Occurrence and morphogenetic characteristics of Gyrodactylus (Monogenea: Gyrodactylidae) from a rainbow trout farm (Lake Ladoga, Russia)

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1 DOI: /ap W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2016, 61(1), ; ISSN Occurrence and morphogenetic characteristics of Gyrodactylus (Monogenea: Gyrodactylidae) from a rainbow trout farm (Lake Ladoga, Russia) Evgeny Ieshko 1, Yulia Barskaya 1, Aleksey Parshukov 1 *, Jaakko Lumme 2 and Oleg Khlunov 3 1 Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Pushkinskaya St. 11, , Petrozavodsk, Russia; 2 Department of biology, University of Oulu, POB 3000, FI-90014, Finland; 3 Petrozavodsk State University, pr. Lenina 33, , Petrozavodsk, Russia Abstract Gyrodactylus parasite infected juveniles on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from a farm in Lake Ladoga were investigated. The observed cases of infection in featured a high prevalence, when almost all of the were infected. However, if an outbreak of the monogenean infection is observed in spring, the intensity of the infection may be low, and when the infection occurs in the ice-covered period (late autumn winter), the number of parasites on the fins of a single may exceed 3000 specimens. Molecular identification of the parasite demonstrated that the infecting clone was identical with rainbow trout specific strain of Gyrodactylus salaris RBT widely spread in Northern Europe, but a small proportion of the parasites were the hybrid clone Gyrodactylus pomeraniae x G. lavareti. Morphological variations of hooks and other opisthaptor parts in the monogenean Gyrodactylus depending on the intensity of infection in rainbow trout were demonstrated. Keywords Gyrodactylus clones, cage aquaculture, rainbow trout Introduction Early in the 1950s the monogenean parasites were first reported from a salmon farm on the Indalsälven River in northern Sweden (Malmberg 1957). It was only 20 years later however that concerns about this parasite upsurged, as it was detected in Norwegian farms and rivers in 1975 (Johnsen 1978; Johnsen & Jensen 1986). These concerns were caused by the fact that Gyrodactylus salaris spread widely across Norwegian rivers and severely affected their natural Atlantic salmon populations by killing heavily infected juveniles (Bakke and MacKenzie 1993). The arrival of G. salaris in the Keret River (White Sea) also resulted in a sharp decline in juvenile Atlantic salmon numbers (Ieshko et al. 2008). Gyrodactylus infection in salmonids has lately become an issue in Karelia, where commercial cage farming in inland lakes has been developing intensively. In the period between 2007 and 2013 alone, farmed rainbow trout output increased from 9,300 to 17,203 tons (State report, 2014), with the potential to grow to 20,000 25,000 tons (Kitaev et al. 2006; Ryzhkov 2008). In Karelia farms annually import rainbow trout fingerlings for commercial rearing from different neighboring regions of Russia (the Leningrad Region, the Republic of North Ossetia-Alania etc.) and Finland. The need for parasitological studies of organisms reared in freshwater aquaculture has become pressing as in 2008 epizootic Gyrodactylus infections of rainbow trout broke out in some cage farms in Lake Onega (Evseeva et al. 2009). Unavoidable escape of infected from hatcheries to the rivers may increase the risk of parasite hybridization and also the invasion to a new host. A risky situation was recorded with G. salaris on rainbow trout (Oncorhynchus mykiss) which was found in Lizhma River, Onega Lake Basin (Barskaya et al., 2009). This paper reports the results of studies of the infection caused by clones of Gyrodactylus salaris (Malmberg 1957) on rainbow trout from a cage farm in Lake Ladoga, and presents the molecular methods needed to accurately identify the parasite strain. Some of the Gyrodactylus species and strains are considered as less dangerous to the endemic salmon pop- *Corresponding author: aleksey.nik.parshukov@gmail.com

