EFFUSE – River Landscape, Volume 2: Guidelines for Experts

PAVOL JOZEF ŠAFÁRIK UNIVERSITY IN KOŠICE Faculty of Science EFFUSE – RIVER LANDSCAPE Volume 2 Guidelines for Experts EFFUSE team of authors Košice 2025 This publication was produced with the financial support of the ENI CBC HUSKROUA program, through grant agreement HUSKROUA/1901/6.1/0075, with a financial contribution from the EU of 364,099.41€. EFFUSE – River Landscape, Volume 2: Guidelines for Experts Educational text Authors: RNDr. Ivana Slepáková, PhD. Department of Microbiology, Faculty of Science, PJ Šafárik University in Košice doc. RNDr. Andrej Mock, PhD. Department of Zoology, Faculty of Science, PJ Šafárik University in Košice RNDr. Peter Ľuptáčik, PhD. Department of Zoology, Faculty of Science, PJ Šafárik University in Košice RNDr. Natália Pipová, PhD. Department of Animal Physiology, Faculty of Science, PJ Šafárik University in Košice RNDr. Monika Balogová, PhD. Department of Zoology, Faculty of Science, PJ Šafárik University in Košice RNDr. Mariana Kolesárová, PhD. Department of Microbiology, Faculty of Science, PJ Šafárik University in Košice RNDr. Lenka Maliničová, PhD. Department of Microbiology, Faculty of Science, PJ Šafárik University in Košice prof. RNDr. Martin Bačkor, DrSc. Department of Plant Biology, Faculty of Science, PJ Šafárik University in Košice doc. RNDr. Michal Goga, PhD. Department of Plant Biology, Faculty of Science, PJ Šafárik University in Košice RNDr. Dajana Kecsey PhD. Department of Plant Biology, Faculty of Science, PJ Šafárik University in Košice doc. RNDr. Yaroslava Hasynets, PhD. Department of Botany, Faculty of Biology, Uzhhorod National University doc. RNDr. Vladyslav Mirutenko. PhD Department of Entomology and Biodiversity Conservation, Faculty of Biology, Uzhhorod National University doc. RNDr. Roman Kish, PhD. Department of Botany, Faculty of Biology, Uzhhorod National University doc. RNDr. Mykhailo Vakerych, PhD. Department of Genetics, Plant Physiology and Microbiology, Faculty of Biology, Uzhhorod National University prof. RNDr. Maryna Kryvtsova, Dr.Sc. Department of Genetics, Plant Physiology and Microbiology, Faculty of Biology, Uzhhorod National University doc. RNDr. Fedir Kurtyak, PhD. Department of Zoology, Faculty of Biology, Uzhhorod National University doc. RNDr. Alexander Mateleshko, PhD. Department of Entomology and Biodiversity Conservation, Faculty of Biology, Uzhhorod National University Editor: RNDr. Beáta Valkay, PhD. This text is published under the Creative Commons 4.0 license - CC BY-NC-SA ("Attribution-NonCommercial-ShareAlike"). The authors are responsible for the professional and linguistic aspects of this publication. The manuscript has not undergone editorial or linguistic editing. Available at: www.unibook.upjs.sk Publication date: 21.10.2025 DOI: https://doi.org/10.33542/EFF-0450-7 ISBN 978-80-574-0450-7 (e-publication) CONTENTS EFFUSE – Guidelines for Experts Why to visit a riverside? 6 Lesson and perspectives 7 Field work 8 Bioindicators – a key to landscape 10 Insects as effective bioindicators 11 Aquatic macrophytes as effective bioindicators 26 Valorization of state parameters and indicators 40 Microbiological analysis of water samples 47 Practise of scientific education 50 Olympus DSX-1000 High End Model use for observation of invertebrates 50 Olympus DSX-1000 High End Model use for observation of reptile and amphibian structures 64 Olympus DSX-1000 High End Model use for observation of algae, lichens and cyanobacteria 82 FOR EXPERTS 6 WHY TO VISIT A RIVERSIDE? River landscape ▪ a unique place, concentrating and connecting landscape phenomena and biodiversity ▪ possibility to understand more comprehensively the environment that surrounds us in space and time ▪ collection of biological data important for the future ▪ excellent destination for excursions and relaxation, but you need to prepare them ▪ our rivers and their surroundings are among the least accessible places in nature and the most forgotten ▪ our rivers and their surroundings are among the most polluted and neglected places FOR EXPERTS 7 Lessons and perspectives Figure 1 Laborec and Uzh, a wonderful, but permanently stressed rivers and environment. ▪ the need for new „literacy“ and enthusiasm in relation to the acceptance of the phenomenon of river and river landscape ▪ cross-border exchange of inspirations and wider cooperation ▪ the need for communication across social and educational layers of society ▪ perhaps we should