Abstract. Key words. Introduction

DOI: 10.2478/s11686-007-0032-1 © 2007 W. Stefañski Institute of Parasitology, PAS Acta Parasitologica, 2007, 52(3), 185–195; ISSN 1230-2821 Identific...
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DOI: 10.2478/s11686-007-0032-1 © 2007 W. Stefañski Institute of Parasitology, PAS Acta Parasitologica, 2007, 52(3), 185–195; ISSN 1230-2821

Identification of Gyrodactylus ectoparasites in Polish salmonid farms by PCR-RFLP of the nuclear ITS segment of ribosomal DNA (Monogenea, Gyrodactylidae) Stefañski Magdalena Rokicka1*, Jaakko Lumme2 and Marek S. Ziêtara1 1Laboratory

of Comparative Biochemistry, Biological Station, Gdañsk University, PL-80-680 Gdañsk-Sobieszewo, Poland; 2Department of Biology, University of Oulu, POB 3000, FI-90014 University of Oulu, Finland

Abstract The Gyrodactylus fauna of 274 fish taken from ten salmonid farms in Poland was sampled in 2006. Four fish species were investigated: rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta (morphs fario, lacustris, and trutta), grayling Thymallus thymallus and huchen Hucho hucho. No parasites were observed on huchen. No indications of gyrodactylosis were observed, but an unexpected parasite species diversity was found. A molecular species identification by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of ITS1 + 5.8S + ITS2 was utilized, with addition of morphometric methods. The most frequent parasite was a new record in Poland, G. teuchis. It was present in two molecular forms on brown trout and rainbow trout, which also carried G. derjavinoides and G. truttae. Three molecular forms of G. salaris/G. thymalli were found, the standard type ITS only on grayling. A heterozygous (or heterogenic) G. salaris type described earlier in Denmark was found in seven farms on rainbow trout, and a complementary homozygous clone which differs from the standard by three nucleotides, in two farms. This homozygous form has not been recorded earlier. The PCR-RFLP results were confirmed by sequencing ITS segment from representative specimens of each type and comparing them with all available salmonid-specific Gyrodactylus sequences in GenBank. The Polish fauna with seven different Gyrodactylus clones separated by PCR-RFLP was the most diverse reported in fish farms in any country so far.

Key words Monogenea, Gyrodactylus, species identification, PCR-RLFP, Polish salmonid farms

Introduction

Skóra

Gyrodactylus Nordmann, 1832 is one of the most species-rich genera of the monogenean flatworms. More than four hundred potentially valid Gyrodactylus species are described from nearly 400 hosts in a wide variety of fish families and orders, but the real species number is estimated to be more than 20 000 (Bakke et al. 2002, Harris et al. 2004). About 20 Gyrodactylus species are recorded as infesting salmonids (Ergens 1983, Malmberg 1993, Bakke et al. 2002). The most infamous Gyrodactylus salaris Malmberg, 1957 was recognized as a virulent pathogen on Atlantic salmon parr populations in Norway (Johnsen and Jensen 1991). Since the introduction of the parasite into Norway from the Baltic Basin (Johnsen and Jensen 1991, Johnsen et al. 1999, Hansen et al. 2003), the economic loss caused by Gyrodactylus salaris amounts to 480 million Euro (Hansen et al. 2005). The parasite has been recorded in 45 Norwegian rivers and 39 freshwater fish farms *Corresponding

(Mo and Norheim 2005). In 1992, gyrodactylosis was also observed among salmon parr in the Russian River Keret’ draining into the White Sea (Ieshko et al. 1993). Therefore, G. salaris is reported as a List III pathogen under the Fish Health Directive 91/67/EEC (Hudson and Hill 1993). The morphological species identification of gyrodactylids is based on the opisthaptoral hard parts (Malmberg 1970). In particular, the shape of the tiny marginal hook sickles is species specific, but unfortunately it is known that the morphology of the attachment organ is variable. Factors like temperature, host and geographical location influence the intraspecific phenotypic variation of the haptors (Malmberg 1970; Ergens 1976; Mo 1991, 1993; Harris 1988). In addition, the large number of species and small size (100 1–15 1–10 1–9 1–8 1>100 1>100 0–6 0–4 1–40 2–30 0 0 0 0–4

Intensity (range) 15 15 20 20 12 15 12 12 15 10 21 22 15 15 40 15

No. of fish

2

28 4

7 12 2 6 4 2 11 1 4

G. derjavinoides

1 1

1 3

1

standard

G. teuchis

1

31 8

34 15

2 7

4 10

cf.

