Sections: BIOLOGY ENVIRONMENTAL ENGINEERING Series: 9 Biology ANNALS OF THE 9 Horticulture UNIVERSITY OF CRAIOVA 9 Food produce processing technology Vol. XXIII (LIX) - 2018 9 Environmental engineering ON THE FRESHWATER TRICLADID FLATWORMS (PLATYHELMINTHES, TRICLADIDA) IN THE URBAN AREAS OF CRAIOVA (ROMANIA) – PRELIMINARY DATA Babalean Anda Felicia1* *University of Craiova, Faculty of Horticulture, e-mail: [email protected] Keywords: Polycelis tenuis, Dendrocoelum lacteum, Girardia tigrina ABSTRACT The paper presents preliminary data on the freshwater tricladid flatworms sampled in some springs and running waters of three urban areas in Craiova municipality: The Botanical Garden, The Romanescu Park and Balta Craioviţei area.The faunistic account comprises: Polycelis tenuis Ijima, 1884; Dendrocoelum lacteum (Müller, 1774) and Girardia tigrina (Girard, 1850) (an asexual population of presumable Girardia tigrina). The gross anatomy of the copulatory complex is presented and briefly discussed for P. tenuis and D. lacteum in relation with the literature. P. tenuis is for the second time reported in Romania. Short notes on the above mentioned species biology are given. INTRODUCTION Craiova is a town located in Oltenia Plain, SW Romania. The literature on the history of Craiova since the medieval period (Ciobotea et al., 1999) mentions numerous springs, watercourses, lakes and marshlands on the present territory of the city: Valea Vlăicii Brook, Stan Jianu Brook and the spring of Hagi Stan Jianu, Valea Orbeţilor Brook, Valea Episcopiei Brook, Valea Tabacilor Brook, Şerca Brook, Belcineanu Pond, Valea Fetii Brook and pool, Bibescu Pond with many springs, Valea Hanului Doctorului Brook, Craioviţa and Geanoglu pools. During the development of the city, there have been elaborated several plans of urban systematization and most watercourses have disappeared as they were integrated in the sewerage system and introduced into the underground. Lakes and marshlands were drained, sanitized, or arranged in parks and recreational areas. Consequently, from the multitude of wetlands, in the urban part of Craiova there were preserved: Jianu Brook and Jianu Spring in the Botanical Garden, Bibescu Pond and Valea Fetii Brook in Romanescu Park and Balta Craioviţei Lake. This paper is a contribution on the Tricladida flatworms in the above mentioned areas for which the author does not know any data to have been reported so far. MATERIAL AND METHODS The worms were removed from the underside of the submerged stones with a paint brush, fixed in Beauchamp 12 + 24 hours and preserved in ethanol 750. Selected specimens underwent paraffin inclusion, sagittal and frontal (horizontal) sections at 5 microns and Haematoxylin-Eosin staining. The collecting sites and moments: 275 Romanescu Park: Bridge no.1: 2.04.2017 – D. lacteum – 1 ind., 5.01.2018 – D. lacteum – 6 ind.; Bridge no. 3: 2.04.2017 – G. tigrina – 20 ind., 5.01.2018 – G. tigrina – 23 ind.; High Bridge: 2.04.2017: D. lacteum – 2 ind., P. tenuis – 2 ind. G. tigrina – 2 ind.; Spring: 2.04.2017 – D. lacteum – 1 mature + 3 youngs, P. tenuis – 18 ind.; Concrete canal: 2.04.2017 – D. lacteum – 2 ind., P. tenuis – 2 ind., G. tigrina – 4 ind., 16.04.2017 – D. lacteum – 2 ind., G. tigrina – 3 ind. Botanical Garden: Lake no. 1: 16.02.2018 – D. lacteum – 1 ind. (deteriorated during fixation); Rivulet no.1: 15.04.2016 – D. lacteum – 1 ind., P. tenuis – 10 ind., 23.03.2017 – D. lacteum – 2 ind., P. tenuis – 13 ind.; Rivulet no.2: 23.03.2017 – D. lacteum – 8 ind., P. tenuis – 3 ind.; Jianu Fountain Spring: 23.03.2017 – P. tenuis – 14 ind. Balta Craiovitei Lake: Zeus spring: 10.04.2017 – P. tenuis – 43 ind. Abbreviations for all figures: ad – adenodactyl; adb – adenodactyl bulb; adpp – adenodactyl papilla; adp – adenodactyl pore; bc – bursal canal; bh – bursal horns; cb – copulatory bursa; cm – circular musculature; com – common atrium; cod – common oviduct; eg – eosinophilic glands; ejd – ejaculatory duct; f – flagellum; fd – folds of the common atrium wall; g – gonopore; ma – male atrium; p – penis; pb – penis bulb; pp – penis papilla; sv – seminal vesicle; spv – spermiducal vesicles; spd – sperm ducts. The histological slides are deposited in author personal collection and may be lend on request. RESULTS AND DISCUSSIONS The systematic account according to Sluys et al. 2009 includes: Subord. Continenticola Carranza et al., 1998 Superfam. Planarioidea Stimpson, 1857 Fam. Planariidae Stimpson, 1857 1. Polycelis tenuis Ijima, 1884 Material examined – serial frontal and sagittal sections on 9 specimens: No. 5 – serial frontal sections on 9 slides, Botanical Garden, 15.04.2016 No. 6 – serial sagittal sections on 35 slides, Botanical Garden, 15.04.2016 No. 7 – serial sagittal sections on 31 slides, Botanical Garden, 15.04.2016 No. 12 – serial frontal sections on 5 slides, Botanical Garden, 15.04.2016 