have the courage to be pioneers in this FOR EXPERTS 8 Field work ▪ samples of animals in the littoral zone (sifting of vegetation and sediment, individual collection) ▪ 2 m2/site examined ▪ sorting of drifted shells (on the Olšava River banks) ▪ collecting of environmental data (type of habitats, pH, temperature, conduction of water) ▪ growing season ▪ incomplete data for the Laborec River (in progress) FOR EXPERTS 15 Trichoptera is a semiaquatic insects order whose larvae live mostly in flowing water bodies. They prefer stony areas of mountain rivers, where they make up the main part of the benthos. Larvae live in "caps", which are built from particles of detritus or other material from the bottom of the reservoir. Imagos live near water bodies and do not feed. Several species of Trichoptera are known from Uzh valley, which are used in bioindication. These are mostly representatives of the genera Rhyacophila, Hydropsyche, Potamophylax Figure 9 Trichoptera FOR EXPERTS 16 The main factor that directly affects most of the characteristics of reservoirs is their flow rate. Species found almost exclusively in flowing water bodies belong to rheobionts. They include the larvae of many dayflies, dragonflies, and freckles. Most of them are also stenoxybionts. Baetis beskidensis, Hydroporus ferrugineus, Orectochilus villosus, Hydraena gracilis, Limnebius truncatellus, Elmis obscura; Figure 10 Hydroporus ferrugineus Figure 11 Plecoptera FOR EXPERTS 17 Rheophiles are restricted to flowing bodies of water, while some dragonflies and water beetles are occasionally found in stagnant water. Moderate rheophiles (reoxenes) are found both in stagnant and weakly flowing water bodies. Callopteryx virgo, Haliplus lineatocollis, Platambus maculatus, Agabus undulatus, Gyrinus substriatus, Helophorus arvernicus, Anacaena globulus, Laccobius obscuratus, Helichus substriatus, Sialis lutaria, Hydropsyche ornatula; Figure 12 Callopteryx virgo Figure 13 Sialis lutaria FOR EXPERTS 18 The species composition of water beetles also depends on the period of existence of the reservoir. Telmatophiles are adapted to life in temporary reservoirs, and therefore, they have a short development cycle. After the reservoir dries up, the insects bury themselves in silt until the next reservoir is filled, while the larvae that did not have time to complete their development and die. They are found mainly in the lowland temporary open swamps. The majority of stagnophilic aquatic insects migrate to other bodies of water during this period. Coelambus confluens, Laccornis kocai, Agabus labiatus, Berosus luridus, B. signaticollis; Figure 14 Laccornis kocai FOR EXPERTS 19 Several species are stenobiont in relation to one or more chemical factors and therefore can serve as natural biological indicators. So, in places of accumulation in reservoirs of species Hydroporus ferrugineus and Limnebius truncatellus, almost always surface water exits are observed. Findings of the species Hydraena pygmaea, Ochthebius metallescens, Riolus cupreus in rivers and streams indicate that they flow over limestone rocks, and the species Hydraena excisa and H. dentipes indicate the probable absence of limestone there. At the same time, the decrease in the number or disappearance of rheobionts and rheophiles, as well as the appearance of stagnophilic forms in flowing water bodies, may indicate their organic pollution. Figure 15 Hydraena excisa FOR EXPERTS 20 The structure of the bottom of the reservoir greatly influences the formation of the insect fauna. Sometimes this factor is considered to be decisive in its influence on the fauna of aquatic organisms. In this relation several groups of aquatic insects can be distinguished. Pelophiles include species that prefer water bodies with silty bottoms. Peltodytes caesus, Haliplus obliquus, H. lineatocollis, H. fluviatilis, H. laminatus, Laccophilus hyalinus, Agabus paludosus, Ilybius fuliginosus, Rhantus notatus, Rh. bistriatus, Ochthebius flavipes, Laccobius gracilis; Figure 16 Halipus obliguus FOR EXPERTS 21 Psammophiles are species confined to water bodies with a sandy bottom: Bidessus delicatulus, Helophorus arvernicus, Laccobius albipes, L. simulatrix; Argillophiles are found mainly in water bodies with a clay bottom: Haliplus laminatus, Scarodytes halensis, Ochthebius rugulosus, O. narentinus; Petrophils are species found in reservoirs with rocky bottoms. Most rheobionts (especially species from the Hydraena genus) belong here as well. Several