bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative analysis of productive qualities of Sables without gray hair and with different degrees of gray hair in JSC «Plemzverosovkhoz» Saltykovsky»
Authors:   Doctor of Аgricultural Sciences, Professor of the Department of Small Animal Husbandry, Moscow State Academy of Veterinary Medicine and Biotechnology – MVA by K. I. Skryabin, Moscow, Russian Federation, Shumilina N. N. , Larina E. E. ,   Candidate of Agricultural Sciences, Assistant of the Department of Small Animal Husbandry, Moscow State Academy of Veterinary Medicine and Biotechnology – MVA by K. I. Skryabin, Moscow, Russian Federation
Published: March 2021

The pride of Russia has always been sable. Sable is unique not only in the quality of its fur, but also in the fact that almost the entire range of its habitat is located on the territory of our country. It was in our country that this animal was first bred in captivity. In this regard, Russia has long had a monopoly on the production of sable skins. In 1969, the black sable breed was approved, and in 1998, the «Saltykov» type of sable was approved. JSC «Saltykovsky Breeding farm» has created a unique herd of dark Sables with evenly spaced gray hair, the skins of which are valued at auctions by 10–30% more expensive [2]. The article presents the results of a comparative assessment of the productive qualities of Sables without gray hair and with different degrees of gray created in JSC «Plemzverosovkhoz » Saltykovsky» livestock. As a result of the analysis, it was found that Sables with gray hair have higher bonitirovki scores (more than 50%). Analyzing the indicators of bonitirovki in males and females with different degrees of gray hair, it was found that the quality of pubescence and color of males with bright gray hair is significantly superior to individuals without gray hair, the same trend is observed in females. In addition, a positive correlation was found (0,79) between the weight of skins in grams, their length and area both in Sables without gray hair, and in individuals with different degrees of gray hair. The most common defect on skins without gray hair is soaking.
Pandemic. Global crisis. Economic state of Russia
Authors:   Academician of the Russian Academy of Sciences, Doctor of Physical and Mathematical Sciences, Scientific Director of named after P. P. Shirshov Institute of Oceanology, Member of the RAS Presidium Moscow, Russia, Nigmatulin R. I. , Nigmatulin B. I. ,   Doctor of Technical Sciences, Professor, Director General of the Institute of Energy Problems Moscow, Russia
Published: March 2021

The article considers scenarios of development of the economic state of Russia against the background of the coronavirus pandemic and the global economic crisis, which provoked a sharp decline in world prices for raw hydrocarbons and fuel.
Makna Ujaran Implisit dalam Vlog Mat Luthfi: Analisis Teori Relevans
Authors:   Universiti Islam Antarabangsa Malaysia, Nopiah Julaina , Nasrong Nur Maizatul Maisarah ,   Universiti Islam Antarabangsa Malaysia
Published: March 2021

This study researches the meanings of implicit utterances in Mat Luthfi’s vlog. This qualitative study employs relevance theory, a linguistics-based theory proposed by Sperber and Wilson drawn from the discipline of pragmatics. This theory foregrounds three main concepts comprising the role of context, cognitive effects and processing efforts. The research data consists of implicit dialogue uttered by characters in the video. Data were identified, transcribed, and subsequently analysed based on Relevance Theory. Library research was also used to further explore the implicit and Relevance Theory-based expressions. The findings show that there are ten utterances in the vlogs with implicit elements. These in turn indicate that the speaker wished to convey specific messages to listeners. This study explains that in the Malay community, utterings are various in meaning and are not merely confined to their literal meanings for correct understanding and interpretation. Hence, the meaning of an utterance is more easily understood when interpreted using the Relevance Theory.
Analisis Makna Konteks Nilai Moral Konfusianisme Ren dan Yi dalam Karya Terjemahan Hikayat Tiga Negara
Authors:   Universiti Sains Malaysia, Oo Jiu Bell , Goh Sang Seong ,   Universiti Sains Malaysia
Published: March 2021

This paper aims at investigating the contextual meaning of the Confucian moral values of “仁ˮ (ren) and “义ˮ (yi) in the situational contexts of hikayat tiga negara. Situational context in a literary text is the episode that describes an event. This story centres on the themes of the values “仁ˮ(ren) and “义ˮ (yi), together with the descriptions of these values through the practices, beliefs and behaviour of characters in the situational contexts. Previous studies have shown that the interpretation of the contextual meaning of Confucian moral values is based on the sentential contexts to convey the general contextual meaning. The other problem is the subjective interpretation of the contextual meaning of the values, based on the motives and cultural contexts of the translators. In order to fill the gap in the studies on the interpretation of the meaning of values, which is confined to sentential contexts and subjective interpretation, this study focuses on the interpretation of the contextual meaning of the values based on their original meaning in the situational contexts. The research employs the method of synchronic system-based contextual meaning analysis to interpret the synchronic contextual meaning of “仁ˮ (ren) and“义ˮ(yi). By classifying the situational contexts into general context, distinctive context and extralinguistic context, the particular contextual meaning can be identified. The findings reveal that the contextual meanings of “仁ˮ (ren) are divided into general meaning, specific meaning and extralinguistic meaning, while the contextual meanings of “义ˮ(yi) are divided into general meaning, pecific meaning, thematic meaning and interpersonal relationship-based meaning upon the classification of situational contexts. Besides this, the interaction between the intralinguistic and extralinguistic situational contexts also affects the interpretation of the contextual meaning of the values. This study presents the importance of discourse context to meaning in recreating Confucian moral values in the situational contexts of hikayat tiga negara
Real-Time Monitoring of Network Devices: Its Effectiveness in Enhancing Network Security
Authors: Ogogo Wycliffe Lamech
Published: March 2021

