Haakon Hansen

Mitochondrial DNA variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling, and rainbow trout in Norway and Sweden

Approximately 800 bp of the mitochondrial cytochrome oxidase I (COI) gene were sequenced from 76 Gyrodactylus specimens of 32 salmonid host populations, i.e. from Salmo salar, Thymallus thymallus, and Oncorhynchus mykiss in Norway, Sweden and Latvia. The COI sequences indicated a substantial intraspecific differentiation of Gyrodactylus salaris and Gyrodactylus thymalli. In total, 12 haplotypes were identified which group into five well supported clades, three clades with parasites from Atlantic salmon and two clades with parasites from grayling. The basal nodes linking the five clades together are only weakly supported. Thus, there is no support for the monophyly of all G. salaris haplotypes and the monophyly of all G. thymalli haplotypes. The lack of monophyly of the mitochondrial haplotypes of G. salaris and G. thymalli may indicate that G. salaris and G. thymalli represent (i). two polytypic species or (ii). one polytypic species, or (iii). refer to a complex of more than two sibling species. The mtDNA data indicate multiple introductions of G. salaris and G. thymalli into Norway. A minimum of three independent introductions of G. salaris and two independent introductions of G. thymalli are supported. This is congruent with earlier hypotheses on the introduction of G. salaris and G. thymalli into Norway.

The effect of various metals on Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon ( Salmo salar )

Atlantic salmon (Salmo salar) parr (age 0+), infected by the ectoparasite Gyrodactylus salaris, were exposed to aqueous aluminium (Al), copper (Cu), zinc (Zn), iron (Fe) and manganese (Mn), at 4 different concentrations. There was a negative correlation between G. salaris infections and metal concentrations in both Zn- and Al-exposed salmon. In the Zn-experiment, all 4 concentrations tested caused a decrease in the G. salaris infections, while in the Al-experiment the G. salaris infection did not decline at the lowest concentration. The number of G. salaris increased continuously during the experiments in all control groups, and in all groups exposed to Cu, Fe and Mn. At the highest concentration, however, copper seemed to impair the growth of G. salaris infection. The results show that aqueous Al and Zn are environmental factors of importance controlling the distribution and abundance of the pathogen G. salaris. Other pollutants might also have an influence on the occurrence of G. salaris. Finally, the results demonstrate that aqueous Al and Zn have a stronger effect on the parasite than on the salmonid host, suggesting that both metals may be used as a pesticide to control ectoparasites such as G. salaris.

Arctic charr (Salvelinus alpinus) is a suitable host for Gyrodactylus salaris (Monogenea, Gyrodactylidae) in Norway.

Gyrodactylus specimens infecting both anadromous Arctic charr (Salvelinus alpinus) from River Signaldalselva (northern Norway) and resident Arctic charr from Lake Pålsbufjorden (southern Norway) were identified as G. salaris using molecular markers and morphometrics. The infection in Pålsbufjorden represents the first record of a viable G. salaris population infecting a host in the wild in the absence of salmon (Salmo salar). G. salaris on charr from Signaldalselva and Pålsbufjorden bear different mitochondrial haplotypes. While parasites infecting charr in Signaldalselva carry the same mitochondrial haplotype as parasites from sympatric Atlantic salmon, G. salaris from charr in Pålsbufjorden bear a haplotype that has previously been found in parasites infecting rainbow trout (Oncorhynchus mykiss) and Atlantic salmon, and an IGS repeat arrangement that is very similar to those observed earlier in parasites infecting rainbow trout. Accordingly, the infection may result from 2 subsequent host-switches (from salmon via rainbow trout to charr). Morphometric analyses revealed significant differences between G. salaris infecting charr in the 2 localities, and between those on sympatric charr and salmon within Signaldalselva. These differences may reflect adaptations to a new host species, different environmental conditions, and/or inherited differences between the G. salaris strains. The discovery of G. salaris on populations of both anadromous and resident charr may have severe implications for Atlantic salmon stock-management as charr may represent a reservoir for infection of salmon.

Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea) parasitising Oreochromis niloticus niloticus (L.) and O. mossambicus (Peters) (Cichlidae)

Three new species of Gyrodactylus are described from two species of Oreochromis (Cichlidae): Gyrodactylus hildae sp. nov. from the Nile tilapia, Oreochromis niloticus niloticus, and from an unconfirmed cichlid in Ethiopia; Gyrodactylus ulinganisus sp. nov. from a South African population of Mozambique tilapia, Oreochromis mossambicus; and, Gyrodactylus yacatli sp. nov. from O. n. niloticus reared in Mexico. The hamuli and marginal hooks of G. hildae sp. nov. and G. yacatli sp. nov. differ notably from G. cichlidarum, a species commonly found on O. n. niloticus. The hooks of G. ulinganisus sp. nov., however, are morphologically similar to those of G. cichlidarum, but the two species were found to differ by 42 nucleotide substitutions (24 within the 342 bp long ITS1; 18 within the 303 bp long ITS2) and by 1 insertion/deletion. This study confirms that Nile and Mozambique tilapia harbour a number of different species of Gyrodactylus, with G. cichlidarum being the most frequently encountered and being associated with mortalities of juvenile O. n. niloticus. This study discusses the host specificity of gyrodactylids on commercial cichlid species and the potential repercussions of their movement on stocks of fish into new environments where cichlids are already present.

Gyrodactylids [Gyrodactylidae, Monogenea] infecting Oreochromis niloticus niloticus [L.] and O. mossambicus [Peters] [Cichlidae]: A pan-global survey

Gyrodactylus infections in intensively-reared populations of Nile tilapia, Oreochromis niloticus niloticus, have been associated world-wide with high mortalities of juvenile fish. In this study, 26 populations of Gyrodactylus parasitising either O. n. niloticus or Mozambique tilapia, Oreochromis mossambicus, were sampled from fourteen countries and compared with type material of Gyrodactylus cichlidarum Paperna, 1968, Gyrodactylus niloticus (syn. of G. cichlidarum) and Gyrodactylus shariffi Cone, Arthur et Bondad-Reantaso, 1995. Representative specimens from each population were bisected, each half being used for morphological and molecular analyses. Principal component analyses (PCA) identified five distinct clusters: (1) a cluster representing G. cichlidarum collected from O. n. niloticus from 13 countries; (2) the G. shariffi paratype; (3) three specimens with pronounced ventral bar processes collected from two populations of Mexican O. n. niloticus (Gyrodactylus sp. 1); (4) four specimens collected from an Ethiopian population nominally identified as O. n. niloticus (Gyrodactylus sp. 2); (5) nine gyrodactylids from South African O. mossambicus (Gyrodactylus sp. 3). Molecular analyses comparing the sequence of the ribosomal transcribed spacer regions (ITS 1 and 2) and the 5.8S gene from the non-hook bearing half of worms representative for each population and for each cluster of parasites, confirmed the presence of G. cichlidarum in most samples analysed. Molecular data also confirmed that the DNA sequence of Gyrodactylus sp. 2 and Gyrodactylus sp. 3 (the morphologically-cryptic group of South African specimens from O. mossambicus) differed from that of G. cichlidarum and therefore represent new species; no sequences were obtained from Gyrodactylus sp. 1. The current study demonstrates that G. cichlidarum is the dominant species infecting O. n. niloticus, being found in 13 of the 15 countries sampled.

Gyrodactylus longipes n. sp. (Monogenea: Gyrodactylidae) from farmed gilthead seabream (Sparus aurata L.) from the Mediterranean

