Identification of species Leucochloridium paradoxum and L ......The ful nucleotidl sequencee of DNs A ribosom clustee orf Leucochloridium parado-xum Carus 183, an5 d L. perturbatum
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ПАРАЗИТОЛОГИЯ, 48, 3, 2014
УДК 576.895.122
IDENTIFICATION OF SPECIES LEUCOCHLORIDIUM PARADOXUM AND L. PERTURBATUM (TREMATODA)
The full nucleotide sequences of DNA ribosome cluster of Leucochloridium parado-xum Carus, 1835 and L. perturbatum Pojmanska, 1967 were obtained. rDNA was extracted from 40 isolates of Leucochloridium sp. and analyzed using specific primers. The intraspe-cific genetically identity of morphologically detected L. paradoxum and L. perturbatum sporocysts was proven. A noticeable interspecific divergence between L. paradoxum and L. perturbatum was indicated. Using rDNA genotyping a case of double infection of snail Succinea sp. with L. paradoxum and L. perturbatum sporocysts was detected.
В исследовании впервые полностью расшифрована нуклеотидная последовательность ри-босомного кластера ДНК трематод Leucochloridium paradoxum Carus, 1835 и L. perturbatum Pojmanska, 1967. С помощью подобранных праймеров проанализировано 40 образцов рДНК трематод, относящихся к этим видам. С использованием молекулярного генотипирования
подтверждены внутривидовая генетическая однородность этих видов и значительные межви-довые различия между ними. Подтвержден случай двойного заражения моллюсков Succinea sp. трематодами двух исследованных видов Leucochloridium.
Leucochloridium sporocysts are well known among biologists for their bro-odsacs which are surprisingly similar to insect's larvae. Vivid colored broodsacs attract different birds that eat them and get metacercariae located in them. Un-fortunately, our knowledge about these trematodes is generally based on the re-sults of old researches (Woodhead, 1935; Ginetsinskaya, 1953, 1968; Pojmans-ka, 1967). The shape and color of broodsacs are the classical species characte-ristics of Leucochloridium sp. trematodes (Pojmanska, 1962, 1967; Lewis et al., 1977; Bakke, 1980).
Few contemporary studies are mainly devoted to the applicability of modern approaches for species identification of these flukes (Bakke, 1980; Casey et al., 2003). In particular, molecular-genetic analysis of sporocysts rDNA (Zhukova et al., 2012) confirmed the objectivity of morphological criteria for identificati-on of L. paradoxum (green broodsacs) and L. perturbatum (brown broodsacs). These data allow uniquely identifying the cases of finding the green and brown broodsacs in the same snail as a result of double infection. It is almost impossib-le to separate stolons of different sporocysts, so this phenomenon was explained as intraspecific polymorphism (Wesenberg-Lund, 1931).
Despite of a high necessity of molecular-genetic approaches to identification of trematode species, and, in particular, development of genetic markers, such as rDNA, full sequence of trematodes ribosome gene cluster is not represented in current databases. The lack of systematic genetic information requires additi-onal efforts to design primers and assembles long sequences from short frag-ments. In addition, available genetic information is based on relatively small sampling. The present study, which is based on information obtained from large sample of snails, confirms objectivity of molecular-genetic criteria.
MATERIAL AND METHODS
Succinea sp. snails, the intermediate hosts of Leucochloridium spp., were collected in Vyritsa and Lyuban towns of Leningrad Oblast (Russia). Forty spo-rocysts (28 with green broodsacs and 12 with brown broodsacs) from all the snails were dissected under a stereomicroscope. Species identification of sporo-cysts was performed basing on two key morphological features: the broodsac shape and color (Pojmanska, 1967; Ginetsinskaya,1968; Bakke, 1982; Ataev et al., 2013a, b).
Trematode chromosome DNA was extracted from nuclei using a standard phenol-chloroform procedure (Maniatis et al., 1989). All DNA specimens were deposited at the DNA Bank of the Laboratory of Experimental Zoology of the Herzen State Pedagogical University of Russia.
