Research Article Genetic Diversity of Freshwater Leeches in Lake Gusinoe (Eastern Siberia, Russia)

Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 619127, 11 pages http://dx.doi.org/10.1155/2014/619127 Research Article Genetic Diversity of Freshwater Leeches in Lake Gusinoe (Eastern Siberia, Russia) Irina A. Kaygorodova,1 Nadezhda Mandzyak,1 Ekaterina Petryaeva,1,2 and Nikolay M. Pronin3 1 Limnological Institute, 3 Ulan-Batorskaja Street, Irkutsk 664033, Russia 2 Irkutsk State University, 5 Sukhe-Bator Street, Irkutsk 664003, Russia 3 Institute of General and Experimental Biology, 6 Sakhyanova Street, Ulan-Ude 670047, Russia Correspondence should be addressed to Irina A. Kaygorodova; [email protected] Received 30 July 2014; Revised 7 November 2014; Accepted 7 November 2014; Published 27 November 2014 Academic Editor: Rafael Toledo Copyright © 2014 Irina A. Kaygorodova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The study of leeches from Lake Gusinoe and its adjacent area offered us the possibility to determine species diversity. Asa result, an updated species list of the Gusinoe Hirudinea fauna (Annelida, Clitellata) has been compiled. There are two orders and three families of leeches in the Gusinoe area: order Rhynchobdellida (families Glossiphoniidae and Piscicolidae) and order Arhynchobdellida (family Erpobdellidae). In total, 6 leech species belonging to 6 genera have been identified. Of these, 3 taxa belonging to the family Glossiphoniidae (Alboglossiphonia heteroclita f. papillosa, Hemiclepsis marginata,andHelobdella stagnalis) and representatives of 3 unidentified species (Glossiphonia sp., Piscicola sp., and Erpobdella sp.) have been recorded. The checklist gives a contemporary overview of the species composition of leeches and information on their hosts or substrates. The validity of morphological identification of each taxon has been verified by phylogenetic approach with a molecular marker adopted foraDNA barcoding of most invertebrates. 2 1. Introduction 0.34 km/km , which is comparable with its great neighbour such as Lake Baikal. The longest tributary of the lake is the LakeGusinoelocated60kmfromthefamousLakeBaikal Zagustay River. Its length and catchment area are 44 km and 2 is one of the largest freshwater bodies in the Baikal basin 382 km ,respectively(Figure 1). andthelargestintheTrans-Baikalregion.Theoriginofthis Taxonomic and ecological diversity of the lake biota has lake goes back to the period of 1740–1749 according to the been studied insufficiently and unsystematically. Thus, only data collected by the exiled Decembrist Nikolay Bestuzhev. preliminary information on the existence of 6 Hirudinea spe- The lake formed as a result of breakthrough of the Temnik cies (Erpobdella octoculata, Glossiphonia complanata, G. het- River runoff (a tributary of the Selenga) towards the Lake eroclita, Helobdella stagnalis, Hemiclepsis marginata, and Pis- Gusinoe depression and the partial filling of the depression cicola geometra) was available without the description of their via Lake Tsaidam by Tsagan-Ghol channel [1]. To date, the 2 biology and ecology [4]. Furthermore, the species identifi- water area of Lake Gusinoe is 164 km ,thevolumeofwater 3 cation was highly doubtful and needed clarification. Mean- mass is 2.4 km , the length is about 24.8 km, the width is while, leeches are an important part of aquatic biota not from5to8.5km,thecoastlineis62km,andmaximaldepth only as an element in the trophic level, but also as parasites is up to 28 m averaging 15 m [2]. The catchment area of the of other hydrobionts. Interest to Hirudinea sp. has been 2 Gusinoe basin is 924 km . It has a well-developed fluvial net. increased in recent years because of its possible relationship In total, there are 72 rivers and creeks with a total length of to transmission of bacterial and viral infections [5–9], as well 312 km [3]. The average density of the river network basin is as hematozoa including trematodes, cestodes, and nematodes 2 The Scientific World Journal Russia Zagustay River Irkutsk region Lake Baikal Buryatia Lake Gusinoe Lake Hovsgol e Mongolia Lake Gusino Lake Tsaidam Figure 1: Geographical location of the study region with indication of the main sampling sites in Lake Gusinoe, Lake Tsaidam, and Zagustay River. [10]andparasiticflagellates[11–13], which are considered piscine leeches were collected directly from the fish caught to be pathogenic organisms for aquatic animals. Moreover, with different fishing tackles such as fishing net and hydrobi- ulceration, hemorrhage, and inflammation associated with ological net or were found on aquatic vegetation (like water leech attachment sites weaken the host undoubtedly and may milfoil, pondweed, and waterweed). predispose