VYTAUTAS MAGNUS UNIVERSITY FACULTY OF NATURAL SCIENCE DEPARTMENT OF BIOLOGY

Saradha Ramesh

PRELIMINARY DATA ON (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)

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TABLE OF CONTENTS

SUMMARY 4 SANTRAUKA 5 INTRODUCTION 6 I. LITERATURE ANALYSIS 7 1.1 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 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 COMPOSITION 20 3.2 NOTES ON PLANARIAN SPECIES 25 3.3 DIVERSITY AND HABITAT DISTRIBUTION 27 CONCLUSIONS 30 REFERENCES 31

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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 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 – , Schmidtea, , Planaria, 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.

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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.

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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 with these characters which lack a definitive anus and a body cavity or . 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.

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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 (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 the number of generations depending on environmental conditions. Self-fertilization is also possible in some species. For example, Mario Bennazi has exhibited auto fecundation in the species Pinguis (Benazzi , 1991). Some terrestrial species have found to lay their eggs in a capsule or in cocoons (Fig. 3) (Ducey et al., 2006). Miniature flatworms that emerge, usually develop directly into adults. A few species are ovoviviparous or have a distinct larval stage. Asexual reproduction occurs seasonally and is the only form of reproduction in a few species. This form of reproduction involves differentiation of the adult body into distinct units (zooids) followed by separation into small flatworms via transverse division of the adult body. This process is aided by the remarkable regenerative abilities of flatworms. (Calow & Woollhead, 1977). Sexually reproducing flatworms can live from a few weeks to a few months, while the life span of asexual species is theoretically indefinite.

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Fig. 3. eggs capsule on stalk (North Carolina Museum of Natural Sciences)

1.1.4 Behavioural Biology A variety of predators feed on flatworms, and they are also attacked by parasites, such as protozoans and nematodes. The predators include fish, predaceous midge larvae, beetle larvae, and other turbellarians (Young, 1973). Some marine planarians are thought to have toxins like tetrodotoxin (TTX) and others camouflage with the surroundings. On the other hand, almost all land planarians are predators and majority of them are considered invasive species and agricultural pests because one of their preferred prey are . Upon encountering an , the flatworm performs a movement called “capping” where it covers the earthworm’s head region, minimizing its escape behaviour, even in individuals significantly larger than the flatworm. Interestingly, a study by Amber Stokes revealed the presence of TTX also in the land planarians adventitium and , indicating that the flatworms use toxins both for defence and attack (Fig. 4) (Stokes et al., 2014)

Fig. 4. Bipalium attacking an earthworm (Stokes et al., 2014)

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1.2 Planarian Regeneration

Planaria are excellent model for regeneration studies. They undergo regeneration in two ways – restorative and physiological (Morgan, 1901). Planarians undergo restorative regeneration due to any type of injury. Blastema, a mass of unpigmented, differentiating cells is formed by continuous cell proliferation. Tissue damaged by injury newly forms from this blastema, producing a fully restored worm in as 1–2 weeks (Fig. 5). This process involves remodelling the pre-existing tissues and integrating them with the newly made anatomy to become original proportions and subsequently, restores function to its organs. In addition, physiological regeneration occurs to repair anatomy as it naturally ages. In the absence of an injury, planarians constantly undergo multiple levels of cell proliferation to replace old, dead tissues. They can maintain this physiological regeneration for tens of years without losing their regenerative ability or developing cancer (Sköld & Obst, 2011). This makes planarians an interesting model for aging research.

Fig. 5. Upon injury, planarians regenerate lost tissues and maintain axial polarity in 1 week; dpa = days post amputation (Wiley Online Library)

Planarians maintain their polarity during regeneration. During regeneration, adult planarians maintain the polarity of their body axes. A small piece of tissue removed from the flank of the conserves the original orientation of the anterior-posterior, dorsal-ventral, and medial-lateral axes during their regrowth. Additionally, experiments rearranging the parts of planaria in opposite direction (Santos, 1929) like transplantation of anterior tissue to posterior region led to abnormal body growth as shown in Fig.6. These results firmly suggested that planarian tissues possess some type of intrinsic positional

10 and polarity information. This could be due to epigenetics, where the planarian might have learnt to orient themselves in the right direction step by step.