3

1 3 3

G. truttae

4

thymalli

1

2

2

1 2 5

4 18

L

G. salaris

1

1

LH

Fish farms: A1-A7 – fish farms in northern Poland, B1-B3 – fish farms in southern Poland were examined from May to October 2006. Age: 0 fry, 0+ one summer old, 1 one year old, 1+ one year and summer old. L – sequence reported by Lindenstrrm et al. (2003), LH – L-homozygote.

B2 B3

B1

A7

A5 A6

Host

Fish farm

Table I. Gyrodactylus species infection on salmonids in fish farms in Poland

186 Magdalena Rokicka et al.

Ziêtara

Identification of Gyrodactylus ectoparasites in Polish salmonid farms

187

Stanis³a mal DNA, a priori identified samples of G. salaris Malmberg, 1957, G. truttae Gläser, 1974 and European isolate of G. derjavini sensu Malmberg (later G. derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007) were readily separated. At present, the internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal RNA genes are the most common molecular marker used to discriminate these parasites (Cunningham 1997; Matìjusová et al. 2001, 2003; Ziêtara et al. 2000; Ziêtara and Lumme 2004). The intergenic spacer (IGS) of ribosomal RNA genes have also been tested for G. salaris and G. thymalli (Collins and Cunningham 2000, Sterud et al. 2002, Hansen et al. 2006). The subunit 1 of the mitochondrial cytochrome oxidase (COI) gene may also be used successfully to differentiate species and local Gyrodactylus strains (Meinilä et al. 2002, 2004; Hansen et al. 2003, 2007). Molecular techniques were necessary to discriminate and describe morphologically almost identical species within several Gyrodactylus groups such as G. teuchis versus G. salaris (Cunningham et al. 2001), G. jussii vs. G. macronychus and G. alexgusevi vs. G. lotae (Ziêtara and Lumme 2003), G. pictae vs. G. turnbulli (Cable et al. 2005), and a number of G. rugiensis-like (Huyse and Volckaert 2002) and G. arcuatus-like species on marine gobies (Huyse et al. 2004) . Molecular discrimination of more than 70 Gyrodactylus species based on the sequences of nuclear rDNA gene cluster encompassing both spacers (ITS1 and ITS2) and 5.8S rRNA gene, proved the region suitable for species identification (Ziêtara 2004, Ziêtara and Lumme 2004). In the present study, this DNA segment was used for diagnosis to screen Gyrodactylus species in Polish salmonid fish farms. A handy PCRRFLP (restriction fragment length polymorphism) method was designed for fast species identification. The method was combined with traditional morphological examination of Gyrodactylus species. This is the first report of such an approach for Gyrodactylus species identification in salmonid farms in Poland.

Materials and methods Study area, fish species studied and Gyrodactylus collection Four salmonid species: rainbow trout Oncorhynchus mykiss Walbaum, 1792; brown trout Salmo trutta L.; grayling Thymallus thymallus (L.) and huchen Hucho hucho (L.) were sampled from May to October in 2006 from fish farms in northern Poland, Pomerania Voivodeship (tributaries of Vistula A6, A7; Pomeranian coast not on Vistula A1-A5) and southern Poland (tributaries of Vistula in Lesser Poland Voivodeship B1-B3). All farms were typical, relatively small salmonid farms with natural waterflow through the ponds. Details of the fish composition are given in Table I. Fish were killed and the body, fins and gills were immediately examined under a stereomicroscope. Infected parts of fish were preserved in 70% (v/v) ethanol. Gyrodactylus specimens were removed from the fish using preparation needles under a ste-