2 152 Evgeny Ieshko et al. ulations in Lakes Ladoga and Onega, but monitoring of the parasite situation is advisable. Materials and Methods Fish and parasite samples From April to August 2010 and from December 2012 to September specimens of juvenile rainbow trout, Oncorhynchus mykiss from the trout farm located in Lake Ladoga, near Island Mäkisalo were studied. Island Mäkisalo lies in the skerries of the Lake Ladoga northern shore (Fig. 1). Live taken from a cage were immobilized and measured for weight and length (AB). Then all monogeneans parasitizing on the fins were counted and fixed in 96% ethanol. Part of the sample was used for further genetic and morphological analysis. The Gyrodactylus specimens were removed from the using preparation needles under a stereomicroscope. In 2013 cages with trout were arranged in groups of 4 6 cages, and were sampled from one cage in each group 25, 39, 4/2, 60, 74. The infection was quantified using the following indices (Bush et al. 1997): 1. prevalence (P) or percentage of the infected population (%): P = (N i x 100%)/N, where N i is the number of infected, N number of examined. 2. mean infection intensity (number of parasites per ), or index of (М): М = n/n, where N is the number of examined, n total number of parasites detected on all examined. Morphological and statistical analyses Karelia Lake Ladoga Fig. 1. Location of the trout farm in Lake Ladoga (Island Mäkisalo) 5 km Microscopic examination of Gyrodactylus specimens was performed using a phase contrast microscope according to Malmberg (1970). The investigated worms were cut in two pieces opisthaptoral hard part and body. The piece with haptors was partially digested by Proteinase K (60 μg/ml) and after that isolated haptors were preserved in ammonium picrate-glycerin (APG) on microscopy slide. The body was placed in a test tube and frozen for later identification by molecular methods. Measurements of each specimen were made and processed using the software Levenhuk ToupView 3.5 (V. Levenhuk, Inc. in Scientific Center) of collective usage platform (Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences). Statistical analysis of the traits of monogenean hooks was carried out with the STATGRAPHICS Centurion XVI software. The ten morphological characteristics of the worms were compared using the discriminant analysis. Further studies are required to assess usefulness of these morphological characteristics for species discrimination. Only individuals for which all opisthaptoral measurements were available were included in the analyses. Molecular identification of the parasites To identify the parasite strain infecting the farm, we used the ADNAM fingerprinting, which rapidly produces a diagnostic phenotypic pattern to be compared with other samples and strains (Ziętara et al. 2006; Kuusela et al. 2007, 2009). The worms were preserved in 96% ethanol. The routine procedure was as follows (Ziętara et al. 2006). Ethanol was removed and evaporated. Digestion in 10 µl 1 x PCR buffer, containing 0.45% Tween, 0.45% IGEPAL and 60 µg ml -1 Proteinase K. Incubation in PCR machine for 25 min at 65 С, followed by 10 min at 95 С to denature the Proteinase K, and completing the procedure by cooling to 4 С. Two microliters of the mixture was used as template for PCR. It is to be pointed out that the procedure is not an extraction of the DNA, just releasing it from histones etc. without any material loss. For the ADNAM fingerprinting, primary PCR was conducted by primers InsF (5 -GATCTGCAATTCATCC- TAAT-3 ) and InsR (5 -TACAATTCGACCAAGGGTAG-3 ). The reaction mixture (20 µl) comprised 2 µl extracted DNA, 1 PCR buffer, 2 mm MgCl 2, 1 µm of each primer, 200 µm dntp (datp, dctp, dttp, dgtp), and 0.5 units Taq-polymerase (Fermentas). Amplification ran as follows: 94 C for 3 min, 30 cycles (94 C for 40 sec, 50 C for 30 sec, and 72 C for 30 sec), 72 C for 7 min, and cooling to 4 C. The PCR product was then checked on 2% agarose gel. Following Exosap (Qiagen) purification, InsF (or InsR) was then used for sequencing the amplified fragment. The sequencing was performed on ABI Prism 3730 DNA analyzer (Applied Biosystems, Hitachi, Japan) with BigDye Terminator Ready Reaction Cycle Sequencing Kit following the vendor s instructions. In the few cases when the primary PCR of ADNAM failed, the sample was identified by complete sequencing of the ITS (Ziętara et al. 2006).