Fig. 6. Experiment to study the polarity during regeneration in Planaria (Santos, 1929)

1.3 Habitat

Planarians live in a great diversity of habitats from permanent ponds, lakes, streams, and rivers to temporary pools and wetlands. Some even thrive within semiaquatic habitats such as among wet mosses at the edge of a stream, or within the film of water coating fallen leaves on a moist forest floor, or even in the capillary water within soils of grassy meadows (Steenkiste et al., 2010). Several terrestrial species have also been discovered including the planarian predator Bipalium kewense, which may grow to 30 cm in length. Shallow waters of lakes and streams support the greatest density and diversity of flatworms, but turbellarians reach the deepest parts of lakes as long as oxygen is sufficient (Kolasa & Jurek, 2001).

According to G. Kriska, planarians especially found in Europe have a characteristic distribution in the streams. This could be due to their temperature tolerances. Crenobia alpina prefers cold temperatures and have very low thermal tolerance; so, they usually live in the upper region of small European brooks. In the middle region occurs Polycelis felina, which has a moderate temperature tolerance while the lower reaches are inhabited by Dugesia gonocephala sometimes together with . They have the broadest range of thermal tolerance (Kriska, 2013).

Most flatworms are benthic, though some species can swim and steer by muscular body waves. Some species possess cilia that helps in their motility (Rompolas, Patel-King, & King, 2009). They 11 commonly rest or hunt in protected areas (e.g., under a stone) during the day and come out when it is dark. Some vernal pool species contain symbiotic algae, and so spend most of their time in the sunniest portions of the pool. R. Pearl had written a book analysing the different movements of freshwater planarians in different habitats. Flatworms typically glide across the bottom by employing cilia for propulsion through a thin layer of mucus they have laid down. Other species may creep along with leech-like movements (Pearl, 1903). Although many species live on the surface of the stream or lake bottoms, others reside interstitially within the substrate where they move among the minute sand grains (Schotz & Talbot, 2011). Under demanding environmental conditions, planarians may enter diapause (a very low metabolic state) and they can encyst the eggs as a whole fragment (Chidester, 1908).

1.4 Economic Importance of Planarians

1.4.1. Regeneration studies

Planarians are capable of profound regenerative feats dependent upon a population of self-renewing adult stem cells called neoblasts. The key features of neoblasts are their capacity for indefinite self- renewal, their totipotency and the ability of their progeny to interpret differentiation and polarity signals and correctly replace lost structures after tissue damage as shown in Fig. 7.

Regeneration in planarians offers a paradigm for understanding the molecular and cellular control. They are used as a model to study the underlying mechanisms of the repair and regeneration of animal tissues and could provide valuable insights for the safe use of stem cells to repair damaged, diseased and ageing human tissues with little or no regenerative capacities and nervous tissue regeneration (Gentile et al., 2011). Another unique aspect of planarian regeneration is that they can regenerate a functional brain from almost any tiny body fragment similar to their evolutionarily primitive brain structure (Umesono & Agata, 2009).

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Fig. 7. Neoblasts as agents of regeneration in Planaria (Stephan-Dubois, 1965)

1.4.2 Germ line regeneration Freshwater planarians, widely known for their remarkable powers of regeneration (Salo & Baguna, 2002) are well suited for studying the mechanisms by which germ cells can be induced. (Newmark, Wang, & Chong, 2008) Classic experiments showed that planarians can regenerate germ cells from body fragments entirely lacking reproductive structures, suggesting that planarian germ cells could be specified by inductive signals. Furthermore, the availability of the genome sequence of a planaria coupled with the animal’s susceptibility to systemic RNA interference (RNAi) can facilitate functional genomic analyses of germ cell development and regeneration.

1.4.3 Ecosystem balance Control of Mosquito According to research done by S. Kar and AK Aditya, planarians living in the water can act as biological controllers of mosquitoes (Kar & Aditya, 2003). Mosquitoes, especially in tropical areas act as a vector for many diseases. To control their population, many Dugesia bengalensis, an aquatic turbellarian that predates on mosquito larva were cultivated. This gave a positive result- a considerable decline in the number of mosquitoes. So, this planaria along with Bacillus thuringiensis israelensis are being used as biological controlling agents against mosquitoes (Fig. 8 and Fig. 9).