The genetic intra- and interspecific polymorphism of rDNA nucleotide sequ-ences was investigated. Complete sequence of Leucochloridium spp. rRNA was amplified using primers designed basing on previously reported sequence infor-mation: fragments of Leucochloridium sp. rDNA cluster (Zhukova et al., 2012)
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T a b l e 1
Specific primers used in the present study
№ Direction Nucleotide sequence ( 5 ' — 3 ' ) Number of bases
1 F ATGCTCTGATGGTATGCTCGTAG 23 R TTTCCTCCGCTTAGTGATATGC 22
2 F TCCTGGTAAGTGCAAGTCATAAG 23 R CTGCACTCTTCATCGACACAC 21
3 F TACTAACATATGAGGTGCCAGATC 24 R TACTCATTGCTGGACTTAGGG 21
4 F TGATGGTAGTGTGTGTCTCGC 21 R CGTTACCCGTTATCACCATG 20
5 F CGGGTTGTTTGTGAATGCAG 20 R CTTAGCCATCAGCAGACCCAC 21
6 F GTACCACCAAGCGTTTGAGC 20 F CGGCCTAGCCGAACACATAA 20
7 R ACAGTCGTGTAAGCGGGATG 20 F GTCAGGGCATAGTGGCATGTA 21
(table 1, lines 2—4, 7), rDNA fragments from GeneBank of Leucochloridium sp. (AY258145.1) (table 1, line 1), Schistosoma japonicum (EU83570) (table 1, line 6) and Caenorhabditis elegans (X03680) (table 1, line 4). Gene Runner (http://www.generunner.com) and Primer 3 (https://www.ncbi.nlm.nih. gov/to-ols/primer-blast) were used for the primers' design. Individual specimens of tre-matodes DNA (n = 40) were used for PCRs by the standard method (Prokhoro-va et al., 2010) with modifications of specific primers annealing temperature and time of elongation.
PCR products were sequenced by genetic analyzer ABI PRISM 310 (Appli-ed Biosystems, USA) in the Laboratory of Neurogenetics of the Pavlov Institute of Physiology of the Russian Academy of Sciences. Sequences were analyzed and aligned using BioEdit (http://www.bioedit.co.uk) and Mega 3.0 (http://me-gasoftware.net/).
RESULTS AND DISCUSSION
According to morphological analysis, all sporocysts isolated from infected snails Succinea sp. were classified as L. paradoxum Carus, 1835 (green brood-sacs) and L. perturbatum Pojmanska, 1967 (brown broodsacs) (fig. 1 ,a,b, see inset). We registered the case of snail double infection: sporocysts with different morphology of broodsacs were revealed in the snail tentacles. It was impossible to separate their stolons one from another (fig. 1, b). Only sporocysts's brood-sacs were used for the molecular analysis.
Information on 7 rDNA fragments (fig. 2, see inset) that span about 5000 bp rDNA fragment was obtained. This fragment includes nucleotide sequences en-coding 18S, 5.8S and 28S rRNA sequences, external transcribed spacers 1 and 2 (ETS1 and ETS2), internal transcribed spacers 1 and 2 (ITS1 and ITS2). As the result, the nucleotide sequence of 4444 bp rDNA was received for each of 40 samples of sporocysts DNA (fig. 3).
Fig. 1. Leucochloridium paradoxum and L. perturbatum sporocysts. a — the double infected Succinea sp. mollusc. Sporosysts's broodsacks of different Leucochliridium species sprouted into tentacles of the snail; b — isolated sporocysts oiL. paradoxum and L. perturbatum intertwined one
with other by their stolons.
К с. 187
1 2 3 4 5 6 М
-«- 1500
-с- 1000
•*— 500
Fig. 2. Agarose gel electrophoresis of PCR products received using Leucochloridium rDNA region specific primers. Ordinal numbers of primers (table 1) are indicated.