The business world has been significantly affected by network intrusion leading to infringement of privacy and unprecedented economic losses. Therefore, real-time monitoring of network devices is important due to the enhanced and complex network systems in organizations and associated cyber threats. Real-time monitoring provides adequate alerts and updates regarding specific networks and their performance as soon as they occur. Constant monitoring of devices also makes it possible for organizations to detect any possible challenges that the networks may be encountering. This paper examines the effectiveness of real-time monitoring of network devices in a bid to enhance network security. The study was an empirical review of recently published research papers, journals, internet sites, and books with relevant content. The findings of this study revealed that Real-time device monitoring has many potential advantages to organizations by securing their systems thereby enhancing their overall performance.
VHL expression in case of thyroid tumor pathology: connection with the prevalence of the disease, expression of transcription, growth factors and components of the AKT / m-TOR signaling pathway
Authors: Spirina Lyidmila , Chizhevskaya Svetlana , Kondakova Irina , Choynzonov Evgeny , Kovaleva Irina
Published: March 2021

Introduction. The oncosuppressor protein VHL plays a decisive or at least important role in the mechanisms of tumor progression. The development of malignant neoplasms of the thyroid gland is associated with the activation of transcriptional and growth factors. However, the role of the VHL gene in the mechanisms of the development of thyroid cancer has hardly been studied.
The study aimed to study the clinical and morphological features, expression of transcription, growth factors, and components of the AKT / m-TOR signaling pathway in patients with papillary thyroid cancer depending on the level of expression of the VHL gene.
Material and methods. The study included 46 patients with tumor pathology of the thyroid gland: 20 patients with benign thyroid tumors and 26 patients with papillary thyroid cancer T1-4N0-2M0. The expression of parameters was determined by PCR in real-time. Mutation BRAF-V600E was determined in allele-specific PCR in real-time.
Results and their discussion. The mRNA level of the VHL gene remained virtually unchanged in the tissue of papillary cancer and benign thyroid tumors. It depended on the prevalence of the disease, the defeat of regional lymph nodes, and the BRAF-V600E status. In the presence of the BRAF-V600E mutation, VHL expression increased 615 times in patients with a mutation compared with patients without this somatic mutation. It was revealed that the expression of NF-κB p65, NF-κB p50, HIF-1, HIF-2, growth factors VEGF and CAIX in patients in the group with a VHL level> 1.0 RU increased compared with patients with a VHL level <1.0 RU. A decrease in PDK, c-RAF, mTOR, 70s 6 kinase, PTEN mRNA levels were also recorded in patients with increased levels of VHL expression compared with patients with VHL expression <1.0 RU.
Conclusion. That the association of the VHL expression with tumor progression in papillary thyroid cancer and the mutant protein bRAF presence was found. Also, associations between the VHL expression and the level of mRNA of transcription and growth factors are noted.
Clinician's modern approaches to molecular genetic diagnosis of glioblastomas
Authors: Ashkhatcava Turna , Tatarinova Marina , Kogoniya Lali , Naskhletashvili David , Zhukov Vadim
Published: March 2021

The article is devoted to the issue of molecular genetic diagnosis of cerebral glioblastomas. Despite significant advances in neurooncology, little progress has been made in prolonging the life of patients with cerebral glioblastoma, and a significant part of the effectiveness of treatment depends on the recognition of two prognostic biomarkers: mutations of the isocitrate dehydrogenase (IDH) promoter and  the methylation of the O6-methylguanine methyl transferase (MGMT) promoter. The article summarizes the data of world and domestic clinical studies, allowing to supplement the histological characteristics of primary glioblastomas with genetic markers: the presence of the TERT mutation, EFGR amplification, loss of PTEN function, LOH 10q, and the presence of the BRAF mutation.
It should be noted that the amplification of EGFR, causing resistance to apoptotic stimuli and alkylating chemotherapy with Temozolomide, attracts much attention as a therapeutic target. The frequency of occurrence of the TERT mutation is 90% of all tumors of various genesis, most often the TERT mutation is found in oligodendroglioma or primary glioblastoma. Loss of heterozygosity in the region of localization of the PTEN gene is observed in many types of sporadic tumors, including more than 40% of glioblastomas. Mutations in this gene are found in tumors of the brain, endometrium, prostate, kidney, and mammary gland. The presence of a PTEN mutation is a poor prognostic factor.
LOH 22q is much more common in secondary glioblastomas (82%) than in primary glioblastomas (41%).
Among brain tumors, the BRAF mutation is most common with pleomorphic xanastrocytoma (60-70%).The BRAF V600E mutation was found in epithelioid glioblastoma, which is a rare and aggressive type of glioblastoma, characterized by an unfavorable prognosis (about 6 months) and frequent leptomeningeal spread.
Thus, knowledge of the molecular mechanisms of carcinogenesis will enable a personalized approach to treatment with glioblastomas of the brain.
Effectiveness of spinal accessory intraoperative nerve monitoring during neck dissections
Authors: Vabalayte Kristina , Romanchishen Anatoly
Published: March 2021

The aim of the study is to evaluate the efficiency and safety of intraoperative neuromonitoring (IONM) of accessory nerves during lateral lymphodissection (LLD).
Materials and Methods. Main group consisted of 63 patients with thyroid cancer (TC) with confirmed metastatic spread into cervical lymph nodes of II-V groups, and which underwent IONM of accessory nerve during LLD. Control group consisted of 60 patients in which no IONM was performed during LLD. Main group consisted of 21 males (33.3%); 42 females (66.7%): mean age 50.7 ± 8,7. Control group consisted of 11 males (18.2%); 49 females (81.8%); mean age 47.2 ± 6,8.
Results. All patients of main group responded to electric stimulation of accessory nerve during IONM. 58 (92.1%) patients did not present new symptom, whereas 4 (6.3%) noted a moderate pain while lifting and stretching the upper extremity during two weeks. In 1 (1.6%) - temporary difficulty in arm abduction up to 90° was observed. In control group, in 5 (8.3%) - damage to accessory nerve was found which manifested as lack of contraction of trapezius muscle in response to electric stimulus. In 12 (20%) - pain during lifting and abduction of upper extremity on respective side was observed up to 3-4 weeks after surgery.
Conclusions. IONM of accessory nerve is a safe and effective way to reduce probability of accessory nerve damage, which, together with improvement in organ-preserving techniques, is demonstrated by decrease of functional loss in upper extremities from 13.3% to 1.6% during the period from 2000 to 2020.
On the Calculation of the Relative Amounts of Endmember Constituents For Garnet
Authors: Hawthorne Frank C.
Published: March 2021

It is commonly accepted that the calculation of the proportions of endmember constituents in garnet is dependent on the particular sequence of calculating the amounts of the endmembers, and this belief has been used to justify avoiding the use of endmembers in the definition of a mineral species. Calculating the amounts of endmember constituents to represent a specific mineral formula involves the solution of a set of simultaneous equations, and hence the idea that the solution is dependent on the order in which the amounts of endmember constituent are determined conflicts with the meaning of the term “simultaneous equations”. Here I examine the data on which these conclusions are based and show that these sequence-dependent results arise because of the use of non-stoichiometric formulae that are not electroneutral. If a garnet formula is adjusted slightly such that it exactly fits the general formula of a garnet, [8]X3[6]Y2[4]Z3O12, and is electroneutral, the simultaneous equations relating its chemical formula to a set of endmember constituents have a single unique solution. Thus, the argument that has been used to justify avoiding the use of endmembers in the definition of a mineral species is specious.
Inquietações, dificuldades e tomadas de decisão de futuros professores que estagiaram e ensinaram matemática na EJA
Authors: Santos Alana Silva dos , Pereira dos Santos Clebison , Saturnino dos Santos Iago Ailton do Vale , Silva Lilian Aragão da
Published: March 2021

Este trabalho tem como objetivo relatar as experiências de futuros professores que ensinaram matemática em turmas da Educação de Jovens e Adultos (EJA). Em particular, essas experiências estão relacionadas às inquietações, dificuldades e tomadas de decisão desses sujeitos quando desenvolveram o estágio de observação e regência na EJA. Como resultados, relatamos inquietações, dificuldades e tomadas de decisão de futuros professores que refletiram aspectos de ordem metodológica, pedagógica ou didática, fruto de problemas inerentes à EJA, a falta de políticas públicas e o silêncio institucional. Além disso, relatamos as dificuldades de estudantes da EJA, em relação a disciplina de matemática, as quais foram enfrentadas pelos futuros professores por meio de tomadas de decisão que resultaram na forma de condução ou mediação da aula e o uso de estratégias didáticas para ensinar matemática.
Palavras-chave: Inquietações. Dificuldades. Tomadas de decisão. Ensino de matemática. EJA.
Research of nitric acid solutions neutralization process with ammonia on the model of a periodic action apparatus
Authors:   Kazan National Research Technological University, Sakharov Ilia Yu. , Makhotkin Alexsey F. ,   Kazan National Research Technological University, Sakharov Yury Yu. ,   Kazan National Research Technological University, Makhotkin Igor A. ,   Kazan National Research Technological University, Durnik Roman V. ,   Kazan National Research Technological University, Sabirov Rauf F. ,   Kazan National Research Technological University
Published: March 2021

