formation of a dense film takes place. The effect of cellulose is not so significant because of formation of a looser surface structure. The sorption characteristics somewhat improve upon further annealing. Good prospects of application of the fabricated sorbents for antibiotics sorption and analytical determination in food products have been demonstrated. Vermiculite, modified with 7% hydrochloric acid, demonstrates a high degree of absorption for chloramphenicol, tetracycline, ciprofloxacin and cefazolin antibiotics. The values of the degrees of absorption show: 73 %, > 99 %, > 99 % and 73 %, respectively. The degree of elution for chloramphenicol is more than 75 %, which allows you to create concentrated cartridges based on this sorbent. The degree of elution is low, which allows the use of a sorbent based on vermiculite for the treatment of waste and natural waters from these antibiotics.
Biochemical study of insects used in fish feed for antioxidant activity
Authors:   Kazan State Energy University, Lapin Anatoly A. , Talan Matvey S. ,   Kazan State Medical University, Dokuchaeva Irina S. ,   Kazan National Research Technological University
Published: March 2021

The article presents the results of biotechnological studies of insects used in fish feed by antioxidant activity. The problem of raw materials of animal origin for feeding fish is an urgent problem of our time. It is aggravated by the shortage, high cost and unstable quality of substrates that are made on the basis of fish meal, meat and bone meal, blood meal, etc. Recently, there has been an increase in research around the world in the search for substitutes for animal feed when growing aquaculture. Orthopteran insects of the cricket species Acheta domesticus are of considerable practical interest as a source of protein and biologically active components necessary for the growth and development of valuable species of fish. Most of the issues related to the technology of growing crickets in artificial conditions and the production of high-quality fodder based on them, remain open today. We chose an optimal diet and identified the most favorable conditions for the breeding of insects in a mini-farm. Studied the qualitative composition of the substrate on the basis of crickets. In order to select the optimal technological regimes for the processing of raw materials on the basis of insects, the total antioxidant activity of aqueous extracts of dried crickets, Daphnia magna Straus crustaceans and Gammarus pulex was determined. For crickets, the activity was 6.288 g of rutin, for daphnia 4.219 g of rutin and for gamarus 6.519 g of rutin per 100 g of absolutely dry sample. It was established that all samples, upon drying dry samples to constant weight at 105 oС, lose the total antioxidant activity.
ELEMENTARY STUDENTS’ CREATIVE THINKING SKILLS IN SCIENCE IN THE MALUKU ISLANDS, INDONESIA
Authors: Leasa Marleny , Batlolona John Rafafy , Talakua Melvie
Published: March 2021

Learning that encourages the development of students’ creative thinking needs to be maximized since the level of primary education, including in the disadvantaged, outermost, and frontier regions that is referred to 3T areas (terdepan, terluar, tertinggal) in Indonesia is still categorized as underdeveloped that requires special attention. The main objective of this research was to diagnose students’ creative thinking skills for four components including fluency, flexibility, originality, and elaboration on students in the islands. The study was conducted on 161 students sitting in fourth grade from 6 elementary schools. The unique thing why this research was conducted because the research location was one of the Maluku Islands, which has abundant sea, air and land in terms of natural resources and is one of the areas that borders directly with Australia, so it can be predicted students’ creative thinking skills will be good. However, the analysis results report that students’ creative thinking skills were still very low and thus require comprehensive learning improvement to improve students’ creative thinking skills. It was hoped that good creative thinking skills of students will support better regional development in the future.
Using single cell/filament PCR method for study non-cultured cyanobacteria
Authors: García Abad Laura , Vondrášková Alžběta , Asencio Martínez Antonia Dolores , Kaštovský Jan , Berrendero Gómez Esther
Published: March 2021