reomicroscope. The haptors were fixed in ammonium picrate glycerine according to Malmberg (1970) for further morphological examination and the rest of the body was transferred to 5 µl of milli-Q water and stored at –20°C until required for DNA analysis. Molecular analysis DNA extraction DNA was released by digesting single specimens without haptor in 10 µl of a lysis solution. Final concentration 1 × PCR buffer, 0.45% (v/v) Tween 20, 0.45% (v/v) NP 40 and 60 µg/ml proteinase K. Tubes were incubated at 65°C for 25 min to allow proteinase K digestion, then 95°C for 10 min to denature the proteinase and cooled down to 4°C. Aliquots of 2 µl of this lysate were used as templates for PCR amplification. PCR amplification The entire ITS region of the ribosomal DNA array (spanning ITS1-5.8S-ITS2 and terminal fragments of 18S and 28S) was amplified with ITS1F (5’-GTTTC CGTAG GTGAA CCT-3’) and ITS2R (5’-GGTAA TCACG CTTGAATC-3’) primers as in Ziêtara et al. (2000). The PCR reaction contained 2 µl of lysate, 1 × PCR buffer, 2 mM MgCl2, 1 µM of each primer, 200 µM of each dNTP and 0.4 unit of Taq polymerase (Fermentas) in final volume of 20 µl. Amplification mixtures were heated for 3 min at 95°C, then subjected to 37 cycles (94°C, 48°C and 72°C for 1 min each), heated for 7 min at 72°C and cooled down to 4°C. PCR products (5 µl) were checked on a 1% agarose gel in the presence of ethidium bromide and visualized under UV light. Design of the restriction method GenBank DNA sequences of Gyrodactylus species expected to be found in fish farms were utilized to plan the RFLP method. The species and sequences were as follows: Gyrodactylus salaris Malmberg, 1957/G. thymalli Z4 itÁan, 1960. Sequences from parasites reported on Salmo salar, Oncorhynchus mykiss, Salvelinus alpinus and Thymallus thymallus in several countries were utilized: Z72477, DQ823390, DQ898302, AJ515912, AJ001847, AF484544, AF328871. Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999. Only one sequence (AJ249350) has been reported from several salmonid species in France, Denmark and Scotland. Gyrodactylus derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007. The European isolates were all originally deposited with a name G. derjavini (accession numbers AF484530, AJ132259, AJ001840). Gyrodactylus derjavini Mikailov, 1975 (DQ323402) was sequenced by us from parasites collected in the Iranian Oncorhynchus mykiss farms by Dr. Behiar Jalali. The sequence is identical with DQ355975 from brown trout in Iran, used in the paper describing G. derjavinoides as a different species (Malmberg et al. 2007).

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Roborzyñski 1

A

fjad kadsææ¿æ

rosbœŸæv 1

2

2

3

3

4

4

B

C

D

1

1

2

2

3

3

4

13

13

13

31

42

42

24

24

4

Fig. 1. The opisthaptoral hard parts from: 1 – Gyrodactylus derjavinoides, 2 – Gyrodactylus salaris, 3 – Gyrodactylus teuchis, 4 – Gyrodactylus truttae. A – hamuli, B – ventral bar, C – marginal hook, D – marginal hook sickle

Gyrodactylus lavareti Malmberg, 1957 from Finnish rainbow trout farms (AF484535). Gyrodactylus truttae Gläser, 1974. The sequence utilized is from Salmo trutta, but the locality of origin remains unknown (AJ132260). The sequences were virtually cut by RestrictionMapper (vers 3.0, shareware program by Peter Blaiklock http://www. restrictionmapper.org/) to select useful restriction enzymes. The enzymes Hinc II (cutting sequence GTYRAC) and Tfi I (GAWTC) proved to separate all the known salmonid sequences and were selected. The predicted restriction fragments were displayed in Figure 2 (Hinc II) and Figure 3 (Tfi I). Digestion by restriction enzymes and recording of the fragment length polymorphisms Aliquots of about 3 µl of PCR products were digested separately for 2 h at 37°C with the restriction enzyme Hinc II and 100 µg /ml BSA (BioLabs) and at 65°C with the restriction enzyme Tfi I (BioLabs) according to the manufacturer’s instructions. The digested DNA fragments were separated on 1.5% agarose gel in the presence of ethidium bromide and