No. 9 – serial frontal sections on 4 slides, Botanical Garden, 23.03.2017 No. 10 – serial sagittal sections on 34 slides, Botanical Garden, 23.03.2017 No. 11 – serial sagittal sections on 19 slides, Botanical Garden, 23.03.2017 No. 13 – serial frontal section on 7 slides, Balta Craioviţei-Zeus spring, 10.04.2017 Pt1 – serial frontal sections on 2 slides, the Romanescu Park (spring), 2.04.2017 The reconstruction of the copulatory apparatus was done using the slides of the specimen no. 10 – Fig. 5. External morphology The general aspect of living adult specimens is presented in Fig. 1 a, b. The mature specimens in fully stretched state are up to 11 mm long and 2 mm wide. The colour of the body is dark brown to black on the dorsal side and brownish on the ventral side in the living worms. The ocular bands include the mid-dorsal part of the head, measure 1/3 of the worm length in fixed specimens and consist of 30 – 35 eyes on each band. The pharynx represents ¼ of the body length and it is located in the posterior half of the body. 276 In all specimens, part of the copulatory complex (the large common atrium) is visible in living animals as a round white area situated in the middle of the posterior third part of the body – Fig. 1 a, b. The pharynx, the copulatory complex and part of the sperm ducts are visible on the ventral side of the fixed worms – Fig. 1c. Fig. 1 Polycelis tenuis – external morphology a – specimen from Zeus spring, 10.04.2017, swimming; b – living specimens from Zeus spring, 10.04.2017, in rest state on the water surface ; c – fixed specimen from the Botanical Garden, 15.04.2016 The copulatory complex – Fig. 2, 3, 4 The sperm ducts present spermiducal vesicles on both sides of the pharynx; they enter the penis bulb separately – Fig. 2a, 4a. The general aspect of the penis is of an elongate cone. The slides of all sagittal and frontal sections show the penis consisting of a well developed muscular, almost spherical bulb and a conic-cylindrical papilla. In most cases the bulb lacks a seminal vesicle, revealing only 2 ducts dichotomically branched surrounded by musculature – Fig. 4a, corresponding to the entrance of the sperm ducts. In few specimens the penis bulb houses a small seminal vesicle – Fig. 2a, 2b. The penis papilla reveals a compartmented/folded ejaculatory duct – Fig. 2a, 2b. The common atrium appears as a large space delimited by a folded wall; the folders take the aspect of adenodactyls– Fig. 2a, 3a. The copulatory bursa is horn shaped. The bursal canal is large and opens into the common atrium. The two oviducts – Fig. 3b, 4b join into a common oviduct which runs between the bursal canal and the male atrium to open into the common atrium. The oviduct is surrounded by branched eosinophilic glands – Fig. 4b. Habitat Polycelis tenuis was found on submerged stones and sand in springs (Fântâna Jianu, Romanescu Park), concrete canals (Romanescu Park), and among vegetation in the spring of Balta Craioviţei-Zeus. Water temperature – 13-14° C in April in Romanescu Park; 18° C in Zeus spring. 277 Fig. 2 Polycelis tenuis – aspects of the copulatory complex in different specimens: a – Botanical Garden, 23.03.2017, frontal section (slide 9.3.4), bar = 200μm; b – Botanical Garden, 15.04.2016, sagittal section (slide 6.25.3), bar = 100μm Fig. 3 Polycelis tenuis – aspects of the copulatory complex in different specimens: 278 a – Botanical Garden, 23.03.2017, sagittal section (slide 10.30.2), bar = 200μm; b – Botanical Garden, 15.04.2016, sagittal section (slide 7.20.3) – the entrance of the common oviduct into the common atrium – in square Discussions The general aspect of the penis and the presence/lack of adenodacyls in P. tenuis is analysed in the English literature (Ball & Reynoldson 1981, Reynoldson & Young 2000). The folded, irregular ejaculatory duct is part of the original description in P. tenuis (Ijima, 1884, Fig. 2 – Bd.XL). This feature was also reported by Năstăsescu (1976). The lack of the seminal vesicles in most specimens might be associated with the physiological state. Instead, the dichotomy of the two vasa deferentia within the penis bulb is a mystery and lead to the idea of more than two entries (sperm ducts) into the penis bulb. The horn shaped bursa present in P. tenuis and P. nigra is discussed elsewhere (Taylor & Reynoldson 1962). Polycelis tenuis was first recorded in the E and SE of Romania - Bucharest and Dobrogea by Năstăsescu (1976). In Oltenia Plain it is the second record of this species in Romania, thus extending to west the species geographical range. Fig. 4 Polycelis tenuis – aspects of the copulatory complex in different specimens: a - Botanical Garden, 15.04.2016, frontal section (slide 5.4.2) – the dichotomy of the sperm-ducts pointed by arrows, bar = 100μm; b – Balta Craioviţei-Zeus, 10.04.2017, frontal section (slide 13.5.2), bar = 100μm 279 Fig.