112, Bull. Eur. Ass. Fish Pathol., 35(3) 2015
ȱȱȱȱǻ DZȱ ¢Ǽȱȱ¢ǰȱ ȱȱ ȱȱMicrogemma tilanpasiri n. sp. ȱȱ ȱ¢ȱTrypauchen vagina
M. A. Freeman1,2*, W. W. Chong1 and K. H. Loh1
1Institute of Ocean and Earth Sciences, University of Malaya, Kuala Lumpur, Malaysia; 2Institute for Experimental Pathology at Keldur, University of Iceland, Reykjavik, Iceland
Abstract ȱ ȱȱȱǰȱMicrogemma tilanpasiriȱǯȱǯǰȱȱȱȱȱ ȱ goby, Trypauchen vaginaǰȱȱ¢ǯȱȱȱȱȱ¡ȱȱȱȱ ȱ ȱȱȱ ȱȱǯȱȱȱȱ¢ȱ¢ȱȱȱȱȱ ȱřǯşŘȹƹȹŖǯŘŗȱΐȱȱȱȱŘǯŞŝȹƹȹŖǯŗŜȱΐȱȱ ǯȱȱȬȱȱ ȱȱ ȱȱ¢ȱȱȱȱȱȱȱȱȱȱŗŘȬŗřȱȱȱȱ ęȱȱęǰȱȱȱ ȱ ǯȱȱȱȱȱȱȱȱȱ ¢ȱȱ ȱȱȱȱȱȱȱȱȱȱMicrogemma. ȱȱȱȱȱȱȱȱȱȱM. carolinusȱ ȱȱ¢ȱȱşşǯŘřƖȱȱ ŗŘşśȱȱȱȱȱȱȱȱǯȱ ǰȱěȱȱȱȱȱȱ ȱȱȱęȱȱ ȱȱȱȱěǰȱȱM. tilanpasiri as a ȱȱǯȱȱȱȱęȱȱȱMicrogemmaȱȱȱȱ ęȱȱȱęȱȱȱ ȱ¢ȱȱęǯȱȱęȱȱȱȱȱȱ ȱȱ ȱȱȱȱȱ ȱM. tilanpasiriDzȱ ȱȱęȱ ȱȱ ȱ Glugea sp. in the visceral mesentery.
ȱȱȱȱȱȱȱ ȱȱȱȱȱȱȱȱ between ¢ȱȱ¡ȬȱȱȱȱMicrogemma and Glugea, that they probably ȱȱȱȱěȱȱȱȱȱ ¢ȱ¢ȱȱȱȱęǯ
Introduction Microsporidians are common intracellular crogemmaǰȱȱȱ ȱȱęȱȱȱ ȱȱęǯȱȱȱȱ¡- ȱǻȱȱǯǰȱŘŖŗŘǼǯȱ ȱȱ¢- sitic complexes, characterised by an extensive ¢ȱȱȱȱ¡ȱȱȱȱ ¢ȱ ȱȱȱǰȱȱȱ ȱȱǰȱȱ¢ȱȱȱȱȱȱ as xenomas. Currently there are six species muscle (Canning et al., 2005). Microsporidians ȱȱȱ¡ȬȱȱMi- ȱȱȱGlugeaȱȱȱ¡ȱȱ
ȘȱȱȂȱȬDZȱřř ȓǯ Bull. Eur. Ass. Fish Pathol., 35(3) 2015, 113
ęǯȱ ǰȱȱȱȱȱ ȱ¢ȱGlugea ȱȱȱȱ ȱȱĚȱȱ species are involved, and the type species, G. ȱ ȱȱȱȱ¢ǯȱȱȱ anomalaǰȱȱȱȱȱȱȱ gobies sampled were transported to the labora- multiple hosts globally (Lovy et al., 2009). ¢ȱȱȱȱȱ£ȱȱǯȱ ȱ ęȱ ȱȱȱ¡ȱȱȱ- ȱȱȱȱȱȱȱȱȱ ȱȱȱǯȱȱ vertebrates, and comprise over 1700 species in ¢ȱȱȱȱȱȱęȱ ȱ¡ȱ ȱȱŘŖŖȱȱȱǰȱȱȱ with an Olympus BX-41 compound microscope ȱęȱǻ ȱȱǰȱŘŖŗŘǼǯȱȱ ȱȱȱȱȱęǯȱ ¢ȱ¢ȱǻȱǼȱȱ Images were taken using a Leica DMLB digital 12 genera and 23 species (Murdy, 2011). Thirty ȱȱȱȱȱ ȱȱŘŖȱȱ ęȱȱȱ¢ȱȱȱȱ¢ȱ ȱȱȱ ǯȱ ȱȱ with seven in the Amblyopinae: Paratrypauchen ǰȱȱȱȱȱȱęȱǰȱ microcephalus, Taenioides anguillaris, T. cirratus, ȱę¡ȱȱşśƖȱȱȱȱ¢ȱȱ T. gracilis, T. nigrimarginatus, Trypauchen vagina ȱ¡ȱę¡ȱȱŘǯśƖȱ¢ǰȱ and T. pelaeos (Ambak et al., 2010; Murdy, 2006; ȱȱȂȱěȱǻŖǯŗǰȱ ȱŝǯŚǼȱ Randall and Lim, 2000). ȱȱȬę¡ȱȱŗƖȱȱ¡ȱ ȱȱȱȱȱ¢ȱȱ Trypauchen vagina, the burrowing goby (locally (Freeman and Kristmundsson, 2013). Semi-thin called tilan pasirǼǰȱȱȱȱȱ¢ȱȱ ȱȱŖǯśȱΐȱȱ ȱȱȱȱ ȱȱȱȱ ȱȱȱ ȱ ȱȱȱȱȱȱȱ- ȱȱȱ Ȭȱęǯȱ¢ȱȱ ȱȱȱ ȱŗƖȱ£ȱ ȱ ȱ¢ȱ ȱȱȱȱȱǰȱȱȱ ŗƖȱ¢ȱȱȱŗƖȱ¡ȱǻśŖDZśŖǼǯ ȱȱȱȱǯȱTaenioides nigrimarginatus,ȱȱęȱȱ¢ȱȱȱ ȱȱ ȱ¡ȱȱȱȱ in similar habitats to T. vagina, typically along using a GeneMATRIX DNA isolation kit (EURx ȱ¢ȱĴȱȱȱȱȱ- Ǽȱ ȱȱȱǯȱȱ¡- ies (Rainboth, 1996). ȱȱ ȱȱȱ¢ȱȱȱ subunit (SSU), internal transcribed spacer (ITS) ȱȱȱ ȱȱȱȱ ȱȱȱȱǻǼȱȱȱȱ ȱȱȱȱT. vaginaȱȱ¢ȱȱ ȱȱǻǼȱȱȱ- report a Glugeaȱǯȱȱȱȱ¢ȱ ans and the mitochondria cytochrome c oxidase in T. nigrimarginatus. We demonstrate that the ȱŗȱȱǻŗǼȱȱȱęǯȱȱȱ ȱȱȱȱȱȱ- ȱȱȱȱȱȱ rowing goby, T. vagina, belongs in the genus previously described (Freeman et al., 2013) with Microgemma and propose the name Microgemma ȱȱȱȱȱȱȱ tilanpasiri n. sp. descriptions. In addition, primer MG-1350r 5´tc- Ĵȱȱȱȱȱȱřȼȱȱ Materials and methods ȱȱǰȱȱęȱȱȱ- ȱ ȱȱȱȱ¢ȱȱȱȱ ǰȱ ȱȱ ȱȱȱ ȱȱ 114, Bull. Eur. Ass. Fish Pathol., 35(3) 2015