petrophilic species from the genus Ochthebius (O. exculptus, O. gibbosus, O. foveolatus, O. sidanus) live mainly at the water-air interface. Figure 17 Halipus obliguus FOR EXPERTS 22 The Uzh River in the village of Kamyanitsa is divided into two sections by a dam. Above the dam, the banks are steep, and the channel is widened and deepened. The entomofauna here is rather poor, insects are represented mostly by eurybionts. Below the dam, the river again acquires a mountainous character with characteristic species. FOR EXPERTS 23 The banks of the river and tributaries are inhabited by numerous insect species. FOR EXPERTS 24 The entomological fauna of the marshy meadows and old shallow rivers is one of the richest in the Uzh valley. Hyadicus transversalis FOR EXPERTS 31 Macrophytes are indicators of the peat state of a standing waters / watercourse Mesotrophic — standing waters / watercourses with an average level of primary production and a moderate content of mineral nutrients. Potamogeton perfoliatus, Myriophyllum verticillatum, Elodea canadensis, Sagittaria sagittifolia, Polygonum amphibium, Sparganum erectum, Nuphar lutea, Ceratophyllum submersum Figure 26 Polygonum amphibium Figure 27 Sagittaria sagittifolia FOR EXPERTS 32 Macrophytes are indicators of the peat state of a standing waters/ watercourse Eutrophic – standing waters / watercourses with a high level of primary production, rich in biogenic elements. Batrachium circinatum, Ceratophyllum demersum, Myriophyllum spicatum, Stuckenia pectinata, Nymphaea alba, Utricularia vulgaris, Hydrocharis morsus-rапае, Salvinia natans, Lemna minor, Spirodela polyrhiza Figure 28 Batrachium circinatum Figure 29 Hydrocharis morsus-ranae circinatum FOR EXPERTS 33 The degree of representativeness and preservation of aquatic and riverine habitats can be used to classify and assess the state of standing waters / watercourses, as well as monitor their current state. FOR EXPERTS 34 Riparian habitats in the lower part of the river Uzh already show significant diversity, however, their representativeness and degree of preservation are average or low, and their distribution is very fragmented and represented in the form of broken loci. FOR EXPERTS 35 In its natural state, the basis of the river floodplain complex at this distance was made up of willow bushes and, mainly, willow-poplar forests galleries. FOR EXPERTS 36 These forests form the willow-poplar forests of floodplains biotope, a priority for protection in the EU (Berne Convention, Resolution 4: G1.11 Riverine Salix woodland; Annex 1 Habitat Directive: 91E0*Alluvial forests with Alnus glutinosa and Fraxinus excelsior (Alno-Padion, Alnion incanae, Salicion albae)), with the participation of Salix alba, S. fragilis, Populus alba, P. nigra. FOR EXPERTS 37 The forests of the habitat in the floodplain form strips, in places up to several dozen meters wide, are well representative, their shrub and grass layers are well developed. As an example, the assessment of the representativeness and preservation of this habitat can be used as an indicator of the state of the river. FOR EXPERTS 38 The evaluation can be carried out according to two criteria universal for all biotopes: area loss - LA (estimated according to IUCN categories) and quality loss - QU (estimated according to IUCN categories), with an additional criterion of regenerability - RE. FOR EXPERTS 39 However, it will be more reliable to evaluate the habitat using parameters that take into account its specific structure and function. The values of indicators of the structure and functions of a natural habitat, specified numerically or descriptively, must be valorized on a three-level scale: FV – favourable; U1 – unfavourableinadequate; U2 – unfavourable - bad (the scale adopted by the European Commission). FOR EXPERTS 40 Valorization of state parameters and indicators of the specific structure and functions natural habitat riparian floodplain forests-galleries Parameter/ Indicator Favourable FV Unfavourableinadequate U1 Unfavourable-bad U2 Habitat area at the site It is not decreasing, it is not anthropogenically fragmented It shows a slow downward trend or is anthropogenically fragmented It shows a rapid downward trend or is highly anthropogenically fragmented Specific structure and functions Characteristic species Typical floristic combination The floristic combination is poor, but based on species typical of the riparian forest Floristic combination dominated by not riparian species, but