Research of nitric acid aqueous solution neutralization process with ammonia gas were performed on model of a periodic action apparatus. This model is implemented on a fundamentally new laboratory unit for analyzing the process of absorption in a gas-liquid system and includes a measuring tank containing selected gas, which is installed vertically and a measuring container with selected liquid, which is connected to the measuring gas container from below. During the process, the numerical values of the piston position are fixed according to the measured time scale and the kinetic curves of the process are constructed, these curves are processed using known algorithms and a conclusion is made about the physicochemical (kinetic and diffusion) process parameters. Investigations have shown that the process of nitric acid aqueous solutions neutralization with ammonia practically does not depend on the concentration of nitric acid in the liquid phase and is directly proportional to the concentration of ammonia in the gas phase. Analysis of the kinetic curves, taking into account the fact that the chemical reaction between ammonia and nitric acid proceeds almost instantly, allows us to make an assumption about the mechanism of ammonia absorption by aqueous solutions of nitric acid - as a diffusion process, the limiting stage of which is the diffusion of ammonia in a thin film of gas at the phase contact surface. At the same time, growth of the phases contact surface area and the rate increase of surface renewal are the main tasks for intensifying the process. A method is proposed to intensify the process of ammonia absorption by nitric acid solutions based on using vortex apparatus with a high degree of gas turbulization and a rapid renewal of the phases contact surface.
Analytical description of the specific electric conductivity of halogenides KHal melts and its calculation for the KAt melt
Authors:   Samara State Technical University, Garkushin Ivan K. , Lavrenteva Olga V. ,   Samara State Technical University, Andreeva Yana A. ,   Samara State Technical University, Gilmanova Karina R. ,   Samara State Technical University
Published: March 2021

In this paper, the analytical description of the specific conductivity of the potassium halogenides melts KHal (Hal – F, Cl, Br, I) is presented. The analitical description is provided on dependence of the specific conductivity on the halogen order number ӕ = f(Z), the ionic radius of halogen-ion ӕ = f(r), the ionic potential ӕ = f(1/r), the electronegativity difference ӕ = f(∆χ) ((∆χ = χ (Hal) – χ(K)). The interrelation of a reduced property with an order number ӕ/Z = f(Z) is considered. According to the obtained analytical dependencies, the calculation of the value of the potassium astatide specific conductivity is given for temperatures above the melting point on 5, 10, 50, 75, 100, 150 и 200°, in literature Information for KAt absent. The calculation was carried out using comparative methods for calculating M.Kh. Karapetyan in the coordinates of "property-parameter" and "property-property." Least squares method was applied for processing the analytical description results with the choice of optimal dependencies on the maximum correlation coefficient and the minimum standard deviation. The analysis of the interrelation of the calculated numerical values with similar characteristics for NaAt и LiAt is presented. Comparison of the specific conductivity obtained numerical values of the astatide potassium melt showed good consistency with the values ӕ obtained from the straight line dependence ӕТпл+n = a∙ ӕТпл+5 (n = 10°…200°) and also with similar characteristics for lithium astatide and sodium astatide. The analytical calculation results allow to describe the temperature dependence of the potassium halogenides specific conductivity, including KAt. The calculation method can be used to describe the melts specific conductivity in the same type series of compounds of alkaline and alkaline-earth elements that make up electrolytes for chemical current sources.
The influence of functional ingredients on the physico-mechanical and operational properties of rubbers for water-swelling sealing elements
Authors:   Chuvash State University  of  I.N. Ulyanov, Egorov Evgeny N. , Ushmarin Nikolay F. ,   Chuvash State University  of  I.N. Ulyanov, Efimov Konstantin V. ,   Chuvash State University  of  I.N. Ulyanov, Sandalov Sergey I. ,   Chuvash State University  of  I.N. Ulyanov, Spiridonov Ivan S. ,   Chuvash State University  of  I.N. Ulyanov, Koltsov Nikolay I. ,   Chuvash State University  of  I.N. Ulyanov
Published: March 2021

The article investigated the effect of caoutchoucs, sevilen 11808-340, vulcanizing groups, fillers, plasticizers, directional ingredients on the physicomechanical (conditional tensile strength, elongation at break, hardness, rebound elasticity, tear resistance) and operational properties of two rubbers (changes of conditional tensile strength of rubbers after exposure to water, weight changes after aging of rubbers in a solution of citric and hydrochloric acids, changes in the volume of rubbers after exposure to water). These rubbers are developed for the manufacture of the outer and inner layers of water-swelling sealing elements (WSSE) for the oil and gas industry. It has been established that rubber for the outer layer of WSSE based on butadiene-nitrile BNKS-18AMN, isoprene SKI-3 and ethylene-propylene SKEPT-40 caoutchoucs, as well as rubber for the inner layer of WSSE based on butadiene-nitrile BNKS-18AMN, butadiene-methyl styrene SKMS-30AR and ethylene-propylene SKEPT-40 caoutchocs possess the required physical-mechanical and operational properties. It was shown that these rubbers containing a vulcanizing group sulfur + thiazole 2 MBS, sevilen 11808-340, a combination of carbon black T 900 with rosil 175, talc and Karelite MK, petroleum resin “Sibplast”, vermiculite and igloprobivnoe cloth, a combination of hydrosorbtional polyacrylamide 639, sodium polyacrylate, perlite and Kometa-R reagent are characterized by improved physicomechanical and operational properties. These rubbers can be recommended as the basis for the manufacture of the outer and inner layers of water-swellable sealing elements.
Guamá's cyber-humor in exile: Aboriginal-Afrocuban identity and politically incorrect translationese working for another Cuba
Authors:   University of Ottowa, Canada, Rodríguez Raúl E. Colón
Published: March 2021