(Direct quotations from source)

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Fig. 8. Comparison of % of feeding of different types of mosquito larvae by the planarian D. bengalensis (Kar & Aditya, 2003)

Fig. 9. Dugesia feeding on mosquito larva (Glime, 2007)

Biological indicators Eutrophication is an increase in nutrients and minerals in lakes and other water bodies due to abnormal growth in algae. This can lead to the degradation of aquatic ecosystem including death of aquatic organisms. Studies have shown that few planarians can act as bioindicators (Maneti & Maneti, 2010). Planarian species like Polycelis feline act as bioindicators, determining the pollution levels in freshwater bodies. In addition, they researchers observed that some aquatic planarians are great

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predators of algae, they feed on them and keep them in check thereby preventing the above-mentioned process.

Biodiversity conservation studies Land planarians have recently been used for identification of biodiversity hotspots due to their strict ecological requirements. The population of land planarians were compared with those obtained in other studies that assessed biodiversity hotspots for taxa on a global scale. They could identify a few global hotspots of diversity that generally do not feature, or only have low rankings like in New Zealand, south-eastern Australia, and Tasmania (Sluys, 1999). Recently, the fauna of these animals is being studied to select conservation priorities in the Atlantic rainforest in Brazil (Álvarez-Presas et al,. 2014).

II. MATERIALS AND METHODS

2.1 Materials and Equipment

The following materials and equipment were used:

i. Microscope MOTIC BA 400 (2004) ii. Camera MOTIC CAM 2000 (2004) iii. Computer with Software program, “ScopeImage 9.0” (H3D) iv. Glass jars with caps v. Metallic pincers vi. Needles vii. Petri dishes viii. Glass slides ix. Glass slips x. Distilled water xi. 70 % ethanol for fixation xii. Moisture absorbent paper xiii. Steel ruler

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2.2 Sampling Sites and Methods

2.2.1 Geographical locations

Planarians were collected from 13 sample sites near freshwater bodies including 5 rivers, 2 lakes and 3 ponds. The capture of planaria were from the rivers Nevėžis river, Neris, Nemunas, Šventoji and Minija river. Different regions along the river were sampled to increase the chance of different species found in varying habitats (Kriska, 2013). Different sites along the Šventoji river flowing in Kretinga and Palanga regions, multiple sites near Neris river in Radikai region, Nevėžis riverbank of Pelėdnagiai and Minija river along Plungė regions gave a good collection of diverse planarians. Along with rivers, other running freshwater bodies were also searched including 2 different sites near Gynia stream (one near Ibėnai and another near central Kauno region), another stream near a pine forest in Palanga and canal of Beaver near Varluva , Kauno region. Lakes and ponds turned out to be the best sampling site as turbellarians prefer shallow water and still water was easier to observe than the running water of the rivers. Rocks from the bottom of the still, freshwater bodies. Also hosted staked cocoons of different species in multiple numbers were found approximately 20 cm from the surface. Lake Vistytis Lake, Lake Kastinio and Kiru pond along Kaunas – Klaipeda highway had an abundant number of planaria. A couple of planarian species were collected from ponds lying next to Dubysa river and the Nemunas river.

Fig. 10. Map of the sampling sites in Lithuania

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2.2.3 Collection of Turbellarians

Freshwater and terrestrial planarians were collected by Prof. dr. Ingrida Šatkauskienė and Jurgita Rutkauskaitė-Sucilienė (Ph.D. student of Vytautas Magnus University). The aquatic planarians were collected by technique of using a shallow bucket. First, the bucket was filled with water from the habitat being explored and then observed for any planarian species. Then, using metallic tweezers, individuals were picked carefully to not hurt them and placed on petri dishes. White plastic is usually best, as the invertebrates show up against the bright surface. It was the easiest and most cost-effective method. They were collected between March and August of 2019. Warmer period was preferred was visibility was high. During the fieldwork, different stations of the same river and lakes were scoured for planarians to increase the habitats and their respective characteristic species that occur at different parts of the river. Extreme caution was taken not to slip in slippery aquatic regions and to not get infected by ticks during the field work. Eggs of some species especially of genus Dendrocoelum and one invasive terrestrial species were collected by upturning rocks and fallen logs near the above mentioned the water bodies and looking underneath them. Since some species needs to be identified by anatomical studies along with their morphological features, it is important to collect sexually mature flatworms. Therefore, the biggest flatworm specimens possible were collected.