3 Gyrodactylus parasite from rainbow trout 153 The molecular work was performed at the University of Oulu, Department of Biology. Results Infection with monogeneans Gyrodactylus salaris RBT on juvenile rainbow trout in a farm in Lake Ladoga was recorded for the first time in the spring of The infection rate in mid-may was low. Further surveys showed the infection rate to grow to a maximum late in May. By June 10 th mean intensity somewhat decreased, but prevalence remained at 100%. Water temperature rose over the period of observations from 4.4 C to nearly 10 C. As the lake warmed up further in June and July, Gyrodactylus infection in rainbow trout was gradually declining. Most of the cage-kept examined on July 7 th contained singular parasites, and only three out of the twenty examined bore 65, 22 and 11 parasite specimens. The checks carried out in August revealed no presence of the parasite (Table I). Next event of Gyrodactylus infestation of rainbow trout was recorded in January As opposed to 2010, the infection must have occurred in late autumn already, since in January all sampled were infected with high intensity. High parasite s, which can be described as an epizootic, persisted nearly until late May. The intensity of the infection varied widely among individuals from 1 to over 3000 worms per. In July however mean intensity dropped sharply to 11 (min and max intensity being 1 and 73 specimens, respectively), but 100% of the remained infected with Gyrodactylus. The intensity of the monogenean infection in rainbow trout declined significantly when the summer began and water temperature in the lake rose above 10ºС (Table II). Morphological analysis Figure 2 illustrates the morphology of median hooks of the Gyrodactylus salaris RBT and G. pomeraniae x G. lavareti hybrids occurring on the rainbow trout reared in cages in Lake Ladoga in We applied discriminant analysis to identify the distinctive morphological features of the monogenean sampled from different cages. This method enabled estimation of the generalized differences between the monogenean from five cages ( 60, 25, 74, 4/2 and 19) taking into account the variability of all the ten analyzed traits. This method helped to reveal which traits of hooks and the opisthaptor contributed the most to the formation of individual parasite clusters. Morphological characteristics of 36 worms collected from five different cages significantly differed in the two coordinates of the canonical equations (Table III). The two discriminating functions with P-values less than 0.05 are statistically significant at the 95.0% confidence level. The most significant division of worm groups belongs to the first discriminant function which gives 80.16% of explained variance. The second canonical axis takes up only 19.84% of the variability. Of ten analyzed signs two indicators of opisthaptoral hard parts of monogeneans allowed to distinguish groups of worms, where the first canonical axis carries the maximum weight of Table I. Gyrodactylus salaris RBT infection in cage-reared juvenile rainbow trout in May August 2010 Date Water temperature, С examined infected W, weight of (g) Р, infection % М, index of parasite Min-max Variance May May June June July August Table II. Gyrodactylus salaris RBT infection in cage-reared juvenile rainbow trout in January July 2013 Date Water temperature, С examined infected W, weight of (g) Р, infection % М, index of parasite Min-max Variance January March May July ,044