Guamá is a graphic humor online publication and an outstanding Cuban cultural phenomenon in exile. It’s creator, Alen Lauzán Falcón, was born in Cuba in 1974, and before he managed to escape the Castros' island, he had already had a successful career in the field of graphic humor. What followed was astonishing for the whole Cuban Diaspora. Upon his arrival in Chile, where he remained, working for and inspired by the graphic humour style of The Clinic, a popular Chilean humoristic publication, he created his own online journal, adopting the name of the most known Cuban aboriginal fighter against Spanish conquistadors. Through incisive and constant “politically incorrect” humour Lauzán Falcón have been ridiculing the majority of the Castroist publications through spicy comments and upturned Cuban propaganda slogans. This became a kind of creative ‘translationese’. His efforts are significantly contributing to criticizing and redirecting the meaning of Castroist ideological indoctrination concepts – efforts enriched with a strong flavour of Cuban Aboriginal (Taíno) and Afro-Cuban humour. Lauzán Falcón aimed to show a critical perspective on Cuban affairs for Cubans, and for anyone else who can feel and understand the Cuban situation and show solidarity with the difficulties of the people living under the longest-running extreme-left-wing regime in Latin America.  In this article, I will first analyze the ‘translationese’ phenomenon from a Complexity point of view, meaning, historically and culturally rebinding of the Cuban study case to the historical antecedents of ‘translationese’. Second, I will analyze ten graphic Guamá ‘front pages’ (satiric imitations of Castroist publications), published by Lauzán Falcón between 2008 and 2014 in his eponymous blog, starting with the main banner of Guamá itself. Third, I will operate a complex rebinding of the results demonstrating that the same spirit of creative resistance that the Taíno and African slaves showed in Cuba during more than five centuries, is still in action today in Cubans’ efforts to deal with the consequences of a long-lasting extreme-left-wing regime.  A selected glossary of Guama’s Afro-Cuban words and expressions analyzed here will appear at the end of the article.
Ciliates as bioindicators of environmental pressure in a karstic river
Authors: Kulaš Antonija , Gulin Vesna , Matoničkin Kepčija Renata , Žutinić Petar , Sertić Perić Mirela , Orlić Sandi , Kajan Katarina , Stoeck Thorsten , Lentendu Guillaume , Čanjevac Ivan , Martinić Ivan , Gligora Udovič Marija
Published: March 2021

Ciliates (protozoa) are a very large and diverse group of microeukaryotes that occupy a central position in the trophic web of freshwater ecosystems. Ciliates exhibit high ecological sensitivity and have tremendous bioindicator potential, but they are largely ignored in routine biomonitoring, mainly due to limitations in morphological diagnosis, which is both time-consuming and costly. In this study, we combined molecular (Illumina sequencing of the hypervariable V9 region of the SSU rRNA gene) and morphological approaches to get a detailed insight into the periphytic ciliate community structure and function within a karst, tufa-precipitating hydrosystem (National Park Krka, Croatia). Periphyton (biofilm) was sampled from light- and dark-exposed lithified tufa/stones at four representative locations within the Krka River, including upstream, midstream, and downstream river sections. We identified hydrological parameters and saprobiological classification of the sampling locations as the main structuring factors for ciliate communities. The molecular approach showed a clear separation of sampling locations in beta diversity analyses, as confirmed by PERMANOVA test. In contrast, alpha diversity was mostly affected by light exposition of the lithified tufa/stones, as confirmed by morphological approach. Environmental conditions consistently affected ciliate community composition at all locations, resulting in clear separation of periphyton samples. Our results indicate that ciliates should be considered as important bioindicators in monitoring tufa-forming rivers and streams, and the molecular approach (V9 region) proved to be applicable because as it efficiently captured the diversity of ciliates in periphyton. Validation of such an approach would lead to significant progress in extending monitoring to a broader range of indicator organisms than those now included in standard monitoring.
eDNA metabarcoding fish in relation to the Water Framework Directive indices
Authors: Herder Jelger , Kranenbarg Jan , Valentini Alice
Published: March 2021

The Water Framework Directive (WFD) recognizes fish as a biological quality element to determine the ecological health and quality of waterbodies. Therefore fish monitoring is carried out using conventional methods like electrofishing and seine fishing following standardized protocols. This data is then used in metrics that calculate ecological quality ratios (EQR) for waterbodies. eDNA-metabarcoding could prove an alternative to this conventional WFD-monitoring by offering high sensitivity, possibly lower costs and less invasive sampling.