Cyanobacteria are photosynthetic prokaryotic organisms, and they are believed to be the Earth’s oldest life form. They were responsible for forming the oxygen on the Earth and from then they have been able to adapt to a multitude of environmental conditions, which allowing to be present in a wide range of habitats. When it comes to perform ecological and/or taxonomic studies one of problems which we must confront is the fact of some cyanobacteria are present in the nature in a few amounts, going unnoticed. Moreover, some of them show large mucilaginous sheaths, which making difficulted its isolation and growth later in culture. All these aspects have hindered the molecular assessment of a large portion of traditional cyanobacterial taxa, as Rivularia and Stigonema, especially necessary evaluation in the taxonomic studies. Rivularia genus grows on submerged stones, rocks, and damp soils near riverbanks, often on calcareous substrates and in clear, unpolluted, and streaming or stagnant waters, but also there are several species known from marine littoral. However, Stigonema genus grows aerophytically or subaerophytically on bark of trees or wet rocks, from lowland to the alpine zone of high mountains, but also several species are known from pools, swamps and moors. In this study, several Rivularia colonies collected in the Croatian Adriatic coast and several Stigonema species from Spanish alpine terrestrial environments are being analysed. The used methodology consists of a number of stages. In the first step, the samples are washed in EDTA disodium salt solution for dissolving the associated of calcium carbonate deposits (STEP 1). For the isolation of single cells and filaments, we are using a modified single filament isolation technique for planktonic cyanobacteria applying a glass capillary under sterile conditions (Zapomělová et al. 2008) described by Mareš et al. 2015 (STEP 2). Then, an optimized protocol we are utilized for amplification of the 16S rRNA gene, the associated 16S–23S internal transcribed spacer (ITS) region and the partially the 23S rRNA gene (2,000-2,500 bp) (Berrendero et al. 2016) (STEP 3). Finally, sequences analysis will carry out to determine the phylogenetic position of the different genotypes found inside a same Rivularia colony to be able to relation them with the morphotypes observed by the microscope. In the case of the Stigonema samples, we have successfully gotten the isolation of several filaments and, at the present, we are working on the amplification the studied region. This methodology, based on a glass capillary isolation technique and a PCR protocol for amplification of specific sequences, has been applied with success in the Rivularia and Stigonema samples without the need to cultivate them. Our future research will focus on the amplification of several taxonomic marker genes at the same time.
Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification
Authors: Sander Mandy , Beermann Arne , Buchner Dominik , Elbrecht Vasco , Haase Peter , Zizka Vera , Leese Florian
Published: March 2021

Environmental DNA (eDNA) metabarcoding is a new, promising, and non-invasive method to detect biodiversity in aquatic environments. So far, it has mainly been used to screen for fish and amphibian diversity and rarely to detect macroinvertebrates. Typically, DNA metabarcoding relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. In comparison to other genes like 16S, COI has a greater taxonomic resolution and availability of an extensive reference database. Benthic stream invertebrates are of critical importance for regulatory biomonitoring, but when using universal primers on eDNA isolated from water, the number of reads and OTUs is “watered down”. This means the target taxa, macroinvertebrates, are underrepresented in comparison to other nontarget taxa, e. g. algae, bacteria, and fungi.

The aim of the project was to design an insect-specific primer, which minimizes nontarget amplification. Therefore, data from a time series of 15 months at the Kinzig (Hesse), a silica-rich low-mountain-range stream, which is part of the Rhine‑Main‑Observatory (LTER site) was generated using the universal primers BF2/BR2. With this data we identified the most abundant nontarget taxa and designed a new reverse primer (EPTDr2n) with 3’ ‐ specificity toward benthic invertebrate taxa. Primer specificity was validated in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. 20 eDNA samples from the Kinzig River and its tributaries were then used to test the new primer in situ together with primer fwhF2.

The new primer combination showed a much higher amplification of benthic invertebrates, insects in particular, than two other universal primer pairs for both, number of target reads (fwhF2/EPTDr2n: 99.6% versus BF2/BR2: 25.89% and fwhF2/fwhR2n: 39.04%; Fig. 1) and number of target species (fwhF2/EPTDr2n: 305 versus BF2/BR2: 113 and fwhF2/fwhR2n: 185). Additionally, the number of benthic invertebrate species exceeded even the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While several taxa reported, like a few trichopteran genera, flatworms, and some crustaceans, were not found, the primer shows greatly improved results for eDNA metabarcoding of benthic invertebrates(Leese et al. 2021).
A pilot for implementing environmental DNA (eDNA) based methods into environmental and biomonitoring
Authors: Laamanen Tiina , Norros Veera , Suikkanen Sanna , Tolkkinen Mikko , Vuorio Kristiina , Vihervaara Petteri , Sirkiä Päivi , Jørgensen Kirsten , Kortelainen Pirkko , Reunamo Anna , Järvinen Marko , Meissner Kristian
Published: March 2021

Environmental DNA (eDNA) and other molecular based approaches are revolutionizing the field of biomonitoring. These approaches undergo rapid modifications, and it is crucial to develop the best practices by sharing the newest information and knowledge. In our ongoing project we:

assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE;

identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring;

promote information exchange on eDNA methods and

advance future research efforts both within SYKE and with our national and international partners.

assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE;

identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring;

promote information exchange on eDNA methods and

advance future research efforts both within SYKE and with our national and international partners.