4 154 Evgeny Ieshko et al. Table III. Standardized coefficients and eigenvalues of significant canonical axis for signs of the monogeneans collected from various cages in the trout farm (Lake Ladoga) Canonical axis 1 2 Standardized coefficients Length of ventral bar Length of ventral bar processes Eigenvalue % of variance Canonical correlation λ χ df 12 5 p Fig. 2. Hard part of the opisthaptor of Gyrodactylus pomeraniae x G. lavareti (A). Hard part of the opisthaptor of Gyrodactylus salaris RBT (B). Scale-bar: 10 µm length of ventral bar processes, and on the second length of ventral bar (Table III, IV). The derived coefficients of the discriminant functions corroborated the accuracy of the morphological descriptions of 59.46% of the 36 worms examined in the study. Noteworthy in terms of morphological differentiation between the identified monogenean groups of Gyrodactylus salaris RBT only from cage 39, 4/2 and 19 where 100% of the specimens were classified correctly (Table V). Morphological distinctions between five groupings of the monogeneans are graphically represented within the axes of two canonical equations (Fig. 3). The graph shows the isolated group of Gyrodactylus salaris RBT, morphologically distinct worms collected from 4/2 cage. The monogeneans from other cages 25, 39, 60 and 74 had similar levels and formed a second distinct group. For comparison in this analysis morphological signs opisthaptoral hard parts monogeneans species were used which status was determined genetically Gyrodactylus salaris (Atlantic salmon, river Kumsa) and hybrid G. pomeraniae x G. lavareti (Fig. 3). Both specimens were morphologically well identified and different from monogeneans defined as Gyrodactylus salaris RBT (Table IV). Ordination Gyrodactylus salaris found on juvenile Atlantic salmon in river Kumsa (basin of Lake Onega) shows significant differences from the studied worms parasitizing on rainbow trout (Fig. 3). Genetically identified hybrid of G. pomeraniae x G. lavareti also morphologically isolated, as shown in Figure 3. It is noteworthy ordination of two monogeneans collected on from cages 25 and 60 which based on the results of discriminant analysis, for their morphological parameters should be classified as G. pomeraniae x G. lavareti (Table IV). However, if a specimen of the parasite from cage 60 has not been investigated genetically, that individual from cage 25 has been identified genetically as Gyrodactylus salaris RBT. Molecular identification In 2010 and 2013 parasite samples from nine cages were identified by molecular methods. In 2010 total number of identified examples of Gyrodactylus was six. In 2013 total number of identified specimens was 81, which represented the standard rainbow trout specific strain of Gyrodactylus salaris, called RBT clone (Kuusela et al. 2007). Among them three specimens were G. pomeraniae x G. lavareti hybrids, described earlier from Finnish farms (Kuusela et al. 2008; Ziętara et al. 2006). Koski and Malmberg (1995) identified the

5 Gyrodactylus parasite from rainbow trout 155 Table IV. Discriminant functions for morphological differentiation of monogeneans Gyrodactylus parasites of rainbow trout in Lake Ladoga (results of forward stepwise linear discriminated analysis) Coefficients of the discriminant functions 25 cage 39 cage 4/2 cage 60 cage 74 cage G_pomer. x G. lavar. G_salaris λ F P Length of ventral bar Length of ventral bar processes CONSTANT Table V. Determination of affiliation of monogeneans Gyrodactylus groups based on two morphometric characteristics of the opisthaptoral hard parts Actual group Group Size Predicted 25 cage 39 cage 4/2 cage 60 cage 74 cage G_pomer. x G. lavar. G_salaris 25 cage 4 1(25.00%) 1(25.00%) 0(0.00%) 0(0.00%) 1(25.00%) 1(25.00%) 0(0.00%) 39 cage 3 0(0.00%) 3(100.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 4/2 cage 10 0(0.00%) 0(0.00%) 10(100.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 60 cage 9 2(22.22%) 0(0.00%) 0(0.00%) 5(55.56%) 1(11.11%) 1(11.11%) 0(0.00%) 74 cage 9 3(33.33%) 3(33.33%) 0(0.00%) 2(22.22%) 1(11.11%) 0(0.00%) 0(0.00%) G_pomer. x G. lavar. 1 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 1(100.00%) 0(0.00%) G_salaris 1 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 1(100.00%)