Gyrodactylus longipes n. sp. (Monogenea, Gyrodactylidae) is described from the gills of farmed juvenile gilthead seabream (Sparus aurata L.) from two sites located in Italy and Bosnia-Herzegovina and represents the second species of Gyrodactylus to be described from S. aurata. Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 was the first gyrodactylid to be described from S. aurata, from populations cultured in Albania and Croatia. In the current study, G. longipes was found in a mixed infection with G. orecchiae on fish maintained in Latina Province, Italy, thus extending the reported distribution of the latter throughout the Mediterranean. The morphology of the opisthaptoral hard parts of G. longipes is compared to those of G. orecchiae, using light and scanning electron microscopy. Gyrodactylus longipes is characterised by having larger, elongated ventral bar processes and long, triangular-shaped toe region to their marginal hook sickles which, by comparison, are rhomboid in G. orecchiae. The marginal hook sickles of G. longipes are almost double the size of G. orecchiae which allows for their rapid discrimination from each other in mixed infections. A comparison of the DNA sequence of the ribosomal internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) of G. longipes with the corresponding sequence from G. orecchiae and with those available in GenBank, supports the separate species status of G. longipes. Part of this study necessitated an overview of the existing Gyrodactylus fauna from Italy and Bosnia-Herzegovina; a summary from each country is provided here to assist future investigations.

Detection of the myxosporean parasite Parvicapsula pseudobranchicola in Atlantic salmon (Salmo salar L.) using in situ hybridization (ISH)

BackgroundParvicapsula pseudobranchicola is a marine myxosporean parasite infecting farmed Atlantic salmon (Salmo salar). A major site for the parasite is the pseudobranch, which may be destroyed in heavily infected fish. Parvicapsulosis may be associated with significant mortality, although the main effect of infections seems to be runting. In situ hybridization (ISH) is, in the absence of specific antibodies, the preferred method for the detection of cell- and tissue tropisms of myxozoans in the early phases of infection of the host, and provides information about the possible association between the pathogen and pathology. A positive diagnosis of parvicapsulosis is based on histopathology and PCR. The aim of the present work was to develop a specific, sensitive and robust ISH assay for the detection of P. pseudobranchicola in tissues.MethodsThe ISH method was designed to specifically target P. pseudobranchicola 18S rDNA/rRNA using a locked nucleic acid (LNA) modified oligonucleotide probe. The method was tested on paraffin embedded P. pseudobranchicola infected pseudobranchs. The infections were confirmed by light microscopy revealing the presence of typical P. pseudobranchicola trophozoites and spores, and the presence of parasite was confirmed with real-time RT-PCR.ResultsSpecific regions stained by ISH overlapped well with the parasitized and degenerated regions in neighbouring HE stained sections. No staining was observed in pseudobranchs of Atlantic salmon which had been held in P. pseudobranchicola-free water.ConclusionsWe report here the development of a sensitive ISH assay for the detection of P. pseudobranchicola in paraffin embedded tissue. The technique will be valuable in the study of host entry, early proliferation, pre-spore development, pathology and tissue tropism in Atlantic salmon.

Gyrodactylus species [Monogenea] infecting alpine bullhead [Cottus poecilopus Heckel, 1837] in Norway and Slovakia, including the description of Gyrodactylus mariannae sp.nov.

Gyrodactylus specimens infecting the skin and fins of two alpine bullhead (Cottus poecilopus) populations from the rivers Signaldalselva (North Norway) and Rena (South-East Norway) were characterized by both morphological and molecular means. Morphometrical differences were minor and the nucleotide sequences of the internal transcribed spacers (ITS) of the nuclear rDNA cluster were identical for parasites from both localities. Based on earlier descriptions, the relatively closest species are Gyrodactylus hrabei Ergens, 1957, described from common bullhead (Cottus gobio) in Slovakia and G. sp. Malmberg, 1973, from alpine bullhead in Sweden. The Norwegian Gyrodactylus specimens from the two alpine bullhead populations were morphometrically different from both the type material of G. hrabei from Slovakia and newly collected Gyrodactylus specimens from alpine bullhead in two Slovakian localities. The Slovakian Gyrodactylus specimens were found to be identical with type material of G. hrabei. The nucleotide sequences of the ITS of the Norwegian Gyrodactylus species were different from the Slovakian material. Hence, the Norwegian Gyrodactylus specimens from the alpine bullhead represent a new species, G. mariannae sp. nov.