Scientific background

Well-functioning and intact natural ecosystems are essential for human well-being, provide a variety of ecosystem services and contain a high diversity of organisms. However, human activities such as eutrophication, pollution, land-use or invasive species, are threatening the state and functioning of ecosystems from local to global scale (e.g. Benateau et al. 2019; Reid et al. 2018; Vörösmarty et al. 2010). New molecular techniques in the field and in the laboratory have enabled sampling and identification of much of terrestrial, marine and freshwater biodiversity. These include environmental DNA (eDNA, e.g. Valentini et al. 2016) and bulk-sample DNA metabarcoding approaches (e.g. Elbrecht et al. 2017) and targeted RNA-based methods (e.g. Mäki and Tiirola 2018). The eDNA technique uses DNA that is released from organisms into their environment, from which a signal of organisms’ presence in the system can be obtained. For example, in aquatic ecosystems, eDNA is typically extracted from sediment or filtered water samples (e.g. Deiner et al. 2016), and this approach is distinguished from bulk DNA metabarcoding, where organisms are directly identified from e.g. complete biological monitoring samples (e.g. Elbrecht et al. 2017). Despite the demonstrated potential of environmental and bulk-sample DNA metabarcoding approaches in recent years, there are still significant bottlenecks to their routine use that need to be addressed (e.g. Pawlowski et al. 2020).

Methods and implementati
on

The project is divided into three work packages: WP1 Gathering existing knowledge, identifying knowledge gaps and proposing best practices, WP2 Roadmap to implementation and WP3 eDNA monitoring pilot. Please see more details in the Fig. 1
Aquatic biofilms can act as natural environmental DNA samplers
Authors: Rivera Sinziana , Vasselon Valentin , Rimet Frederic , Bouchez Agnès
Published: March 2021

Diatoms, macroinvertebrates and fish communities are widely used for the assessment of the ecological status of rivers and lakes. Metabarcoding studies of these communities are usually performed from “bulk” samples in the case of diatoms and macroinvertebrates; and from water samples in the case of fish.

Recent studies, suggest that aquatic biofilms can physically act as environmental catchers of environmental DNA (eDNA) (e.g. Mariani et al. 2019). Thus, we propose an alternative metabarcoding approach to study macroinvertebrates and fishes directly from this matrix.

The capacity of aquatic biofilms to catch macroinvertebrate eDNA was tested from a previous study in Mayotte Island were both biofilm samples and macroinvertebrate morphological inventories were available at same river sites (Rivera et al. 2021). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. Our results showed that both methods provided comparable structures and diversities for macroinvertebrate communities when using unassigned OTUs suggesting that macroinvertebrate DNA is present in biofilms and representative of the communities. However, after taxonomic assignment of OTUs, diversity and richness were no longer correlated. Indeed, many constraints were observed as the need for: a) more specific primers to avoid co-amplification of untargeted taxa inhabiting biofilms, b) primers targeting shorter barcodes to sequence more easily degraded eDNA that may be captured in biofilms, and c) a reference database well adapted to our tropical study sites. Finally, even if the results of this first study were encouraging, we wanted to test the biofilm approach on organisms that do not inhabit this environmental matrix in order to be able to distinguish between intra or extra-cellular DNA.

Based on these observations, a second study looking for a fish eDNA signal in aquatic biofilms was performed. Environmental biofilm and water samples were collected in parallel at littoral sites at Lake Geneva. DNA was extracted from these samples, and fish communities were targeted using a standard 12S barcode. The molecular inventories derived from the biofilm and the water samples were compared. Both methods provide comparable floristic lists, providing a novel approach for ecological studies related to fish phenology using eDNA in biofilms.