visualized under UV light; 100 DNA ladder (BioLabs) was used to estimate the DNA fragments sizes. Some representative samples from each species and RFLP types were sequenced by standard methods (Ziêtara and Lumme 2004). The sequences are compared with the available salmonid parasite sequences in Figure 5, which is a maximum parsimony tree produced by MEGA3 (Kumar et al. 2004). Morphological analysis Microscopic examination of Gyrodactylus specimens was performed using a phase contrast microscope as in Malmberg (1970). Only the measurements used in the identification key by Gusev et al. (1985) were scored. The Malmberg preparation method was slightly modified to better flattening the haptoral sclerites, which were preserved previously in 70% ethanol. The isolated haptors were soaked in 10% SDS and washed with distilled water before they were preserved in ammonium picrate glycerine. From 10 to 100 specimens were measured per species. Measurements of opisthaptoral hard parts were performed by digital camera and image analysis

189

Identification of Gyrodactylus ectoparasites in Polish salmonid farms

(Tayama CCD moticamera 1000, Motic Images Plus vers 2.0 program). The haptoral dimensions were estimated by interactive measuring on the computer screen. One of the smallest and the largest specimens for each species were drawn. The images and measurements were compared to drawings and dimensions from Gyrodactylus identification key of Gusev et al. (1985), or to the original description. Drawings of opisthaptoral hard parts are shown in Figure 1 and the measurements are given in Table II.

Results Molecular identification Prior to the molecular identification of the parasite specimens by PCR-RFLP, the fragment lengths of the known European salmonid parasites were predicted on the basis of deposited GenBank entries (Figs 2 and 3). The total length of the amplified region is variable, around 1300 base pairs. The fragment pattern obtained with restriction enzyme Hinc II was expected to differentiate all candidate Gyrodactylus species (Figs 2 and 4A). In Table III, multiple GenBank entries of G. salaris and G. derjavinoides were compared, to see if the observed variation could cause additional restriction fragment polymor-

G. salaris

G. salaris*

G. lavareti

G. teuchis

phism. There are altogether ten variable sites reported in G. salaris, and two in G. derjavinoides. The previously known variable sites of G. derjavinoides had no effect to the predicted restriction pattern, and no RFLP variation was observed among the 83 specimens. However, the ITS1 + 5.8S rDNA + ITS2 sequenced (accession number EF445939) differed from Swedish and UK isolates by one nucleotide. Also the variable sites reported in G. salaris mostly remain cryptic with our enzymes, but Hinc II enzyme differentiated the most common standard sequence of G. salaris and the unusual heterogeneous or heterozygous ITS genotype reported from Denmark (AJ515912, Lindenstrrm et al. 2003). In the standard G. salaris, there were 4 DNA fragments (143, 275, 298, 552 bp) predicted and observed (Fig. 4A, lane 1). The Danish G. salaris type, having the ambiguous nucleotide C/T (Y) at position 278, was visible as a heterozygous loss of one restriction site resulting in fusion 275 + 143 = 418 bp fragment (Figs 2 and 4A, lane 2). The intensity of the remaining 275 band was also clearly weaker than the 275 as homozygous. The heterozygous sequence contained all three ambiguous nucleotides reported in Denmark and is deposited by accession number EF464676. The RFLP type corresponding to the hypothetical homozygote 278 (T/T), should have only three DNA fragments (298, 418, 552 bp), and such pattern was indeed observed in

275 bp

143 bp

298 bp

552 bp

275 bp

143 bp

298 bp

552 bp

418 bp 450 bp 450bp

298 bp

552 bp

482 bp

299 bp

552 bp

446 bp

301 bp

552 bp

G. truttae 403 bp

300 bp

552 bp

G. derjavinoides Fig. 2. Diagram of Hinc II restriction sites of ITS rDNA region: G. salaris (Z72477, DQ823390, DQ898302, AJ001847, AF484544, AF328871), G. salaris* (AJ515912, Lindenstrrm et al. 2003), G. “lavareti” (AF484535), G. teuchis (AJ249350), G. truttae (AJ132260), G. derjavinoides (AF484530, AJ132259, AJ001840), G. derjavini (DQ323402, DQ355975)

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Magdalena Rokicka et al.