Fig. 3. The alignment of rDNA sequences of Leucochloridium paradoxum and L. perturbatum. 1—1837 : fragment encoding 18S rRNA, 1838—2521 — internal transcribed spacer (ITS)l, 2522—2681 — fragment encoding 5.8S rRNA, 2682—3039 ITS2, 3040^1451 — fragment encoding 28S rRNA. Sequences of internal transcribed spacers (ITS1 and ITS2), are indicated by extra bold. Differences in nucleotide sequences are designated. The numbering system used in the alignment represents nucleotides positions in the analyzed sequen-
LPAR 2(70-TCTG-TGTGT AAGTTCAGCG GGTAATCCCA CCAGATGTTG CTATCCTACT AATCTACCAG TCATGCTTAG LPER 2(70 - TCTGATG-GT ATGCTC - GTA G ATT AT - -ТА П А - П А П А ITATTATAST AAACTACCAG TCATGCTTGG
LPAR 23 (0 -CCGCCTCAn TGTTGTTATT П А А П А С Т А П А П А С А С Т GTnAAGCTA ACTAAAnAG АТПААТТСТ LPER 23(0 - CCGCCTCATT TGTTGTTAAT TTAATTACTA ТГАТТАСАСТ GTTTAAGCTA ACTAAATTAG ATTTAATTCT
LPAR 2380-GATTTTGTTA GTTGAATAAT GGCATGCACC TGATTGCTAG TCAATTGGAC TGCATGTGAC GATCGCCTAG LPER 2380- G A T m G n A GTTGACTTAT GGCATGCAC С TGATTGCTAG TCAATTGGAC TGCATGTGAC GATCGCCTAG
LPAR 2730-CCAGCTGGCA TGATTTCCT| AATGTAGTA- Е т д П А А Т А TACATACATA TGAGGTGCCA GATCTATGGC LPER 2730-CCAGCTGGCA TGATTTCCTC AATGTAGTAT TTAATTAATA TACTAACATA TGAGGTGCCA GATCTATGGC
LPAR 2800-TCCGTCCTAA TGTATCCGGT TACAGCCAAG TCTATATTTA TTATAATATT A A A T G ^ A A ATTGCTGTA-LPER 2800-TCCATCCTAA TGTATCCGGT TACAGCCAAG TCTATATTTA TTAT --TATT AAATGATAAA ATTGCTGTAT
LPAR 2870 - JA^TGGATT A TGCTCAGGTC GTGGCTCAAT GATTTTGAAC ACGCTTGATG TTATAAT- ^ J T A T A J T ^ A J T LPER 2870-TACTGGATTA TGCTCAGGTC GTGGCTCAAT GATTTTGAAC ACGCTTGATG ТТАТААТС£Д ТАТАТТАА Т T
LPAR 3010-TG ATTTAT AT GATACCCTAA TT ATA TAT AT TATGACCCTG ACCTCGGATC AGACGTGATT ACCCGCTGAA LPER 3010 - TGATTTATAT GATACCCTAA TTATATATAT TATGACCCTG ACCTCGGATC AGACGTGATT ACCCGCTGAA
Рис. 3 (продолжение).
189
LPAR 3080-CTTAAGCATA TCACTAAGCG GAGGAAAAGA A A C T A A C C A G GATTCCCCCA G T A A C G G C G A GTGAACGGGG LPER 3080-CTTAAGCATA TCACTAAGCG GAGGAAAAGA A A C T A A C C A G GATTCCCCCA G T A A C G G C G A GTGAACGGGG
LPAR 3150- ATTAGCCCAG C A C C G A A G C C TGTGACCATT TGGTTACTAG GCAATGTGGT GTTTAGGTCA ACTTATGGCA
LPER 3150- ATTAGCCCAG C A C C G A A G C C TGTGACCATT TGGTTACTAG GCAATGTGGT GTTTAGGTCA A C H A T G G C A
LPAR 3220-TTATTGCTCTACCCTAAGTC CAGCAATGAG T A C G G C T T A C T G G A A T G G C C C A T A G A G G G T G A A A G G C C C G
LPER 3220-TTATTGCTCTACCCTAAGTC CAGCAATGAG T A C G G C T T A C T G G A A T G G C C C A T A G A G G G T G A A A G G C C C G
LPAR 3360-CCAAAATGGG TGGTAAACTC CATCTAAGGC T M A T A C T G A C A C G A G T C C G ATAGCGAACA AGTACCGTGA
LPER 3360-CCAAAATGGG TGGTAAACTC CATCTAAGGC T M A T A C T G A C A C G A G T C C G ATAGCGAACA AGTACCGTGA
LPAR 3430-GGGAAAGTTG A A A A G T A C T T T G A A G A G A G G GTAAACAGTG C G T G A A A C C G CTTAAAGGTA A A C G G G T G G A LPER 3430- GGGAAAGTTG AAAAGTACTT TGAAGAGAGG GTAAACAGTG C G T G A A A C C G CTTAAAGGTA A A C G G G T G G A
LPAR 3990-CACTAAATTG GGCCAATAGT CTGTGGTGTT GCGGTAGACG A T C C A C C T G A CCCGTCTTGA A A C A C G G A C C
LPER 3990-CACTAAATTG GGCCAATAGT CTGTGGTGTT GCGGTAGACG ATCCACCTGA CCCGTCTTGA A A C A C G G A C C