6 156 Evgeny Ieshko et al. Plot of Discriminant Functions Axis 2 Axis 1 Fig. 3. Ordination of Gyrodactylus pomeraniae x G. lavareti, Gyrodactylus salaris and five groups of its hybrids, parasitizing on rainbow trout farms of Lake Ladoga hybrid as G. lavareti. The parasite composition in this study corresponds to that commonly found in Finnish rainbow trout farms. Discussion Rainbow trout infections in cage farms in Karelia have been studied since When trout farming was developing actively in , few events of infestation with Gyrodactylus salaris were detected. Starting 2008 however, the number of trout farms where G. salaris infections of rainbow trout were of epizootic scope has been growing. According to published data on 17 trout farms surveyed in Karelia in (Evseeva 2009; Evseeva et al. 2009), seven of the eight farms operating on Lake Onega demonstrated cases of high infection with G. salaris in spring. Only in two of the three trout farms on Lake Ladoga all were infected with the monogeneans. As for the rest of the farms, rainbow trout gyrodactylosis was found in three of the six farms surveyed. The results of parasitological monitoring in the trout farm on Lake Ladoga presented here prove that monogenean infections on rainbow trout have become quite frequent. It is demonstrated that infection may happen both in spring (Table I) and in the autumn-winter season, when the infection intensity would be high (Table II). Previously published data on Gyrodactylus hybrids on trout from farms of Karelia (Evseeva 2013) are corroborated by our findings, where both G. salaris RBT and G. pomeraniae x G. lavareti were identified among the worms examined in We now have a description of both monogenean forms, including their origin, occurrence, morphology, and molecular-genetic properties (Ziętara et al. 2006; Ieshko et al. 2015). These results suggest that G. salaris clones have spread widely and established firmly on rainbow trout reared in Karelia. The majority of the 81 worms used in genetic analysis were identified as G. salaris RBT, and only one of the three preserved specimen identified as G. pomeraniae x G. lavareti. The morphology of the median hooks and opisthaptoral hard parts monogeneans G. salaris, parasites of juvenile Atlantic salmon is significantly different from the hybrid forms collected at trout farms (Ieshko et al. 2015). Parasitological monitoring of rainbow trout on a farm in Lake Ladoga revealed variability in the morphology of the opisthaptoral of worms Gyrodactylus. In four out of five studied cages collected monogeneans had similar parameters of signs of median hooks and monogeneans from cage 4/2 genetically identified as G. salaris RBT, morphologically were significantly separated (Fig. 3, Table IV). A possible explanation for the observed difference is due to the fact that the trout in cages 4/2 had a high intensity of infection (more than 1000 examples/), and in other cages infection was low (about 20 examples/). Conclusions The studies have shown that the RBT clone of the monogenean Gyrodactylus salaris frequently occurs on farm-reared rainbow trout in Karelia. Morphological variability of the median hooks and opisthaptor of G. salaris RBT, which may to a certain extent depend on the infection intensity, was demonstrated. The infection indices and the distribution of parasite s within the host population were found to closely correlate with season. One can assume that a spring rise in the infection rates

7 Gyrodactylus parasite from rainbow trout 157 will not induce a dangerous epizootic event or result in high kill due to the monogenean infection. On the other hand, an outbreak of the parasitic infection in the autumn-winter season may cause mass mortalities in rainbow trout. Acknowledgements. The study was supported by Russian Presidential grant for state support to leading scientific schools in ( ). The study was carried out under state order (project ) and the Programme of fundamental researches of Biology Department of RAS ( ).The authors wish to thank Dar ya Lebedeva for help in analyzing parasitological material. References