119 bp 105/77/12 bp

627 bp

311 bp

17 bp

G. salaris 1283 bp

G. lavareti

G. teuchis

G. truttae

641 bp

342 bp

629 bp

298 bp

G. derjavinoides

G. derjavini

364 bp

80 bp

629 bp

868 bp

17 bp

311 bp

311 bp

311 bp

311 bp

17 bp

17 bp

17 bp

17 bp

Fig. 3. Diagram of Tfi I restriction sites of ITS rDNA region: G. salaris (Z72477, DQ823390, DQ898302, AJ001847, AF484544, AF328871, *AJ515912), G .“lavareti” (AF484535), G. teuchis (AJ249350), G. truttae (AJ132260), G. derjavinoides (AF484530, AJ132259, AJ001840), G. derjavini (DQ323402, DQ355975)

two specimens (Fig. 4A, lane 8), from separate farms on Oncorhynchus mykiss. This homozygous ITS type differing from the standard G. salaris sequence by three nucleotides has not been reported before, and the sequence of ITS1 + 5.8S rDNA + ITS2 was deposited in GenBank with accession number EF464677. Standard G. salaris ITS was not found in Polish farms on rainbow trout, but only as four specimens of G. salaris/ G. thymalli on grayling (deposited by accession number EF464678). Within the samples analyzed, we found one more RFLP pattern, not predicted by the a priori sequences (Fig. 4A, lane 4). It looks like a derivative of G. teuchis because it shares all its restriction fragments, but there is an additional recognition site producing two additional DNA fragments, one little over 400 and the other little less than 400 bp (Fig. 4A, lane 4). Curiously, the total length of fragments is longer than the amplification product adjusted to diploid compilation. Some of the fragments are weaker than others suggesting heterozygosity. The observations do not exclude triploidy, but prior to more detailed sequence analysis, we leave the case open and call the type on Figure 4 lane 4 as G. cf. teuchis. Interestingly, this type was much more common (112 specimens) than the standard G. teuchis (seven specimens, Fig. 4A, lane 3), and was found in almost all farms on Oncorhynchus mykiss. G. cf. teuchis

was also the most common parasite among Salmo trutta. Standard G. teuchis was rare and found on rainbow and brown trout (Table I). We sequenced the ITS fragment of these worms, and the G. cf. teuchis sequence shows five ambiguous (heterozygous) nucleotides in ITS2, which is a relatively high number for ITS2 (deposited by accession number EF464680). However, large proportion of the ITS1 of G. cf. teuchis from direct sequencing was unreadable and unalignable, and is left to be solved later. The ITS segment of the G. teuchis proper was identical with the sequence described in Western Europe (deposited by accession number EF464679). The RFLP patterns corresponding the predictions of G. truttae (lane 5) were found, without any variants. Also, the ITS sequence of this type was perfectly identical with the published sequence (Cunningham et al. 2000). For a record, the ITS sequence of G. truttae was deposited by accession number EF464681. The pattern of G. lavareti (lane 6) was not found in Polish farms; the reference sample in Figure 4 was from a rainbow trout farm from Finland. Digestion with the restriction enzyme Tfi I (Fig. 4B) was used to confirm the identification. The patterns in lane 3, 4 and 5 in Figure 4B are quite identical. The RFLP cannot differentiate G. teuchis, G. cf. teuchis, and G. truttae because the 364 and 342 fragments are too similar on a 1.5% agarose gel. Also, the three variants of G. salaris/G. thymalli are all identical