2.2.4 Handling and Storage

The collected turbellarians were placed in cleaned glass jars with caps. Wide containers with a flat bottom and removable lid were ideal (Fig. 11) Large and small flatworms were maintained indoors in small glass containers with perfectly clean water, well oxygenated, and around a temperature of 7– 12°C following the recommendations of Winsor (Winsor, 1998). Pond water or distilled water were preferred over tap water as it contains traces of chlorine (Kolasa & Jurek, 2001). Some species of planaria from family were identified soon after being collected. For prolonged storage up to four weeks, they can be refrigerated but the water must be recycled regularly along with the supply of food (McDonald & Jones, 2007). The feed including a mix of insect larva and small oligochaetes collected from under wet leaves were fed to the stored planarians.

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Fig. 11. Storage of the collected planarians in glass jars at 8 °C (photo from personal archive)

2.2.5 Fixation and Mounting

Planarians are very delicate and can be distorted easily. Therefore, necessary care was taken to preserve the anterior head and the posterior end which is important for taxonomic identification. If fixated wrongly, they discolour rapidly along with strong contractions (Reynoldson & Young, 2000). Many fixation methods are available like Hyman recommends placing them in small water and killing them with 2% Nitric acid followed by fixing them in 70% alcohol (Hyman, 1967) and Kenk suggests placing a sagittal section on a slide and studying them under a microscope with suitable homological stain according to the observer (Kenk,1974). For our fixation, unpigmented and some pigmented planarians mostly from family Dendrociliidae were fixated by washing them with cold distilled water in a petri dish and later placing them in 70% ethanol. The pigments were not distorted. They were then placed on glass slide with few drops of distilled water and covered with a glass slip. These fixated specimens were not fixed permanently and were discarded after their identification under the microscope. For some highly pigmented planaria, fixation was not applied as the pigments could be easily dissolved. Instead, squash mounts (Hobart, 2019) were made to hold them until identification. Squash mounts were prepared by mounting the planaria on a clean glass slide with a few drops of water. A cover slip was placed on top and the excess water was removed by pipetting allowing the pressure to secure the planaria to the slide. The squeezing pressure was adjusted by moderating the water levels. Such mounts, with appropriate pressure and focus revealed the internal organs of the flatworms. Very active individuals were placed in diluted alcohol to reduce their activity levels before mounting in the above-mentioned way. The choice of the stain depends on the researcher (Kenk,1974). But the species that we collected, did not require any stain of dye. The planarians mounted by squash mounts were still alive and were released back into a stream. 18

2.2.6 Identification

Identification of the aquatic species required both anatomical and morphological studies while for the terrestrial Microplana, just physical analysis provided results. After mounting the flatworms on glass slides, they were placed under the microscope MOTIC BA 400 (2004). They were then adjusted for focussing under objective lens of 4x magnification with further magnification to 10x if needed to observe the pharynx shape. The microscope was connected to camera MOTIC CAM 2000 (2004), which was further annexed to the computer installed with the software program ScopeImage 9.0 (H3D). Using this software, detailed images of the eyes number, distance between the eyes, the structure and branching of intestine, the distance of the intestine from anterior end, the location and size of proboscis on the dorsal surface, presence of auricles around the end and the presence of cilia if present in any species were captured after proper modifications to the light intensity and relative colour adjustments. The distance between the eyes and the distance between the eyes and body margins are the features best maintained on fixed specimens (Basquin et al., 2015).

For their morphological study, they were placed in petri dishes with distilled water. Following the guidance from (Knezović et al, 2015), the morphological features used in identification were: the coloration of both the dorsal and ventral side of the body followed by the pigment distribution, the presence of tentacles around the head if any and their location, number and position of the eyes in respect to each other and the body margin at both the anterior and posterior sides and the shape of the head. The shape of the head was the important determining factor for many aquatic species. Additionally, internal pharynx structure and shape of a penis were used in identification of Polycelis sp., along with the outer morphological feature of presence of multiple dark eye spots along the margins on their anterior end were crucial in distinguishing them. Some specimens of Dendrocoelum sp., and had more than two eyes. In specimens of genus Dendrocoelum the number of eyes feature was important for species determination (Stocchino et al., 2013). The land planarian Microplana terrestris was completely identified morphologically based on its colour on both ventral and dorsal side along with the measurement of its size.