Barskaya J., Lebedeva D., Lumme J., Veselov A Problems of salmon river: parasitological view. In: Proceedings of the 3rd Symposium of Scandinavian Baltic Society of Parasitology, April, 2009, Riga, Latvia, 12 Bakke T.A., MacKenzie K Comparative susceptibility of native Scottish and Norwegian stocks of Atlantic salmon, Salmo salar L., to Gyrodactylus salaris Malmberg: laboratory experiments. Fisheries Research, 17, DOI: / (93) U Bush A.O., Lafferty K.D., Lotz J.M., Shostak A.W Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology, 83, DOI: / Evseeva N.V. Cross-border transport as a pathway for the dispersal of alien parasites. In: Proceedings of the 3 rd Interregional Conference of Parasitologists of Siberia and Far East devoted to the 80 th anniversary of Professor K.P. Fyodorov. Novosibirsk, 2009, Evseeva N.V. Parasitic monogeneans of genera Gyrodactylus and Discocotyle inducing diseases in cage aquaculture in Karelia. In: Proceedings of the 29 th International Conference Biological Resources of the White Sea and Internal Water Bodies of the European North. Murmansk: PINRO publishers, 2013, Evseeva N.V., Barskaya J.J., Lebedeva D.I The first case of gyrodactylosis of the rainbow trout in aquaculture of Karelia. In: Proceedings of GosNIORH December, 2009, St. Petersburg, Ieshko E.P., Lebedeva D.I., Lumme J A new Gyrodactylus strain on brown trout (Salmo trutta) in Jänisjärvi, Russian Karelia, and a literature revision of salmonid parasites of the genus Gyrodactylus in North-Western Russia and adjacent areas. Acta Parasitologica, 60, Ieshko E.P., Shulman B.S., Shurov I.L., Barskaya Y.Y Longterm changes of epizootic juvenile salmon (Salmo salar L.) in the river Keret (the White Sea basin) caused by Gyrodactylus salaris Malmberg, Parasitologiya, 42, Johnsen B.O., Jensen A.J Infestations of Atlantic salmon (Salmo salar) by Gyrodactylus salaris in Norwegian rivers. Journal of Fish Biology, 29, DOI: /j tb04941.x Johnsen B.O., The effect of an attack by the parasite Gyrodactylus salaris on the population of salmon parr in the river Lakselva, Misvaer in Northern Norway. Astarte 11, 7 9 Kitaev S.P., Ilmast N.V., Sterligova O.P Methods for assessing the nutrient load of trout farms on aquatic ecosystems. Karelian Research Centre, Petrozavodsk, Russia, 39 p. Koski P., Malmberg G Occurrence of Gyrodactylus (Monogenea) on salmon and rainbow trout in farms in northern Finland. Bulletin of the Scandinavian Society for Parasitology, 5, Kuusela J., Holopainen R., Meinila M., Anttila P., Koski P., Ziętara M.S., Veselov A., Primmer C.R., Lumme J Clonal structure of salmon parasite Gyrodactylus salaris on a coevolutionary gradient on Fennoscandian salmon (Salmo salar). Annales Zoologici Fennici, 46, Kuusela J., Ziętara M.S., Lumme J Hybrid origin of Baltic salmon-specific parasite Gyrodactylus salaris: a model for speciation by host switch for hemiclonal organisms. Molecular Ecology, 16, DOI: /j X x Kuusela J., Ziętara M. S., Lumme J Description of three new European cryptic species of Gyrodactylus Nordmann, 1832 supported by nuclear and mitochondrial phylogenetic characterization. Acta Parasitologica, 53, /s x Malmberg G On the occurrence of Gyrodactylus on Swedish es. Skrifter utgivna av Södra Sveriges Fiskeriförening, Arsskrift 1956, Ryzhkov L.P Cage aquaculture programme of action. In: Proceedings of the scientific conference Cage Aquaculture. Technology of cultivation. Feeding the and the preservation of their health, October, 2008, Petrozavodsk, 3 6 State Report on the Environment of Karelia in Ministry of Nature Use and the Environment of the Republic of Karelia, Petrozavodsk, 328 pp. Ziętara M.S., Kuusela J, Lumme J Escape from an evolutionary dead-end: a triploid clone of Gyrodactylus salaris is able to revert to sex and switch host (Platyhelminthes, Monogenea, Gyrodactylidae). Hereditas, 143, DOI: / j Received: July 9, 2014 Revised: August 14, 2015 Accepted for publication: October 12, 2015