191

5.9–7.0 6.2 7 6.2–8.5 7.8 8–9 6.8–7.5 7.1 7–9 6.3–7.4 7.0 6–8 30.1–33.0 31.4 29–34 36.0-42.2 37.4 34–41 30.2-36.9 35.1 34–39 29.6–36.1 32.3 29–34 11.2–15.8 12.2 12–17 17.1–21.1 18.7 15–16 14.4–17.6 16.3 14–22 14.1–17.1 16.1 10–17 6.4–8.1 7.2 7–10 8.9–12.1 10.4 7–13 7.7–10.3 9.1 10–16 7.2–10.4 8.9 7–9 24.0–27.6 26.0 22–29 28.3–38.1 31.2 22–27 26.3–32.1 29.4 26–32 523.1–33.1 27.8 24–32 16.7–19.1 18.5 15–20 22.8–31.3 25.3 20–21 17.0–24.6 20.6 17–27 16.1–23.4 20.35 16–21 1Prost

G. truttae2

G. teuchis3 G. truttae

G. salaris2 G. teuchis

G. derjavinoides1 G. salaris

(1991), 2Gusev et al. (1985), 3Lautraite et al. (1999).

24.8–31.7 29.3 26–32 30.0–45.4 37.2 30–37 33.4–36.6 35.0 31–41 27.7–38.1 33.8 29–34 36.2–43.7 39.4 37–47 50.0–67.6 58.1 48–49 47.4–55.6 52.4 44–56 39.6–59.6 50.0 40–49 51.7–63.3 54.6 53–64 69.1–87.9 76.4 61–69 64.5–71.8 68.7 61–74 52.8–74.8 65.75 54–65 G. derjavinoides

total length (µm) point length (µm) shaft length (µm) total length (µm) Species

with Tfi I. Other species are readily separated, especially G. lavareti by having no visible restriction sites at all. The ITS sequences of the specimens analyzed in this study are compared with other salmonid parasite sequences available in GenBank by constructing a phylogenetic hypothesis (Fig. 5). Because the ambiguous nucleotides are removed from the comparisons, the heterozygous G. salaris is not included in the tree, and the G. cf. teuchis is represented by the hypothetical “partner” of the standard G. teuchis needed to produce such a deviant heterozygous or heterogenic phenotype.

Anchor

Fig. 4. The RLFP patterns for ITS rDNA region: A – fragments digested with restriction enzyme Hinc II; B – fragments digested with restriction enzyme Tfi I. Lines: 1 – G. salaris (standard), 2 – G. salaris (Lindenstrrm et al. 2003), 3 – G. teuchis, 4 – G. cf. teuchis, 5 – G. truttae, 6 – G. “lavareti”, 7 – G. derjavinoides, 8 – G. salaris (Lindenstrrm et al. 2003) homozygote; M – 100 bp marker

Table II. Dimensions of the opisthaptoral hard parts of the Gyrodactylus species

root length (µm)

length (µm)

Ventral bar

width (µm)

membrane length (µm)

Marginal hook

stickle length (µm)

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Magdalena Rokicka et al.

Fig. 5. Maximum parsimony tree of the ITS2 sequences produced in the present study, as compared with GenBank entries of known salmonid parasites. The compared and perfectly aligned sequence was 426 nucleotides long, because the G. brachymystacis (from Oncorhynchus mykiss in China) was that long. The detailed pairwise comparisons of the full length sequences are presented in the results. The “partner predicted by subtraction”, nearest to G. teuchis, is presented for scaling the five substitutions along the ITS2 of G. cf. teuchis. The bootstrap values of 500 repeats are presented along the nodes. The tree was contructed in MEGA3.1 (Kumar et al. 2004). The accession numbers of the ITS sequences produced in this study are given in the text

Summary of the occurrence of Gyrodactylus infection among fish farms Four salmonid species from 10 fish farms were investigated. Seven were from northern Poland, three from south. A total of 274 fish were checked: 188 rainbow trout (O. mykiss), 46 brown trout (Salmo trutta) of anadromous, lacustrine and brook morphs, 25 grayling (Thymallus thymallus) and 15 huchens (Hucho hucho). Gyrodactylus parasites were found on all examined fish species except huchen. One grayling out of 25 examined carried four specimens of G salaris/G. thymalli. The highest prevalence and intensity of Gyrodactylus infection were found on rainbow and brown trout in the northern fish farms. The distribution of the Gyrodactylus infections are shown in Table I. On the basis of ITS-PCR-RFLP identification, four different Gyrodactylus species were observed: European G. derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007, which is recently separated from the Caspian G. derjavini Mikailov, 1975 (DQ323402), G. truttae Gläser, 1974, three different RFLP types of G. salaris Malmberg, 1957, and two different variants of G. teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999. The variant G. cf. teuchis may be another species, but quite closely related.

