VYTAUTAS MAGNUS UNIVERSITY FACULTY OF NATURAL SCIENCE DEPARTMENT OF BIOLOGY Saradha Ramesh PRELIMINARY DATA ON PLANARIA (TURBELLARIA) FROM LITHUANIA Bachelor thesis Biology and genetics study program, State Code 612C10003, Biological Sciences Supervisor: Prof. dr.Ingrida Šatkauskienė __________ __________ (signature) (date) Defense: Prof. Dr. Saulius Mickevičius ___________ _________ (signature) (date) Kaunas, 2020 Experimental work was done: During autumn semester 2019- spring semester 2020, Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University. Reviewer of the Bachelor’s thesis: Vytautas Mažeika Defence of the Bachelor’s thesis: Defence of the thesis will be held on Thursday, June 18th, 2020, at 10:00 at a remote public meeting of the Bachelor's Thesis Defense Commission, Vytautas Magnus University, Department of Biology. Address of the Department: Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos street 8, LT-44404, Kaunas, Republic of Lithuania. Bachelors’s thesis is defended by: Saradha Ramesh (Signature) Supervisor: Prof. dr.Ingrida Šatkauskienė (Signature) Head of the Department: Prof. Dr. habil. Algimantas Paulauskas (Signature) 2 TABLE OF CONTENTS SUMMARY 4 SANTRAUKA 5 INTRODUCTION 6 I. LITERATURE ANALYSIS 7 1.1 PLANARIAN BIOLOGY 7 1.1.1 ANATOMY 7 1.1.2 PHYSIOLOGY 7 1.1.3 REPRODUCTION AND LIFE HISTORY 8 1.1.4 BEHAVIOURAL BIOLOGY 9 1.2 PLANARIAN REGENERATION 10 1.3 HABITAT 11 1.4 ECONOMIC IMPORTANCE OF PLANARIANS 12 1.4.1. REGENERATION STUDIES 12 1.4.2 GERM LINE REGENERATION 13 1.4.3 ECOSYSTEM BALANCE 13 II. MATERIALS AND METHODS 15 2.1 MATERIALS AND EQUIPMENT 15 2.2 SAMPLING SITES AND METHODS 16 2.2.1 GEOGRAPHICAL LOCATIONS 16 2.2.3 COLLECTION OF TURBELLARIANS 17 2.2.4 HANDLING AND STORAGE 17 2.2.5 FIXATION AND MOUNTING 18 2.2.6 IDENTIFICATION 19 III. RESULTS AND DISCUSSIONS 20 3.1 PLANARIAN SPECIES COMPOSITION 20 3.2 NOTES ON PLANARIAN SPECIES 25 3.3 GENUS DIVERSITY AND HABITAT DISTRIBUTION 27 CONCLUSIONS 30 REFERENCES 31 3 SUMMARY Author of diploma paper: Saradha Ramesh Full title of diploma paper: Preliminary data on Planaria (Turbellaria) in Lithuania Diploma paper advisor: Assoc. Prof. dr.Ingrida Šatkauskienė Presented at: Vytautas Magnus University, Faculty of Natural Science, Kaunas, 2020 Number of pages: 35 Number of tables: 1 Number of pictures: 13 Planaria are one of the leading model organisms for many modern scientific experiments on regeneration and stem cell research. However, the distribution and diversity of these freshwater and terrestrial flatworms has not yet been established in the Baltic states. In consideration with the economic importance of planaria and the lack there of any sources on its distribution in Lithuania, this research paper focuses on different methods for sampling, collecting, fixation and identification of freshwater and terrestrial planaria from different sampling sites across Lithuania. The obtained results show the preliminary data of different planarian species, the diversity in their genus and the corresponding relationship with different habitats of rivers, lakes, ponds and streams. Analysis of anatomical and morphological features of the flatworms were required to know the planrian classification. The shape of the head, the number and position of eyes in relation with the anterior and posterior margins, the colouration of both the ventral and dorsal were the important determining factors for most genus identification as well as species differentiation. In individuals, with difficulty in identification of the exact species name, only the genus was identified. A total of six genus were identified – Polycelis, Schmidtea, Dendrocoelum, Planaria, Microplana and Phagocota. A difference in the number of individuals from each each genus was seen. Same habitat as river Sventoji had three different genus of planaria, coexisting at the same time. More number of individuals were also found in lakes than rivers. Two planarian species were also found to be invasive to Lithuania – Polycelis nigra and Micoplana terrestris. 4 SANTRAUKA Diplominio darbo autorius: Saradha Ramesh Visas diplomo darbo pavadinimas: Preliminary data on Planaria (Turbellaria) in Lithuania Diplominio darbo vadovas: Assoc. Prof. dr.Ingrida Šatkauskienė Pristatyta: Vytauto Didžiojo universitetas, Gamtos mokslų fakultetas, Kaunas, 2020 m Puslapių skaičius: 35 Lentelių skaičius: 1 Paveikslų skaičius: 13 Planarijos yra vienas iš svarbiausių modelinių organizmų moksliniuose eksperimentuose, susijusiuose su regeneracija ir kamieninių ląstelių tyrimais. Tačiau šių gėlavandenių ir sausumos plokščiųjų kirmėlių paplitimas ir įvairovė Baltijos šalyse dar nėra nustatyta. Apie Lietuvos planarijų rūšis yra tik fragmentiniai duomenys. Todėl šio darbo tikslas planarijų paieška įvairiuose vandens telkiniuose ir rastų rūšių identifikacija. Surastos planarijos buvo identikuotos iki rūšies ar genties, remiantis morfologiniais požymiais: galvos forms, akių skaičiumi ir išsidėstymu, kūno spalva. Iš viso buvo identifikuotos šešios gentys - Polycelis, Schmidtea, Dendrocoelum, Planaria, Microplana ir Phagocota. Nustatytas skirtingas rastų individų gausumas tarp kiekvienos genties. Šventosios upėje buvo rastos tris skirtingos planarijų gentys. Dvi, iš rastų planarijų rūšių, yra priskirtos invazinėms: - Polycelis nigra ir Micoplana terrestris. 