Nucleospora cyclopteri is a common microsporidian parasite infecting lumpfish around Iceland. Fish with visible clinical signs, like those infected with N. cyclopteri, were found at 12 of 43 sites around the country. Although all fish were not screened for the microsporidian, the cause of the clinical signs observed is assumed to be N. cyclopteri as all other fish screened with equivalent signs were infected with this microsporidian. Even though the prevalence data are limited to a few fish examined at each sampling site, the geographic distribution is wide. It causes severe clinical signs and extensive histopathological changes.
This is the first report of intranuclear microsporidiosis in wild lumpfish, Cyclopterus lumpus. However, a microsporidian infection has previously been reported from lumpfish reared in a recirculation facility in Canada, which was associated with chronic fish mortalities. That paper describes the clinical, histopathological and ultrastructural features of an intranuclear microsporidian. Mullins et al. suggested that the lumpfish microsporidian they found belonged to the genus Enterocytozoon based on similarities to the previously described species Enterocytozoon bieneusi and E. salmonis (syn. Nucleospora salmonis), both with regard to morphology and the type of immune response, but they did not make a formal species description. The former species is a non-intranuclear intestinal parasite of humans and numerous higher vertebrates[22, 23], the latter is a well documented intranuclear microsporidian pathogen which has been identified in various salmonid species[24, 25] and also from Atlantic halibut, Hippoglossus hippoglossus.
Phylogenetic analyses (Figure 7) and a percentage identity matrix (Table 2) show that N. cyclopteri is most closely related to isolates of N. salmonis with an identity of approximately 96% to all isolates. Intraspecific variation of N. salmonis between four separate isolates, amplified from different host fish, is between 99.1–99.84% (Table 2), indicating that a similarity of 96% would represent a different species and not another isolate of N. salmonis. In addition to this, the spore size of 3.12 × 1.30 μm and evenly oval shape in N. cyclopteri is untypical for the family group that generally have smaller more ovoid to rounded spores with sizes recorded for Nucleospora spp. of 1.6 × 0.8 μm and 2.0 × 1.0 μm; Enterocytozoon spp. of 1.5 × 0.8 μm and 1.1 × 0.7 μm; Enterospora 1.3 × 0.7 μm and Desmozoon 2.84 × 1.83 μm.
Comparing the microsporidian infection described in this paper to the one from Canada, some similarities are evident; macroscopic clinical signs include renomegaly and exophthalmos, lymphocytes and lymphoblasts are the target cells in both cases and infections are detected in all organs examined, via vascular migration of infected cells. Other things are more difficult to compare, such as the histopathology and the morphology and size of the microsporidian spores. The histopathological description made by Mullins et al. is quite brief; mainly describing an infiltration of lymphocyte like cells, morphological features of the target cells and their presence in all organs examined, but no actual tissue damage associated with infections. Histopathological changes observed in the lumpfish in the present paper were in many cases considerable including extensive degeneration and necrosis of kidney tubules and vacuolar degeneration of the haematopoietic tissue. It seems that a lymphocytotsis or lymphoblastosis type of inflammatory reaction, with a subsequent prominent hyperplasia, and affected cells being lymphocytes or lymphocyte precursor cells, is a relatively common feature of intranuclear microsporidiosis[28–31]. Except for one fish, all the 13 fish, which where thoroughly examined in the present study, had either light subclinical infections or severe infection with considerable or extreme clinical signs. As the extent of the clinical signs of the fish examined by Mullins et al., such as renomegaly, is not obvious, it is possible that these fish had lighter infections and hence lacked severe histopathological changes, which might occur at later stages of infection in wild fish. Comparing the spore morphology observed in these two studies, the shape is very similar, i.e. uniformly oval and not tapered at one end, which is a common feature of some microsporidian spores. However, the spore size is considerably different, i.e. 2.1 μm x 1.0 μm in the previous study compared to a mean size of 3.12 μm x 1.30 μm in the present study. Mullins et al. made spore measurements using ultrathin sections on a TEM but fresh spores were used in the present study. Hence, the spores from the Canadian lumpfish had been fixed in glutaraldehyde and processed for TEM and might have shrunk during this procedure. Furthermore, when using ultrathin sections, one cannot be sure that the plane of section is through the entire length and width of the spore. Consequently, the spore size in these two studies is not suitable for direct comparison and fresh spores from Canadian lumpfish should be analysed. Although some similarities are apparent between the microsporidians described in these two studies, DNA sequence data is required from Canadian lumpfish to confirm their conspecificity.