Our results open the door to the study of diatoms, macroinvertebrates and fish communities through metabarcoding from a single matrix reducing sampling efforts and costs.
RPA-based point-of-need detection assay for the invasive Mediterranean fanworm Sabella spallanzanii
Authors: Zirngibl Martin , von Ammon Ulla , Pochon Xavier , Zaiko Anastasija
Published: March 2021

The Mediterranean fanworm Sabella spallanzanii was first sighted in New Zealand in 2009 (most likely introduced via hull fouling) and has spread across multiple coastal locations. The species presents significant risks to ecological, economic, and societal values, and therefore is subject to targeted surveillance in 11 major ports and marinas, that were identified as high-risk marine biosecurity sites. Great effort and financial resources are put into bi-annual diving surveys that include removal of individuals to contain population growth and spread. In that context, sensitive molecular detection techniques gain great interest and are being increasingly tested for the fanworm detection in New Zealand and Australia. However, conventional molecular detection via PCR assays from environmental DNA (eDNA) samples requires specific laboratory resources and scientific expertise, which restricts the applicability of this approach by biosecurity practitioners or citizen scientists. In order to provide end-users with a fast, easy, and highly specific way to detect S. spallanzanii at the site of interest, a recombinase polymerase amplification assay (RPA) was designed for read-out with lateral flow detection. RPA generates amplification within 20 minutes at 39°C, which makes it possible to run assays hand-held. Because of this, RPA presents an excellent tool for the point-of-need application in targeted biosecurity surveillance. Here we present the results of the successful in silico and in vitro validation of the newly designed RPA assay for S. spallanzanii.
Diat.barcode: a DNA tool to decipher diatom communities for the evaluation environmental pressures
Authors: Rimet Frederic , Chonova Teofana , Gassiole Gilles , Kahlert Maria , Keck François , Kelly Martyn , Kulikovskiy Maxim , Mann David , Pfannkuchen Martin , Baričević Ana , Trobajo Rosa , Vasselon Valentin , Zimmermann Jonas , Wetzel Carlos , Bouchez Agnès
Published: March 2021

Diatoms (Bacillariophyta) are ubiquitous microalgae, which present a huge taxonomic diversity, changing in correlation with differing environmental conditions. This makes them excellent ecological indicators for various ecosystems and ecological problematics (ecotoxicology, biomonitoring, paleo-environmental reconstruction …).

Current standardized methodologies for diatoms are based on microscopic determinations, which is time consuming and prone to identification uncertainties. DNA metabarcoding has been proposed as a way to avoid these flaws, enabling the sequencing of a large quantity of barcodes from natural samples. A taxonomic identity is given to these barcodes by comparing their sequences to a barcoding reference library. However, to identify environmental sequences correctly, the reference database should contain a representative number of reference sequences to ensure a good coverage of diatom diversity. Moreover, the reference database needs to be carefully taxonomically curated by experts, as its content has an obvious impact on species detection.

Diat.barcode is an open-access library for diatoms linking diatom taxonomic identities to rbcL barcode sequences (a chloroplast marker suitable for species-level identification of diatoms), which has been maintained since 2012. Data are accumulated from three sources: (1) the NCBI nucleotide database, (2) unpublished sequencing data of culture collections and more recently (3) environmental sequences. Since 2017, an international network of experts in diatom taxonomy curate this library. The last version of the database (version 9.2), includes 8066 entries that correspond to more than 280 different genera and 1490 different species. In addition to the taxonomic information, morphological features (e.g. biovolumes, chloroplasts, etc.), life-forms (mobility, colony-type) and ecological features (taxa preferences to pollution) are given.

The database can be downloaded from the website (www6.inrae.fr/carrtel-collection/Barcoding-database/) or directly through the R package diatbarcode. Ready-to-use files for commonly used metabarcoding pipelines (Mothur and DADA2) are also available.
How many Non-Indigenous Species occurring in the Mediterranean Sea can actually be identified through environmental DNA? A gap analysis of NIS DNA barcodes in the reference libraries
Authors: Pinna Maurizio , Zangaro Francesco , Tzafesta Eftychia , Specchia Valeria
Published: March 2021

The Mediterranean Sea is known for hosting a high diversity of species, with more than 17,000 reported marine species, one fifth which are endemic. Moreover, the number of non-indigenous species (NIS) in the Mediterranean Sea is recently reported to be increasing. The monitoring and the distribution of NIS is a key requirement for the conservation and management of the ecosystems, as also foreseen by the regulation (EU) No 1143/2014 of the European Parliament and of the Council of 22 October 2014 on the prevention and management of the introduction and spread of invasive alien species. A promising innovative approach for the identification of species and biodiversity is the use of molecular tools (DNA barcoding and metabarcoding). Combined with environmental DNA (eDNA) extracted from environmental samples like water or sediment, traces of species can be identified. Currently, the main limitation in the use of molecular tools (DNA barcoding and metabarcoding, eDNA) for species identification is the incompleteness of the DNA barcode reference databases. We assessed the extent of gaps in DNA barcode reference libraries, using the most updated inventory of 666 confirmed alien species occurring in the Mediterranean Sea. Also, the availability of primers for the amplification process was investigated. 32.58% of these species lack DNA barcodes in the reference libraries. Also, 70% of the retrieved NIS in the DNA barcode libraries lack the public availability of the primers used for the amplification process.