Electrocotyle whittingtoni n. gen., n. sp. (Monogenea: Monocotylidae: Heterocotylinae) from the gills of a captive onefin electric ray, Narke capensis (Narkidae) at Two Oceans Aquarium, Cape Town, South Africa.

Electrocotyle whittingtoni n. gen., n. sp. (Monogenea: Monocotylidae) is described from the gills of a captive female onefin electric ray, Narke capensis, collected for exhibition at Two Oceans Aquarium in Cape Town, South Africa. Electrocotyle n. gen. is most similar to the heterocotyline genera Heterocotyle and Potamotrygonocotyle but could not be accommodated easily in either of these groups. The new genus is characterised by a haptor with one central and eight peripheral loculi, four unsclerotised structures on the dorsal surface of the haptor, a single unsclerotised non-sinous ridge on the ventral surface of the haptoral septa, large hamuli with a long handle and reduced guard, a vagina with sclerotised walls, and tetrahedral eggs. Molecular phylogenetic analyses based on 28S sequences strongly support the separate genus status of Electrocotyle n. gen and thus support our morphological conclusion. The Heterocotylinae is amended to accommodate the new genus, and the new species is fully described and illustrated herein. This is the first record of a monocotylid from the Narkidae. Electrocotyle whittingtoni n. gen. n. sp. is considered potentially pathogenic given its negative impact on the health of its captive host kept in the quarantine facility at Two Oceans Aquarium.

Infections with Gyrodactylus spp. (Monogenea) in Romanian fish farms: Gyrodactylus salaris Malmberg, 1957 extends its range

BackgroundThe salmon parasite Gyrodactylus salaris Malmberg, 1957 has caused high mortalities in many Atlantic salmon, Salmo salar, populations, mainly in Norway. The parasite is also present in several countries across mainland Europe, principally on rainbow trout, Oncorhynchus mykiss, where infections do not seem to result in mortalities. There are still European countries where there are potential salmonid hosts for G. salaris but where the occurrence of G. salaris is unknown, mainly due to lack of investigations and surveillance. Gyrodactylus salaris is frequently present on rainbow trout in low numbers and pose a risk of infection to local salmonid populations if these fish are subsequently translocated to new localities.MethodsFarmed rainbow trout Oncorhynchus mykiss (n = 340), brook trout, Salvelinus fontinalis (n = 186), and brown trout, Salmo trutta (n = 7), and wild brown trout (n = 10) from one river in Romania were sampled in 2008 and examined for the presence of Gyrodactylus spp. Alltogether 187 specimens of Gyrodactylus spp. were recovered from the fish. A subsample of 76 specimens representing the different fish species and localities were subjected to species identification and genetic characterization through sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1).ResultsTwo species of Gyrodactylus were found, G. salaris and G. truttae Gläser, 1974. This is the first time G. salaris is diagnosed in Romania. Gyrodactylus salaris was found to infect rainbow trout, brown trout and brook trout in eight of the 12 farms examined. The prevalence and intensity of infections were generally low in all farms. Gyrodactylus truttae was present on brook trout in one farm and on wild brown trout in the river studied. This also represents the first record of this parasite in Romania. Analyses of sequences of the cox1 gene of G. salaris from Romania revealed four haplotypes, all previously undescribed. While it is not unlikely that the infections in Romanian fish farms originate directly from imported rainbow trout, the current data is not sufficient to conclude on this and does not exclude that the infections can originate from hosts in the local water systems. The study shows that there are still unknown populations and variants (haplotypes) of G. salaris present in European rainbow trout aquaculture, all or many of them with unknown biological characteristics such as host specificity and virulence. As some strains might be pathogenic to Atlantic salmon, the importance of carrying out surveillance and keeping a high focus on control with import and export of live fish for aquaculture purposes is important.ConclusionsGyrodactylus salaris and G. truttae are for the first time found on salmonids in Romania. All mitochondrial haplotypes recovered were previously undescribed and this indicates that there is still an unknown diversity of this parasite present in localities not previously examined. The virulence of the haplotypes found in Romania is unknown and requires establishing.