LPAR 4060- AAGGAGTTTA ACATGTGCGC GAGTCATTGG GCGTTACGAA A C C C A A A G G C GTAGTGAAAG TAAAGGCTTA
LPER 4060- AAGGAGTTTA ACATGTGCGC GAGTCATTGG GCGTTACGAA A C C C A A A G G C GTAGTGAAAG TAAAGGCTTA
LPAR 4130-GCTTGTCTAA GCTGTGGTGA G A T C C T G C C G TTTCTCGTGC CTGGTACCAC CAAGCGTTTG A G C G G T A G G C LPER 4130-GCTTGTCTAA GCTGTGGTGA G A T C C T G C C G TTTCTCGTGC CTGGTACCAC CAAGCGTTTG AGC GGTAGGC
LPAR 4200-GCATCACCAG CCCGTCTCAT GGTGTAGTCA TGTAACTTGTTATATGCATC A C C G G G G C G G AGCTTGAGCG LPER 4200-GCATCACCAG CCCGTCTCAT GGTGTAGTCA TGTAACTTGT TATATGCATC A C C G G G G C G G AGCTTGAGCG
LPAR 4270- TACACGTTGA G A C C C G A A A G ATGGTGAACT ATGCTTGCGC AGGTTGAAGC CAGAGGAAAC TCTGGTGGAG
LPER 4270-TACACGTTGA G A C C C G A A A G ATGGTGAACT ATGCTTGCGC AGGTTGAAGC CAGAGGAAAC TCTGGTGGAG
LPAR 4340- GACCGTAGCG ATTCTGACGT GCAAATCGAT CGTCTAACGT G A G T A T A G G G G C G A A A G A C T AATCGAACCA
LPER 4340- GACCGTAGCG ATTCTGACGT GCAAATCGAT CGTCTAACGT G A G T A T A G G G G C G A A A G A C T AATCGAACCA
LPAR 4410- TCTAGTAGCT GGTTCCCTCC GAAGTTTCCC TCAGGATAGC A
LPER 4410- TCTAGTAGCT GGTTCCCTCC GAAGTTTCCC TCAGGATAGC A
Рис. 3 (продолжение).
190
T a b l e 2
ITS rDNAs sequences polymorphism detected among Leucochloridium paradoxum and L. perturbatum
rDNA region Deletions/insertions Substitutions One nucleotide polymorphisms
ITSl ITS2
11 17
21 7
13 5
To the best of our knowledge, the present study reports the longest nucleoti-de sequence of trematodes rRNA genes cluster. The alignment demonstrated a full intrapopulation homology of tested rDNA regions from all L. paradoxum (n = 28) sporocysts. rDNA sequences of all L. perturbatum (n = 12) sporocysts were also identical.
The differences in rDNA sequences between L. paradoxum and L. perturba-tum constituted about 1.31 %. As expected, sequences encoding 18S, 5,8S and 28S rRNA were completely (100 %) identical. The highest degree of divergence was observed among internal transcribed spacers: 9.03 and 7.21 % for the ITSl and ITS2 respectively. All identified polymorphism was analyzed; the results of this analysis are presented in table 2 and fig. 3.
As such, the use of molecular markers allows differentiation of L. parado-xum and L. perturbatum. Molecular data are congruent with morphological cri-teria (the shape and color of broodsacs), traditionally used for identification of Leucochloridium species.
Together with the sequence analysis, simultaneous presence of green and brown broodsacs (fig. 1, a) in the same snail indicates simultaneous invasion by two different trematode species (double infection): L. paradoxum and L. pertur-batum. These data also suggest a possibility of simultaneous development of different species of Leucochloridium in the same snail. These finding may exp-lain the reason why many mature broodsacs are detected sometimes in a single snail (Wesenberg-Lund, 1931).
ACKNOWLEDGMENTS
This study was financially supported by the Russian Foundation for Basic Research, project 10-04-00938-a and a Grant of the President of Russian Fede-ration (MK 2935. 2013.4).
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