A large project was set up in the Netherlands together with STOWA and the waterboards to explore the possibilities to use eDNA-metabarcoding as an alternative way to carry out the WFD-monitoring for fish. In 2015 in a total of 55 WFD transects (250 meter) were sampled using eDNA-metabarcoding in parallel to the conventional WFD-monitoring. On average 60% mores species were detected with eDNA-metabarcoding. In 2016 a total of 9 whole waterbodies (small rivers/ lakes / canals) were samples in parallel. A sample strategy that mixed water collected from 3 long transects was compared to a strategy that sampled the 3 transects individually.

The results showed that the strategy with mixed water samples resulted in nearly the same results at far lower costs. Furthermore a first comparison was made between the relative abundance in eDNA sequences and the relative abundance in biomass and numbers caught. Building on this project between 2018 and 2020 a total of 73 waterbodies were sampled with eDNA-metabarcoding in parallel to conventional WFD-monitoring. Those included streams, rivers, canals, lakes and brackish waterbodies.

The final goal is to assess if eDNA-metabarcoding fish can provide similar EQR-scores compared to the conventional WFD-monitoring. This can be done by either renovating the current metrics used or to build new ones based on eDNA data.

This presentation will discuss the advantages and disadvantages of both options and gives some insight in the steps still needed for implementation of eDNA-metabarcoding methods in standardized WFD-monitoring.
A quantitative eDNA-based method to monitor spawning activity of two emblematic fish species in lake Geneva
Authors: Vautier Marine , Chardon Cécile , Goulon Chloé , Guillard Jean , Domaizon Isabelle
Published: March 2021

Anthropogenic pressures and more recently climatic change have increased the interest to study the impact of environmental changes on the key stages of fish life cycle. In lake Geneva, a deep peri-alpine lake, climate change and phosphorous level are known to have consequences on salmonid and percid populations, including key species for recreational and commercial fisheries, whose stocks are subject to significant fluctuations. To follow these stock variations, the spawning activity of European perch (Perca fluviatilis) and whitefish (Coregonus lavaretus) is monitored in this lake since several years using traditional methods, unfortunately mostly destructive or damaging (e.g. gillnetting and collection of fertilized eggs).

DNA isolated from the environment (eDNA) has been widely developed for the detection of specific species or whole biological communities, and this non-invasive method offers an alternative to conventional surveying tools. Until recently, the methods used for eDNA analysis (e.g. qPCR, metabarcoding) could be limited by their sensitivity, quantification limit or price, but the emergence of new methods, such as the droplet digital PCR (ddPCR), offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a lower cost.

Here, we show for the first time the applicability of an eDNA method to monitor the spawning activity of two fish species in a lake by using ddPCR.

During two spawning seasons for whitefish and one spawning season for European perch, water samples were collected every week from the subsurface, simultaneously to traditional monitoring sampling, and filtered through sterile cartridges. The eDNA was then extracted and analyzed using ddPCR, targeting the mitochondrial DNA of the two fish species.

The results demonstrate the efficiency of eDNA coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for whitefish and European perch in Lake Geneva. This study shows that we have reached an operational level to use this non-invasive eDNA monitoring of the spawning activity of these fish species in lakes.
Identifying genome level variation in generalist and specialist bacteria and their relationship to abiotic conditions in European freshwater ecosystems
Authors: Shah Manan , Bornemann Till , Nuy Julia , Probst Alexander , Beisser Daniela , Boenigk Jens
Published: March 2021

Microbial biodiversity is one of the increasingly studied fields, yet we are unable to accurately pinpoint the intricacies of how the biotic landscape changes with its abiotic counterpart. Every organism reacts differently to physical changes in its surrounding(Zeglin (2015)). While there are some generalist microbes that can grow in limited capacity in many different types of environment, there are also some specialists that can flourish in very specific environments(Monard et al. (2016)).

We hypothesize that both generalists and specialists have developed advanced strategies that allow them thrive under diverse conditions or in very specific niches, and that these changes should be traceable on the genomic level. We expect to see variations in factors like genome sizes, number of genes, GC content, metabolic pathways that yield growth and replication advantages in nutrient deficient niches and defense mechanisms. We also expect to identify genomic streamlining in organisms that survive in nutrient deficient environments.

We are currently studying the genetic basis of why these generalists are so universally present though in smaller numbers, and what allows the specialists to efficiently exploit the abiotic factors of their specific environment in order to flourish. To pursue this, we have sampled and sequenced metagenomes from 47 lakes spread across Europe with strongly diverging environmental conditions such as pH, temperature, organic and nutrient content, elevation, and conductivity.