The length and width of the body and pharynx were carefully noted down as well. The measurement was done by placing them on a plain surface with a clean and white background. They were then measured using a steel metal ruler when they were at their maximum relaxation state (complete stretch of the body). The measured specimens were immediately returned to the petri dishes with water. Additionally, the worms were observed for their movement in the petri dishes with water. While some species moved by gliding, others propelled their body by contractile motion or leech like movements like mentioned by Pearl (Pearl, 1903).

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For some individuals, the identification of exact species was found difficult. So, only their Genus was recorded.

The collected planarians were identified using the following literary identification keys: • Kriska György. (2013). Freshwater invertebrates in Central Europe: a field guide. Wien: Springer.

• Reynoldson, T. B., & Young, J. O. (2000). A key to the freshwater triclads of Britain and Ireland with notes on their ecology. Ambleside: FBA.

III. RESULTS AND DISCUSSIONS

3.1 Planarian species composition

In the given table (Table 1), data of planaria species that were collected from different parts of Lithuania is given, mentioned along with the date of capture and with photo identification of some planaria from personal archive.

Table 1. Planarian species in Lithuania

Species Photo Nr.of Locality date Notes indivi duals Genus Polycelis Polycelis nigra 1 Grybaulios 2019 05 11 pond

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Polycelis 2 Canal of 2019 05 20 Narrow canal tenius beaver beside mixed forest. (Varluva, Canal almost Kaunas outgrowed by reg.) aquatic vegetation; water flow very slowly, almost standing. Shallow.

Polycelis sp. 6 Vistytis 2017 07 22 Many black eyes Lake along the margin; (Vilkaviskis region)

Polycelis sp. 4 Kastinio 2019 05 10 Lake

Polycelis sp. 1 Gynia 2019 05 23 Flowing streams stream and standing in (Middle) many places. Deep about 15 -20 cm in sampling point beside village Ibėnai. Water semi -transparent.

Polycelis sp. 2 Minija river 2019 08 06 1adult and 1 (Plungė reg) juvenile were collected

Polycelis sp. 5 Gruslauke 2019 08 17 Size: 5.1; 5.2; 5.2; pond 4; 4; 4 mm (Kretinga region)

Polycelis sp. 1 Šventoji 2019 07 29 river (Kretinga reg.)

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Genus Schmidtea Schmidtea 3 Grybaulios 2019 05 11 3 individuals of polychora pond varying length between 11-20 mm were collected.

Schmidtea 7 Nemunas 2019 08 20 Brownish grey lugubris river with two eyes (3 individuals; size in mm: 7; 10;8) Spotted grey with two eyes (4 individuals, size in mm: 13, 10, 7; 5 )

Schmidtea 1 Neris river 2019 08 02 polychora (beside Radikiai; Kaunas reg.)

Genus Planaria Planaria torva 2 Gynia 2019 05 23 Few eggs of Stream turbellarians were (Ibėnai) found at this time on stones. Stream is narrow , about 1 m width, abundant aquatic vegetaion and whole shores are covered by plants, therefore very difficult to come close to water.

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Planaria sp. 1 Šventoji 2019 07 29 River in this River locality is about 3 (beside m width, flow Laukdvaris) slowly through the Palanga pine forest with mixture deciduas trees. Bottom sandy, covered by stones. Abundant aquatic vegetation.

Numerous Neris river 2019 07 09 t-a water: 20-22C eggs of (beside t air: 26-27C Planaria sp. Radikiai; Very warm water, Kaunas dominated Lemna reg.) sp., grass like ‚sea salad“. Bottom formed by stones.

Genus Dendrocoelum Dendrocoelum 1 Šventoji 2019 07 29 River in this lacteum River (near locality is about 3 Laukdvaris, m width, flow Palanga) slowly through the pine forest with mixture deciduas trees. Bottom sandy, covered by stones. Abundant aquatic vegetation.

Dendrocoelum 6 Vištytis 2019 07 22 Bottom covered by lacteum Lake, stones, water (Vilkaviškis transparent. About reg) 20 cm deep in point of collection.