The results of this study allow us to direct scientific efforts towards specific taxonomic groups in order to complete the NIS DNA barcode reference libraries and enable effective application of eDNA in investigations of the occurrence and the distribution of NIS in the Mediterranean Sea, and in the first sighting of still unknown NIS.
Species sensitivity analysis as a tool for interpreting diatom metabarcoding for WFD bioassessment
Authors: Trobajo Rosa , Pérez Burillo Javier , Vasselon Valentin , Rimet Frederic , Bouchez Agnès , Mann David
Published: March 2021

Recent metabarcoding work in European rivers, including our own studies of Mediterranean rivers in Catalonia (Pérez-Burillo et al. 2020), has shown promising agreement between morphology- (LM) and DNA-based (HTS) assessments of diatom periphyton for the Water Framework Directive. However, in 10 out of the 164 Catalan sites we analysed, the ecological status class was downgraded from “Good”/ “High” with LM to “Moderate”/ “Poor”/ “Bad”by HTS. We call these the “critical” sites and they are especially important because the WFD requires remedial action to be taken by water managers for any river stretch with Moderate or lower status. In order to discover the reasons for downgrading we investigated the contribution of each species to the Indice de Polluosensibilité Spécifique [Specific Pollution-sensitivity Index, IPS] using a “leave-one-out” sensitivity analysis, paying special attention to the critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of its very weakly-silicified frustules during sample preparation for LM. Another important bias was brought about by differences between species in the rbcL copy number per cell, which for example affected the relative abundance obtained by HTS for Nitzschia inconspicua and Ulnaria ulna; these species were also identified by the sensitivity analysis as important for the WFD. However, blanket application of a correction factor based on cell volume did not lead to a clear improvement in the agreement between HTS and LM assessments. Only minor IPS discrepancies could be attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a valuable method for identifying priority species for isolation and barcoding that are currently not, or only poorly, represented in the DNA reference database.
Phytoplankton assessment and Non-Indigenous species monitoring through eDNA metabarcoding in marine ecosystems
Authors: Fernandez Sara , Miller Dulaney , Mirimin Luca
Published: March 2021

Invasive Alien Species (IAS) are one of the most contributing constituents of biodiversity loss with the distribution range of an increasing number of IAS being artificially re-shaped by human activities. Rapid and effective tools are therefore needed in order to monitor changes in biodiversity and the dispersal of IAS. Molecular tools such as environmental DNA metabarcoding are being progressively included in the tool kit for IAS monitoring, however, the literature is largely focused on metazoans markers targeting regions such as COI and 18S and scarce studies include a marker for plants or algae. Here, we have tested a marker in ribulose-1,5-bisphosphate carboxylase-oxygenase chloroplast region to assess phytoplankton through environmental DNA metabarcoding. Several sample types (low and high-volume water filtration, sediment grabs, net tows) were deployed at twelve study sites along the Irish coast to determine which technique would perform better for the targeted group. A total of eleven NIS were detected during the study. Low-volume surface water filtering exceeded in NIS detection and recovered the highest diversity with 123 species detected. High volume samplers were the ones recovering the lowest number of OTUs (N= 53). Western sites (Galway Bay) had greater diversity and the highest number of total NIS compared to eastern sites in Carlingford, Dublin and Waterford. These results have shown the need for a specific IAS surveillance and has served as a basis to implement the use of eDNA metabarcoding and algae markers in future monitoring programs.
Towards an eDNA assay for decapodiform cephalopods
Authors: Sanchez Gustavo , Fernández-Álvarez Fernando
Published: March 2021

Assessing the distribution and diversity of cephalopods using traditional approaches (standard fishing, market collection, and DNA barcoding) is time-consuming. Environmental DNA (eDNA) assays are non-invasive, fast, and can capture the diversity of the species of interest using a specific primer-set. For Cephalopods, standardization of such primers has been challenging because of a poor database of several markers, and the absence of complete mitochondrial genomes for its design. In this fast-talk, I will briefly talk about our new 87 complete mitochondrial genomes from specimens correctly identify and with museum vouchers, and how these mitogenomes promise to resolve the database limitation for eDNA assays in decapodiform cephalopods.