We’ve identified reads from more than 9000 taxonomically unique organisms, of which 700 were identified as generalists and 1200 as specialists, based on niche width permutations. In total we have assembled 313 high quality Metagenome Assembled Genomes (MAGs). Which now allow us to get a more detailed insight into the genetic basis and specific adaptations of these organisms.
Optimization and Application of direct PCR in community metabarcoding
Authors: Roeder Nina , Schwenk Klaus
Published: March 2021

Direct PCR allows the amplification of DNA from animal or plant tissue samples without the need for DNA extraction and purification steps. For this procedure, dry tissue is homogenized, dissolved in water and subsequently amplified, thus, its successful application largely depends on the absence of PCR inhibitors. Although this method has been successfully applied in barcoding approaches of invertebrates, it has not yet been attempted in metabarcoding approaches. We used nonbiting midges (Diptera: Chironomidae) to test if amplicons produced by direct PCR could be used for next-generation sequencing. To access whether direct PCR is applicable for a variety of chironomid species, we tested 236 adult specimens randomly selected from emergence traps of an artificial pond mesocosm. We used ground tissue, corresponding to 0.1% of the specimens’ biomass, and a direct PCR protocol following Wong et al. (2014) for amplification. In total, 98 % of the samples were successfully amplified and we found a diverse community comprised of 20 different genera. In order to compare direct PCR and ´traditional´ DNA isolation-PCR, we created mock communities (14 species) and used both approaches for the amplification of a 421 bp COI fragment. After a second PCR for indexing and adapter ligation, samples were sequenced on an Illumina sequencer. We found only slightly lower recovery rates for mock communities with the direct PCR approach compared to traditional protocols. These recovery rates were further improved for both methods when an equal biomass (ca. 0.006 mg) of chironomid specimens was used. With our approach, it was possible to detect species which constituted only 1% of the entire biomass of a sample. Generally, direct PCR did not have a large effect on sequence read abundance. However, read abundance varied strongly between species. We are currently investigating whether this was caused by primer bias or an artifact of differently degraded tissue. This study is a proof of principle that the amplicons produced by direct PCR can be used for next-generation sequencing, with possible applications for future biomonitoring projects and portable laboratory technologies. We are currently using this technique to monitor a large-scale chironomid community experiment (artificial pond mesocosm facility) covering weekly samples taken over two summer half-years.
PEMA v2: addressing metabarcoding bioinformatics analysis challenges
Authors: Zafeiropoulos Haris , Pavloudi Christina , Pafilis Evangelos
Published: March 2021

Environmental DNA (eDNA) and metabarcoding have launched a new era in bio- and eco-assessment over the last years (Ruppert et al. 2019). The simultaneous identification, at the lowest taxonomic level possible, of a mixture of taxa from a great range of samples is now feasible; thus, the number of eDNA metabarcoding studies has increased radically (Deiner and 2017). While the experimental part of eDNA metabarcoding can be rather challenging depending on the special characteristics of the different studies, computational issues are considered to be its major bottlenecks. Among the latter, the bioinformatics analysis of metabarcoding data and especially the taxonomy assignment of the sequences are fundamental challenges.

Many steps are required to obtain taxonomically assigned matrices from raw data. For most of these, a plethora of tools are available. However, each tool's execution parameters need to be tailored to reflect each experiment's idiosyncrasy; thus, tuning bioinformatics analysis has proved itself fundamental (Kamenova 2020). The computation capacity of high-performance computing systems (HPC) is frequently required for such analyses. On top of that, the non perfect completeness and correctness of the reference taxonomy databases is another important issue (Loos et al. 2020).

Based on third-party tools, we have developed the Pipeline for Environmental Metabarcoding Analysis (PEMA), a HPC-centered, containerized assembly of key metabarcoding analysis tools. PEMA combines state-of-the art technologies and algorithms with an easy to get-set-use framework, allowing researchers to tune thoroughly each study thanks to roll-back checkpoints and on-demand partial pipeline execution features (Zafeiropoulos 2020).

Once PEMA was released, there were two main pitfalls soon to be highlighted by users. PEMA supported 4 marker genes and was bounded by specific reference databases. In this new version of PEMA the analysis of any marker gene is now available since a new feature was added, allowing classifiers to train a user-provided reference database and use it for taxonomic assignment. Fig. 1 shows the taxonomy assignment related PEMA modules; all those out of the dashed box have been developed for this new PEMA release. As shown, the RDPClassifier has been trained with Midori reference 2 and has been added as an option, classifying not only metazoans but sequences from all taxonomic groups of Eukaryotes for the case of the COI marker gene. A PEMA documentation site is now also available. PEMA.v2 containers are available via the DockerHub and SingularityHub as well as through the Elixir Greece AAI Service. It has also been selected to be part of the LifeWatch ERIC Internal Joint Initiative for the analysis of ARMS data and soon will be available through the Tesseract VRE.
Applications of environmental DNA methods for charophyte biodiversity
Authors: Nowak Petra , Wiebe Christina , Karez Rolf , Schubert Hendrik
Published: March 2021

The use of environmental DNA (eDNA) for qualitative species inventories offers great potential as a cost-effective tool for species identification. This requires that the target species release DNA, reference information is available and detection methods exist. Environmental DNA analyses are currently used routinely to inventory fish fauna (Wang et al. 2021), molluscs (Klymus et al. 2017) or insects (Uchida et al. 2020). For other groups, such as macrophytes, there is not much information available (Scriver et al. 2015). In plants, identifying suitable eDNA markers been much more challenging, as no single DNA region has been accepted for the purposes of barcoding.