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Dendrocoelum 9 Stream in 2019 04 19 Bottom has sand sp. pine forest and stones. Many (Palanga) submerged branches, wood. Water clean , transparent, flow rapidly and low temperature. Width of stream 1.5 m. Numerous eggs of these Genus Phagocata 1 Nevėžis 2019 07 06 Whitish, vitta river microscopic, 2 eyes. Body shape like drop

Phagocata sp. 2 Pond Kiru 2019 08 11 Dark grey with 2 (beside eyes. No clear Dubysa proboscis and gut. river, Truncate head. Ariogala in Raseiniai region) Genus Microplana Microplana 1 Pine forest 2019 03 12 Invasive to terrestris in Palanga Lihtuania

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3.2 Notes on planarian species Family: Planariidae Polycelis nigra (Muller, 1774) Diagnosis: Brownish-black flatworm of 10 mm length and 1.3 mm width. The head was almost straight frontal margin with laterally rounded edges followed by a minute narrowing of the neck. 2 distinct eyes. They also have 10 eyes distributed along the anterior edge.

Habitat: Present in very varied environments from artificial ponds to rivers, lakes, and canals.

Interspecific competition for food between triclads is a major factor driving its distribution (Reynoldson and Young, 2000). This species mostly undergoes sexual reproduction, releasing unstalked spherical cocooned eggs.

Distribution: They are found mostly in Northern European countries of United Kingdom, Austria, Germany and Sweden. They are invasive species as no prior registered occurrences were observed in Eastern Europe. In Lithuania, they were collected from from Grybalulia pond in Varana region.

Polycelis tenius (Ijima, 1884)

Diagnosis: A brownish flatworm of 7mm length and 3.3 mm width. The pigments were more non uniform than P. nigris, giving a mottled appearance. Head was truncate with two small and separated eyes. A distinct barrel shaped penis in the centre was. A row of posterior eye spots were distributed parallelly along posterior margins.

Habitat: Predominantly found in small running water bodies like streams and creeks. They were collected from a slow-moving narrow canal beside a forest. The canal was almost outgrown by aquatic vegetation and the planaria was found in the shallow part of it.

Distribution: They are present in most of Western Europe in the north up to Finland. The species is abundant also in the British Islands and Northern Italy. In the east the species has been reported in Russia and Hungary (Kriska György,2013). They were found in Canal of beaver, Varluva in Kaunas region of Lithuania.

Planaria torva (Muller, 1773)

Family: Planariidae

Description: A darkish brown, elongated flatworm of 7mm in length. The head was slightly rounded. Both ventral and dorsal surface were similarly coloured. Two eyes situated close together on either side of the midline. A highly branched gut is present along the body. 25

Habitat: They occur in diverse freshwater bodies like lakes, canals and ponds. This species was found in a narrow stream of 1-meter width. Abundant vegetation was seen surrounding the stream and along the shore. Few eggs were collected from rock surface at around 23-25ºC, that were attached to the surface with stalks.

Distribution: P. torva is known from most of Western Europe, British Islands and Scandinavia and is reported to range as far east as the river Volga in Russia (Reynoldson and Young, 2000). They were observed in Gvynia stream near Ibenai in Lithuania.

Family:

Schmidtea polychroa (Schmidt, 1861)

Description: They were reddish brown or greyish brown with protruding head. They lacked tentacles and auricles around the head. A great variability in body size from different sampling site was seen betweem 10-20 mm. They had two clear eyes and the distance between the eyes was greater than the distance and the margin. The movement was by gliding. The dorsal side was darker than the ventral side.

Habitat: Schmidtea polychroa inhabits shallow lakes and rivers. They were found gliding along the bottom surface.

Distribution: The species are spread widely across lakes, basins, and rivers across Europe. They exist from the Iberian Peninsula to Southern Sweden in the North. Many sexually active species are also found in Central Europe and Hungary (Pongratz et al. 2003). It is also present in North-Africa and has been introduced into North America. They are found in Neries river in Kauno region and Grybaulio.

Family:

Dendrocoelum lacteum (Muller, 1774)

Description: The body is elongated, milky white and 18 mm in length and 3 mm in width. The head shape is blunt with two lateral auricular lobes and an adhesive organ. The adhesive organ makes the planaria move by contractile motion. The two eyes are set very wide apart. The individual has a highly branched gut of pinkish red colour and it widely changes because of the food particles present init.