hosts to bacterial infections. The leech specimens were directly fixed in 80% ethanol. Sequencing of particular mitochondrial genes in animals, Alcoholised leech tissues retain flexibility, making investiga- such as Folmer’s fragment of cytochrome oxidase subunit I tion of annulation and allocation of genital openings easier. (COI) gene, can yield phylogenetic information and aid in the Moreover, such material is suitable for further molecular identification of species. For leeches, DNA barcoding could analysis. be particularly useful, as their identification using stand- Morphological analysis was performed using a stereomi- ardtaxonomictechniquescanoftenbeambiguous[14]. In croscope MSP-2 var. 2 (LOMO). [15–17]. Reference spec- this study, COI sequences of leech species were obtained for imens were deposited in the zoological collection of the specimens collected in different sites of Lake Gusinoe and in Laboratory of Molecular Systematics, Limnological Institute. eastern Ukraine, resulting in the addition of 14 leech sequences to the GenBank database. Using COI sequences of closely 2.2. DNA Sequencing. Sequences of the mitochondrial cyto- related species that already existed in GenBank, phylogenetic chrome oxidase subunit I (COI) were newly generated from interrelationships were traced out. Genetic diversity was 12 specimens from the Lake Gusinoe area and two individuals revealed and evaluated. Morphological data were used to of Glossiphonia concolor were from Ukraine (Table 1). corroborate DNA identification. Total DNA was extracted from a small portion of the pos- terior sucker of the leeches according to a slightly modified 2. Material and Methods method using cetavlon [18]. This tissue was selected in order 2.1. Sample Collection. Biological material was collected dur- to avoid host blood contamination. COI gene fragments were ing expeditions to Lake Gusinoe in 2012-2013 (Figure 1). amplified with oligonucleotide primers universal for most Additional samples were collected in the adjacent water bod- invertebrates [19]using1.5 Lofprimersat10 M, 4 Lof ies, the Zagustay River flowing into the lake in its northern dNTP-mix at 10 M, 0.25 LofTaqpolymerase,5 Lbuffer 10x, 1-2 L DNA sample, and MilliQ H2Oforatotalvolume part and Lake Tsaidam adjoining to the southeastern shore of ∘ of 50 L. Reaction mixtures were preheated to 94 Cfor Lake Gusinoe and connecting with the latter by a channel. ∘ To collect leeches, we have inspected various underwater 1 min followed by 30 cycles of denaturation at 94 C(30s), ∘ ∘ objects (macrophytes, rotten wood, driftwood, snags, stones, annealing at 47 C (40 s), and extension at 72 C(80s),and ∘ etc.) to which hirudinids could be attached. In most cases, final extension at 72 C(7min). The Scientific World Journal 3 PCR fragments were sequenced at the CJSC “Syntol” Order RHYNCHOBDELLEA Blanchard, 1894 (Moscow, Russia). The homologous gene sequences of 44 Family GLOSSIPHONIIDAE Vaillant, 1890 related Hirudinea species available from previous studies were also included in the present analyses for comparative Genus Hemiclepsis Vejdowsky, 1884. purposes. GenBank accession numbers of all representative (1) Hemiclepsis marginata (Muller,¨ 1774) is as follows. species are provided in Table 1. Local host: Esociformes: Esocidae: Esox lucius Linnaeus, 1758. invertebrates. 2.3. Bioinformatic Analyses. The sequences were aligned with Locality: Lake Gusinoe, Lake Tsaidam. the ClustalW [20] using default parameters and then were verified by hand. The final dataset matrix included 70 ter- A common Palaearctic species: bloodsucker of fish, tad- minals and 709 aligned nucleotide characters. Phylogenetic poles, and amphipods. Representatives of this species were analyses were carried out using maximum likelihood (ML) numerous in the littoral part of these lakes. They were found method implemented in MEGA V6.06 [21]. All distance val- on stones and in washout from aquatic vegetation. Living ues among COI sequences were calculated in MEGA V6.06 leeches are green and brownish-green with a length of 10– using a model of molecular evolution that was considered to 12 mm and 2.5–3 mm in width. Alcohol fixed specimens describe the substitution pattern the best. Pairwise distance rapidly lose their beautiful intravital colouring. analyses were conducted using the Tajima-Nei model [22]. The differences in the composition bias among sequences Genus Helobdella Blanchard, 1876, is as follows. were considered in evolutionary comparisons [23]. The (2) One considers Helobdella stagnalis (Linnaeus, 1758). percentage of trees in which the associated taxa clustered together was estimated using 500 bootstrap replications. Ini- Local host: small

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