Within this project, we assessed if stoneworts (Charophytes, Characeae) can be detected by using eDNA analysis and if it can be used to support macrophyte monitoring. Charophytes are macroscopic green algae which, because of their role as habitat engineers, are of special importance for aquatic ecosystems. Many charophyte species are bound to clean, nutrient-poor fresh and brackish waters (e.g. Melzer 1999) and are regarded bioindicators for water quality by national and international directives (e.g. Habitats Directive, EU Water Framework Directive). Being sensitive to anthropogenic pressures, a drastic decline in populations with increasing eutrophication has been reported (Sand-Jensen et al. 2017) . However, the diversity of Characeae is often underestimated due to difficulties in morphological determination, and the genetic identification of charophytes has been established only in the recent few years (e.g. Nowak et al. 2016).

We assessed the potential utility of eDNA to document the diversity of charophyte species. eDNA from a fresh water lake (Dreetzsee, Germany, 2018) and from a brackish water site (Darß-Zingst Lagoon System, Germany, 2018) was extracted from filtered or ethanol‐precipitated water samples, and we designed and tested eDNA markers based on four regions of the chloroplast genome - atpB, rbcL, psbC, and matK. Of the four regions, matK and rbcL were most likely to amplify DNA from charophyte species. Both sites exhibit a diverse charophyte flora, which we successfully could identify to species/group level by eDNA analysis.

In a current study, the developed eDNA markers are used to scrutinize the charophyte population of the Schlei estuary (Germany, Schleswig-Holstein). Since conventional monitoring can only be carried out once a year at a few sites, Characeae have not been observed in recent years, or only very sporadically. As it is not possible to survey the entire Schlei, especially due to high water turbidity, the eDNA methodology is tested to assess the presence of Characeae species.
The value of DNA barcoding in a hotspot area: an example of Rhyacophila tristis (Trichoptera) in the Western Carpathians
Authors: Bozáňová Jana , Čiampor Jr Fedor , Mamos Tomasz , Grabowski Michal , Čiamporová-Zaťovičová Zuzana
Published: March 2021

DNA barcoding has proven to be an essential tool in providing molecular tags for animal species. In addition, the value of DNA barcoding undoubtedly consists in giving information about intraspecific genetic diversity, which is of great importance for biodiversity monitoring and conservation assessments. Such data are especially valuable in case of biodiversity hot-spots. Therefore, the aim of our study was to expand the knowledge of the genetic patterns and distribution of the caddisfly Rhyacophila tristis (Trichoptera, Rhyacophilidae) population in one such biodiversity hotspot - The Western Carpathians.

The W Carpathians include rich freshwater systems of springs and streams, where molecular diversity and phylogeographic patterns of aquatic fauna are yet to be fully explored. Based on the mitochondrial DNA barcoding fragment (COI-5P) of 161 sequences, two BINs representing distinct lineages within R. tristis were identified. BIN BOLD:AAD5574 occurred in 16 localities to the west and BIN BOLD:ADL4166 in 44 localities more to the east, with contact zone in the middle of the mountain system (Fig. 1). BIN BOLD:AAD5574 occurred at a significantly narrower altitudinal interval compared to BIN BOLD:ADL4166, but we did not record significant differences in molecular diversity between BINs. Likewise, past population growth was found in both lineages. Both BINs started to expand demographically at the beginning of the Last Glacial Maximum, however BIN BOLD: ADL4166 increased its demography more sharply compared to BIN BOLD:AAD5574, moreover the effective population size of BIN BOLD:ADL4166 was much higher. BIN BOLD:ADL4166, showing a significantly wider range of altitude, has probably found higher potential for dispersal to various mountain units in the area of the W Carpathians. Our results showed also that BIN BOLD:ADL4166 is more closely related to the separate R. tristis BIN BOLD: ADL4367 recognized in Bulgaria than to the BIN BOLD:AAD5574 occurring geographically in the same mountain system. Additionally, different patterns of population expansion of BIN BOLD:ADL4166 between springs and streams were found. These differences may have occurred due to specific environmental conditions of the karstic springs, which are considered as relatively isolated aquatic habitats.

Our initial study of R. tristis phylogeography in W Carpathians opens several new important questions: Is it possible that BIN BOLD:ADL4166 is expanding from the eastern part of Europe (Bulgaria) to the colder streams in the W Carpathians during the LGM? What role do the W Carpathian springs play in maintaining the genetic diversity and sustainability of R. tristis? Could these relatively isolated aquatic habitats serve as postglacial refugia for R. tristis species? And, maybe also, are they two separate species? This contribution was partially supported by the project VEGA 2/0084/21 and VEGA 1/0127/20.