In farm environment, the Gyrodactylus species observed here did not show strict host specificity. Rainbow trout was infected by all four parasite species and altogether, seven different RFLP patterns were observed. Only grayling was infected by only one species namely G. salaris/G. thymalli. G. derjavinoides was the most common species and was found in all infected fish farms, not only on S. trutta but on O. mykiss as well. Numbers of Gyrodactylus species in each fish farms and hosts studied are shown in Table I. Morphometrical comparisons Microscopic slides were prepared from all parasite specimens as the first step after the catching, prior to molecular study. After identifying the parasites on the basis of PCR-RFLP, nine morphometric characters were measured from altogether 253 individual haptors mounted in ammonium picrate glycerine. The measurements are presented in Table II, as compared with values from literature. The dimensions of central hooks, ventral bars, and marginal hooks show a low degree of variability in overall morphology. The ranges of measurements overlap between different Gyrodactylus species, causing the morphometric identification very difficult if not impossible. The largest hamuli were observed in G. salaris,

Identification of Gyrodactylus ectoparasites in Polish salmonid farms

193

Table III. Comparison of ITS rDNA region sequences of G. salaris and G. derjavinoides Nucleotide position

Gyrodactylus species 18S Accession number G. salaris Z72477 G. salaris DQ823390, DQ898302 G. salaris AJ515912 G. salaris AJ001847 G. salaris AF484544 G. salaris AF328871 Accession number G. derjavinoides AF484530 G. derjavinoides AJ132259 G. derjavinoides AJ001840

22 G G G – G G

ITS1 104 R A R A A A 391 C Y T

278 C C Y C C C 635 G G S

5.8S 424 T T T T C T

751 G G G T G G

ITS2 913 C C Y C C C

1092 A A W A A A

1101 G A G G G G

28S 1112 – – – C – –

1245 – – – C – –

Nucleotides counted as amplified with ITS1F and ITS2R primers.

and the smallest ones in G. derjavinoides, but also in G. truttae (Table II).

Discussion Salmonids have been cultured for more than 100 years. In fish farms, the ectoparasitic Gyrodactylus species are of less concern than many other diseases. Without the spread of G. salaris into the Atlantic and White Sea salmon populations, with fatal consequences, this parasite genus would scarcely be mentioned among pathogens. However, frequent trading and transports of farmed salmonids implies a risk of parasite introduction and transmission. Therefore, some knowledge about the presence of Gyrodactylus species on salmonids in fish farms and natural waters is urgently needed. The traditional identification of Gyrodactylus species by morphology and morphometrics is very difficult and causes numerous ambiguities. A ‘species recognition hypothesis’ based on morphology can not be tested by morphology. Molecular analyses shed new light on Gyrodactylus species discrimination. There are about 21 Gyrodactylus species described from salmonids in the world. At least eight of them, namely G. birmani Konovalov, G. derjavini Mikailov, G. lavareti Malmberg, G. truttae Gläser, G. salaris Malmberg, G. bohemicus Ergens, G. caledoniensis Shinn, Sommerville et Gibson (listed by Ergens 1983, Malmberg 1993) and G. teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle are mentioned in the European fauna. Five of them (G. derjavini, G. derjavinoides, G. lavareti, G. salaris, G. teuchis and G. truttae) were already tagged with DNA markers and placed in a wageneri species group sensu Ziêtara and Lumme (2002). In Poland, the gyrodactylid fauna on salmonids has not been extensively studied. In a previous study, a sample of fish was collected in southern Poland from two rivers So³a and Czarna, and from a fish farm (Prost 1991). On the basis on morphology, two species, G. derjavinoides (G. derjavini at that time) and G. truttae were recorded. G. derjavinoides was