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Habitat: They live closer to the bottom of still waterbody or running water. The river that they were collected from was 3 m wide, with water flowing slowly through the pine forest with a mixture of deciduous trees. They were also collected from a transparent lake. Both the river and lake had sandy bottom, covered by stones. An abundant number of D. lacteum eggs were found on the surface of these stones.

Distribution: They are found in most of the European countries north of the Pyrenees mountain where it is widely distributed. It is also present in the British Islands. In the Mediterranean, it is present in Italy but not in Iberian Peninsula (Reynoldson and Young, 2000). Dendrocoelum is widely spread across Lithuania, especially the species D. lacteum is seen in river Sventoji and Lake Vystitis.

Family:

Microplana terrestris (Muller, 1773)

Description: A darkish grey with a blunt anterior end. Has a shiny texture to it. It is of 1.5 cm in length. It is dorso-ventrally flattened, soft bodies and unsegmented and creeps along with the whole of its ventral surface in contact with the substrate. A mucus like trail was seen as they moved.

Habitat: They are completely terrestrial and are free living. They were found in pine forest underneath a fallen branch. They grow well in moist habitats.

Distribution: It is found in Western Europe. Its range extends from Sweden in the north to the United Kingdom and Ireland and France, to Greece in the east (Sluys, 1999). So, they are invasive to Lithuania and were seen in Palanga.

(Species note on species of Schmidtea lugubris and Phagacota vitta is not presented due to insufficient data availability on its identification and distribution.)

3.3 Genus diversity and Habitat distribution

From the results, we can see a difference in the number of individuals from each genus in addition to species diversity (Fig. 12). As seen from Table 1, Polycelis sp., has the highest number of individuals followed by Dendrocoelum sp. Also, while Polycelis sp., were found in both running and still freshwater bodies, the latter were predominantly found in shallow running water bodies of rivers and 27 streams. But unlike Planaria sp., many egg cocoons of Dendrocoelum sp., were collected from the surface of rocks from the bottom of these strems. The eggs were distinguishable based on the adult form collected along with it and their cocoon colour. Planaria eggs were a darker shade than eggs of Dendrocoelum sp. Interestingly, Schmidtea and Planaria which were more abundant in Western Europe (Kriska György,2013) are comparatively lower in Europe. Multiple individuals of S. lugubris was identified from Nemunas river, the individuals identified belonging to S. polychora were more concentrated in still water body of Grybaulios pond. This could indicate their preferance of different habitats from the same biotype. Phagocata vitta collected from Nevėžis river were mature while Phagocata from pond Kiru were still in their juvenile form determined the reduced branching of pharynx, making it harder for species identification.

In general, more planarians were collected from lakes, ponds, and streams than from rivers as seen from Fig. 13. This could be due to the difficulty in catching them in flowing waters. Additionally, genus diversity was observed from a same habitat type. For example, planarians from genus Polycelis, Dendrocoelum and Planaria were found together in Sventoji river or two of them were seen coexisting together in Lake Vistytis and Gynia stream. This could reflect their behavioural biology of their similar temperature tolerance and low competition for resources between them. Only species of land planaria – Microplana terrestris, which is considered invasive to Lithuanian soil was also collected from a fallen branch of pine tree.

Genus Diversity

29% 39%

5% 2% 5% 20%

Polycelis Schmidtea Planaria Microplana Phagocata Dendrocoelum

Fig. 12. Diversity of planarian genus in Lithuania

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Habitat Distribution

12

No. Individuals No. of 7 5 3 3 4 4

1 1 2 1 2 2

KIRU

NERIS

GYNIA

MINIJA

NEVĖŽIS

VIŠTYTIS

KASTINIO

ŠVENTOJI

VARLUVA

PALANGA

NEMUNAS

GUSLAUKE GRYBAULIOS RIVER STREAM PONDS LAKE HABITAT

Fig. 13. Number of individuals collected from different habitat

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CONCLUSIONS

1. A total of six genus of free living planaria were identified in Lithuania – Dendrocoelum, Polycels, Schmidtea, Planaria, Microplana and Phagocata. 2. Dendrocoelum sp. and Polycelis sp. were found in a wide range of habitats while Schmidtea sp. were predominantly found in one habitat type of rivers. Genus diversity was also observed in habitats of Sventoji river and Lake Vistytis.. Many individuals were found more in lakes and ponds than in running water podies. Polycelis nigra and Microplana terrestris were both identified as invasive to Lithuania.

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