Currently, the life cycle of Nucleospora cyclopteri and its route of infection/transmission is not known. However, many microsporidians infecting fish have a simple direct life cycle and no vector or intermediate host is required. This simple form of direct transmission between fish is known to occur in the congener Nucleospora salmonis, which is the closest known relative to N. cyclopteri in our phylogenetic analyses. Indeed, from our histology and PCR results, we found that the kidney and gills were very common sites of infection, indicating that these organs could be the sites for direct transmission to occur, being excreted from the kidney and entering another fish host via the gills.
Another close relative, and the robustly supported sister taxon in the phylogenetic tree, Desmozoon lepeophtherii, is a hyperparasite of the salmon louse Lepeophtheirus salmonis and also an intranuclear parasite of Atlantic salmon[27, 33, 34]. It is possible that multiple hosts or a complicated life cycle could occur in N. cyclopteri infections in lumpfish. Several things could support this: 1) According to Heuch et al. lumpfish are a preferred host for the parasitic copepod Caligus elongatus and might serve as a reservoir for caligid infections of other fish species. 2) One of the clinical signs detected in our study were small lesions on the skin similar to those caused by parasitic copepods. 3) Histological examination revealed microsporidian spores in the most outer layers of lumpfish skin. At present this remains speculative, and additional studies are needed to examine whether C. elongatus are infected with N. cyclopteri and how transmission of the parasite might take place. In the present study spores of N. cyclopteri were also found in close association with the eggs suggesting that spores may also be vertically transmitted. Some microsporidia that infect crustaceans are known to be transovarially (vertically) transmitted[36, 37] and some pathogens that enter the microphyle of the egg before its shell fully matures are known to cause bacterial infection such as Renibacterium salmoninarum. Screening larval lumpfish soon after hatching, using the nested PCR developed in this study, would be a simple method to check for the presence of vertical transmission as a route of infection for this microsporidian. It will become necessary to evaluate such routes of infection for important pathogens of lumpfish, in order to help limit their spread and impact in captive fish. Lumpfish are becoming increasingly more important for the aquaculture industry, being used as cleaner fish in Atlantic salmon cages for the removal of salmon lice. However, their culture is problematic as they are susceptible to numerous pathogens.
The extreme pathology seen in some fish, which looks highly unlikely to be reversible, raises the question whether infections could have an impact upon lumpfish populations. More complete data on prevalence and severity of infections, with regard to fish age, geographic distribution and time of year, are required to determine any potential mortality rates.
Phylum: Microsporidia (Balbiani, 1882)
Class: Microsporea (Levine & Corliss, 1963)
Order: Microsporida (Balbiani, 1882)
Family: Enterocytozoonidae (Cali & Owen, 1990)
Fresh spores are evenly oval in shape, length 2.9–3.5 μm width 1.1–1.5 μm, with two lines of symmetry being evenly rounded at both ends. Mature spores, numbering between 1–14, are found inside lymphocyte nuclei in haematopoietic and other tissues. SSU rDNA sequence data confirms a close association with other Nucleospora species and robustly places N. cyclopteri within the Enterocytozoonidae.
Type host: Atlantic lumpfish (Cyclopterus lumpus, L. 1758)
Location: Coastal waters of Iceland
Type location: Skagafjordur, northern Iceland (66° 4'34.90"N, 19° 9'22.28"W).
Site of infection: The nucleus of lymphocytes and lymphocyte precursor cells. Infected lymphocytes infiltrate haematopoietic tissues (kidney and spleen), heart myocardium, gills and skin.
Etymology: The specific name cyclopteri refers to the generic assignment of the host fish.
Type material: Two stained wet mount slides and two histological sections have been submitted to the collections of the Natural History Museum, London, and assigned the accession numbers: (kidney tissue) NHMUK 2013.1.15.1, (heart tissue) NHMUK 2013.1.15.2, (kidney imprint) NHMUK 2013.1.15.3, (kidney imprint) NHMUK 2013.1.15.4. DNA sequence data has been submitted to GenBank with the accession number [KC203457].