5 INTRODUCTION Phylum Platyhelminthes traditionally include 4 classes- Turbellarians and 3 parasitic groups – Trematodes, Monogeneans and Cestodes. Turbellarians are free living, dorsoventrally flattened worms, found in fresh and marine water or in damp soil. These flatworms can be categorized as the simplest organisms to be triploblastic and bilateral symmetry. They are the only animals with these characters which lack a definitive anus and a body cavity or coelom. These flatworms also exhibit organ level complexity- two or more type of tissue cells combine to form an organ of specific function. Two common names for freshwater macro turbellarians are triclads and planarians. Planarians are widely used in laboratories to study simple behaviour and tissue regeneration. Planarians are best known for their regenerative abilities with astonishing capability to regenerate completely from tiny tissue pieces. Planarians are also of key interest to stem cell research and reproductive strategy dependent ageing phenomena and food-supply dependent growth. Many research is possible due to the easy maintenance of the planarians in the laboratory. Hundreds of planarian species exist worldwide. Some are known to be regeneration impaired or even entirely regeneration-deficient and life spans range from seemingly unlimited in asexual strains to a few months in few species. So, the knowledge of the Planarian diversity in a country can be used to collect them to study for more scientific importance. In this study, we focus on a free living turbellarians and try to understand their biology, distribution, importance and methods of collecting. This is applied to form a database of the planarians found in Lithuania as there are no current information on their distribution and diversity. The aim of this study – on the base of scientific literature and adapted methods to determine species diversity of planarians in some regions of Lithuania Tasks of this study: 1. To identify planarian by applying the methods for planarian sampling, storage, and fixation. 2. To analyse the collected planarians and evaluate the species composition. 6 I. LITERATURE ANALYSIS 1.1 Planarian Biology 1.1.1 Anatomy Planarians are the most primitive multicellular organisms with bilateral symmetry on earth. They have an incomplete gut with only one opening and lack coelom, which is common in higher organisms. These worms are unsegmented and are triploblastic. In other words, they arise from three germ cell layers - ectoderm, mesoderm, and endoderm (Russell-Hunter, 1968). All species lack a hard skeleton. They are generally elongated with varying thickness. Their colour, that depends on epidermal pigments, differs from nearly colourless to white or shades of red, yellow, blue, green, brown, black or a mixture of these colour. The body tends to be tapered at both ends and may have lateral flaps in the anterior cerebral region giving the appearance of a distinct head (Fig. 1) (Kobayashi et al., 2007). Fig. 1. A simple model of Dugesia (Pearl, 1903). 1.1.2 Physiology Studies have found the presence of organ level organisation in planaria (Roberts-Galbraith & Newmark, 2015).They use a variety of sensory structures to obtain environmental stimuli related to touch, pressure, chemical conditions, and light. These sensory receptors are spread across the body surface or organized into sensory complexes (Agata & Umesono, 2008). One or more pairs of primitive eye spots (ocelli) are present anteriorly in many species. They help the flatworms 7 detect light intensity but cannot form images. Statocysts are sensitive organelles present to orient their bodies. Exchange of gases occur across the body surface because no discrete respiratory organs are present. Their excretory system is protonephridia that bare similarities to the vertebrate kidneys AS as shown in Fig. 2(Scimone et al., 2011). Fig. 2. Physiology of Planaria (Accorsi et al., 2017) 1.1.3 Reproduction and Life History Most species reproduce sexually and almost all of them are hermaphroditic. Some flatworms, especially those living in temporary aquatic habitats, reproduce once per year (univoltine), while other species are multivoltine, with