ŞŝŖȱśȼĴĴȱȱȱȱęȱ ȱȱ ȱȱǁşşǯşƖȱȱȱȱ ȱȱȱǯȱŗȱȱȱęȱȱ ŗȱȱǻ ŖŖŖŘřŜǼȱȱȱȱȱ ȱęȱȱȱȱȬŗȱȧȱŗȱ- T. vaginaȱȱȱ ȱǯȱȱŗȱ- scribed by Ward et al. (2005). Positive PCR prod- ȱȱȱ ȱT. nigrimarginartus were ȱ ȱęȱȱGeneMATRIX PCR puri- ȱȱȱŜşşȱȱȱȱȱȱ ęȱȱǻ¡ȱǼǰȱȱȱȱ were most closely related to the CO1 sequence ȱȱȱ¢ȱȱ ǻ ŗŞŖŝŘşǼȱȱOdontamblyopus rubicundus (Go- Cycle Sequencing chemistry utilising the same DZȱ¢Ǽȱȱȱ ȱȱşŗƖȱ ǯȱȱȱ ȱȱȱ ¢ǯȱȱŗȱȱȱȱ ȱȱȱȱȱȱȱȱ ȱ¢ȱȱȱĴȱȱ ȱ the expected sizes, and compared to sequences under the accession numbers KJ865405-7. available in the GenBank databases. CLUSTAL_X ȱȱȱȱȱȱȱ- ȱęȱT. vagina had numerous round to oval- centage divergence matrices were constructed shaped macroscopic cysts randomly distributed using the neighbour-joining method based on throughout the liver tissue, some visible at the the Kimura 2-parameter model (Saitou and Nei, ȱ ȱȱȱ¢ȱȱȱ ŗşŞŝǼǯȱ¢ȱ¢ȱ ȱȱ ȱǯȱȱ¢ȱȱȱ¢ȱȱ using the maximum likelihood methodology in ȱȱȱ¢ȱ¢ȱȱȬ PhyML (Guindon et al., 2010) with the general shaped microsporidian spores (Figure 1A.). time-reversible substitution model selected and ¢ȱȱȱŚȱęȱǻȱƽȱŞŖǼȱȱřǯřȱ ŗŖŖŖȱȱǰȱȱ¢ȱȱ ȬȱŚǯŚΐȱǻȹƹȹǯǯȹƽȹřǯşŘȹƹȹŖǯŘŗǼȱȱȱȱ analysis using MrBayes v. 3.2.2 (Ronquist and ŘǯśȱȬȱřǯřȱΐȱǻŘǯŞŝȹƹȹŖǯŗŜǼȱ ȱǻȱŗǼǯȱȱ Huelsenbeck, 2003). For the BI analysis models ranged in size to over 1 mm in diameter and ȱȱȱ ȱęȱȱȱ could be discrete or in small clusters. In semi- the alignment using MrModeltest v. 2.2 (Nylander thin histological sections, xenomas packed with et al., 2004). ȱ ȱȱ ȱȱȱ¢ȱ ȱȱ ȱĴȱȱȱȱęȱ Results ȱȱȱǯȱ ȱȱ¡ȱ ȱȱȱśȱȱǻŗŜŝȬŗŞŗȱȱǼȱȱT. had a relatively thin xenoma wall, which was vaginaȱ ȱȱȱęȱ- ¢ȱȱȱǻȱŗǯǼǯȱ ȱ ¢ȱȱDZȱȱȱȱȱęȱśŚȬśŞȱ ęȱ ȱȱȱȱȱǰȱ ǻȱśśǯŘǼDzȱȱȱȱȱęȱŚřȬŚşȱ ǰȱȱȱęȱ ȱȱȱ ǻȱŚŜǯŞǼǯȱȱȱȱŗśȱȱǻŗŚşȬŘŝŞȱ ǯȱ¢ȱȱȱȱT. nigrimar- ȱǼȱȱT. nigrimarginatus were caught and ginartus had a ȱǰȱ ȱȱ ęȱ¢ȱDZȱȱȱȱȱ single