found on Salmo trutta, Oncorhynchus mykiss and Salvelinus fontinalis. G. truttae was reported only from Salmo trutta. Here, by utilizing a molecular approach for the species identification – the PCR-RFLP technique – it was revealed that Polish Gyrodactylus fauna in salmonid farms consists of at least two additional species: G. teuchis and G. salaris/G. thymalli, with two and three separable subtypes, respectively. It is not the first time when molecular methods enabled discrimination of Gyrodactylus species. G. teuchis in France was initially identified as G. salaris before a molecular approach was introduced (Cunningham et al. 2001). Until present, the dangerous species G. salaris has been molecularly confirmed mainly from the Northern Europe, i.e. Norway, Sweden, Denmark, Finland and Russia (Cable et al. 1999; Cunningham et al. 2000; Ziêtara and Lumme 2002; Hansen et al. 2003, 2007; Lindenstrrm et al. 2003; Meinilä et al. 2004; Ziêtara et al. 2006; Robertsen et al. 2007; Kania et al. 2007). The ITS rDNA sequences are highly conserved revealing only a few nucleotides substitutions. One of the G. salaris variants reported, the genotype from rainbow trout farms in Denmark (Lindenstrrm et al. 2003) could be easily recognised using Hinc II restriction enzyme. This genotype 278 (C/T) was the most common one in the investigated Polish farms on rainbow trout, and it was also observed on brown trout. We also found two worms, which possessed all the three divergent nucleotides as homozygous, either a candidate to be the other parent of the heterozygous genotype reported by Lindenstrrm et al. (2003), or a homozygous progeny from the heterozygous parents. Only the four G. salaris/G. thymalli worms collected from grayling in one farm in northern Poland had the standard ITS, while this standard ITS type is the most common found in Norway, Sweden, Finland and Russia, in three host species (O. mykiss, S. salar, T. thymallus) (Cable et al. 1999, Cunningham et al. 2000, Ziêtara and Lumme 2002, Ziêtara et al. 2002, Meinilä et al. 2004). Other types, divergent and homozygous for one nucleotide substitutions are reported on O. mykiss (Kania et al. 2007), on Salvelinus alpinus in Norway

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(Robertsen et al. 2007) and on T. thymallus in the White Sea basin (Ziêtara and Lumme 2002). Gyrodactylus teuchis was so far reported (and confirmed molecularly) only from France, Denmark and Scotland (Cunningham et al. 2001). The Polish observations of two different RFLP types add considerably to the distribution area of this interesting parasite species, and solving the suggested triploid heterozygosity is planned. The European G. derjavinoides has been molecularly identified from Scotland, Denmark and Sweden (Cable et al. 1999; Cunningham et al. 2000; Ziêtara and Lumme 2002, 2004). The predicted RFLP pattern seen here confirms the observation by Prost (1991). It is an important observation that G. truttae from Britain (Cunningham et al. 2000) was also found in Poland, thus confirming the observation by Prost (1991). G. truttae has been reported in several countries, but these are the only molecular confirmations so far. To separate G. derjavinoides and G. truttae by the less/more curved shape of the marginal hook sickles indeed is very difficult (Fig. 1, parts 1 and 4). Size variation seems to overlap widely. It is interesting that the two dominant Gyrodactylus parasite clones in northern Finland, the rainbow trout specific clone of G. salaris (Ziêtara et al. 2006), and another farm specific clone named as G. lavareti (used as a reference in Fig. 4) were not observed in the Polish farms. The Caspian G. derjavini was not found either, and G. brachymystacis is still recorded only from China. In conclusion, this work indicates that modern identification and understanding of Gyrodactylus species should be necessarily accompanied with DNA analyses. The simple PCRRFLP of the ribosomal DNA utilized here offers a reliable and repeatable method to screen large numbers of single individuals from this very species-rich genus. Acknowledgements. We would like to thank all the people who made their farms available for us and Behiar J. Jalali for the sample of G. derjavini from Iran. The project was supported by a grant from the Ministry of Science via Gdañsk University (BWZ 127/01/E335/S/2006 and 3856/P01/2007/32).

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(Accepted March 8, 2007)