meadow or ruderal species FOR EXPERTS 47 Microbiological analysis of water samples (near the intake of water from treatment facilities) Microbiological analysis of a water sample - taken near the drain - on nutrient media to determine the sanitary and hygienic condition FOR EXPERTS 48 Storozhniva village Kamjanista village FOR EXPERTS 49 Escherichia coli Enterococcus faecalis FOR EXPERTS 50 PRACTISE OF SCIENTIFIC EDUCATION Possibilities of Olympus DSX-1000 High End Model Olympus DSX-1000 High End Model use for observation of invertebrates FOR EXPERTS 51 BF (bright field) DF (dark field) MIX (BF + DF) OBQ (olique light) PO (polarized light) DIC (differential interference contrast) Six observation modes allow different view on biological objects: The microscope allows observation of objects in 6 different observation modes. Changing the observing mode is done by simply clicking on the selected mode on the control panel of the operating software. FOR EXPERTS 52 All observation modes can be seen at once on one screen. Thus, it is possible to easily and quickly choose the most suitable observation mode. FOR EXPERTS 53 Observation of objects in liquids or mounted into microscopic slides Different possibilities for wet and dry samples Observation of specimens embedded in medium or mounted in a slide with a coverslip is limited. It seems that the only suitable observation mode for such preparations is the dark field (DF). Amphipoda – wet sample. FOR EXPERTS 54 Observation of objects in liquids or mounted into microscopic slides Different possibilities for wet and dry samples Observation of specimens embedded in medium or mounted in a slide with a coverslip is limited. It seems that the only suitable observation mode for such preparations is the dark field (DF). Gastropoda, shell detail – dry sample. FOR EXPERTS 55 Magnification range: 20x – 7000x Suitable for observation of very small but also big organisms Lymnaea peregra The magnification range allows for an overall view of the entire object as well as a detail of small structures on the surface. There is no need to specially prepare the sample for detail photography. 20x 144x 280x Combination of streomicroscope and routine microscope, objectives must be changed manually. FOR EXPERTS 56 Amphipoda Before photographing, it is advisable to stick the object to a mat (dry samples) or cover it in a liquid with a glass plate (wet samples) to prevent movement of the sample when the microscope stage is moving during photographing. 20x 144x 280x FOR EXPERTS 63 The image stitching method makes it possible to obtain images of objects that exceed the field of view of the selected lens. At the same time, the image stitched together from several 3D images has a great depth of field. However, the light built into the objectives can cause uneven lighting in the resulting image when composing 3D images. In such cases, it is useful to use external lighting. Image stitching FOR EXPERTS 64 Olympus DSX-1000 High End Model use for observation of reptile and amphibian structures FOR EXPERTS 65 Exuvia (shed epidermis) of the snake from Colubridae family FOR EXPERTS 66 6 observation modes of a snake pileus A set of scales on the top of snake or lizard head FOR EXPERTS 67 6 observation modes of Colubrid snake scales FOR EXPERTS 68 Process of making photos of exuvia parts FOR EXPERTS 69 Detailed view of the toad warts Bombina genus FOR EXPERTS 70 6 observation modes of the warts FOR EXPERTS 71 Detailed view on the dorsal side of the toad Bombina genus The outlines of the vertebrae clearly visible FOR EXPERTS 72 6 observation modes of the dorsal side of the toad from Bombina genus FOR EXPERTS 79 6 observation modes of the turtle scutes FOR EXPERTS 80 Detailed view on the yellow spot on the belly of the Bombina toad FOR EXPERTS 81 6 observation modes of the Bombina spot on the belly FOR EXPERTS 82 Olympus DSX-1000 High End Model use for observation of algae and lichens and cyanobacteria FOR EXPERTS 83 Observation of the lichen and demonstrating the presence of the secondary metabolite parietin Xantoria parietina FOR EXPERTS 84 Qualitative evidence of the presence of the secondary metabolite parietin in lichen Parietin reacts with potassium hydroxide (KOH) to form an intense magenta-red color. Demonstration video available here. FOR EXPERTS 85 Observation of the lichens Stereocaulon FOR EXPERTS 86 Polytrychon FOR EXPERTS 87 Umbilicaria FOR EXPERTS 88 Observation of unicelular organism Cyanobacteria Synechocystys