small xenoma (<1mm in diameter) visible ęȱśŖȬśśȱǻȱśŗǯśǼDzȱȱȱȱȱęȱ on the visceral mesentery, not embedded in the 43-48 (mean 44.2). CO1 sequences were gener- ǯȱȱ¡ȱ ȱȱȱȱ¢ǯ ȱȱ ȱęȱȱȱǯȱȱ ȱȱ ȱȱȱT. vaginaȱěȱȱřȱ On the TEM sections no developing stages were ȱȱȱŜşŞȱȱȱȱ ǰȱȱȱȱ ȱȱȱěȱ Bull. Eur. Ass. Fish Pathol., 35(3) 2015, 115
a b
Figure 1.ȱǻǼȱȱȱȱMicrogemma tilanpasiri ǯȱǯȱǻǼȱȬȱȱȱȱȱȱ ȱ ȱ¡ȱęȱ ȱȱǯȱǻȱ¢ȱ£Ǽǯ
Table 1.ȱȱȱTrypauchen vaginaȱȱȱȱȱǻŘŖȱȱęǼǯ
Host Spore measurements (μm) Sample Total length (cm) Length Width Fish 1 18.1 3.9 ± 0.17 2.8 ± 0.16 Fish 2 17.5 3.9 ± 0.23 2.9 ± 0.13 Fish 3 17.7 3.9 ± 0.19 2.9 ± 0.2 Fish 4 16.7 4.0 ± 0.23 2.9 ± 0.13 Mean (n=80) 3.9 ± 0.21 2.9 ± 0.16
ȱȱȱȱŗŘȬŗřȱȱȱȱȱ 2)Dzȱȱȱ¢ȱ¡ȱȱMicrogemma ęǰȱȱȱ ȱ ȱǻȱŘǯǼǯȱ- ǯȱȱ ȱȱȱȱȱȱǯȱȱ veloping stages, such as meronts, are sometimes ȱȱȱŚşřȱȱ ȱȱȱȱ ȱȱȱȱ¡ǰȱ ǰȱȱȱ ȱ¡ȱȱT. nigrimarginatus, which ȱȱȱ£ȱȱȱȱ- ȱȱȱřȼȱȱȱȱȱȱ ȱȱȱȱȱǯ ȱȱŞŝŖȦ ȬŗřśŖǯȱȱȱ ȱ¢ȱȱȱǻǁşşƖǼȱȱȱ ȱȱȱȱȱŗŞśŚȱȱ ȱ Glugea isolates: G. anomala (AF056016), G. her- ȱȱŚȱȱȱȱT. vagina which twigi (GQ203287), G. stephani (AF056015), G. included the SSU, complete ITS and partial plecoglossi (AB623035), G. atherinae (U15987) ǯȱȱęȱȱ ȱȱ and Glugea sp. GS1 (AJ295325). Microsporidian ȱȱȱȱȱęǯȱ ȱȱȱȱȱ¢ȱ ȱȱȱȱşşƖȱ¢ȱȱ ȱȱĴȱȱ ȱȱȱ ȱȱȱȱMicrogemma over the accession numbers KJ865404 (M. tilanpasiri) ȱȱȱȱ ȱȱȱ(Table and KJ865408 (Glugea sp.). 116, Bull. Eur. Ass. Fish Pathol., 35(3) 2015
Figure 2.ȱȱȱȱȱȱȱȱȱMicrogemma tilanpasiri n. sp. showing the ȱęȱ ȱŗŘȬŗřȱȱȱȱ ȱ ǰȱȱƽȱŗȱΐǯȱǻȱ¢ȱ£Ǽǯ
Table 2:ȱȱȱȱȱȱǰȱȱǰȱȱȱȱȱǰȱ ȱǰȱȱȱȱȱȱ
(1) (2) (3) (4) (5) (6) (1) oaȱaa - 99.23 98.51 93.19 98.80 94.35 (2) oaȱao 1295 - 98.84 93.28 99.03 94.61 (3) oaȱa 1343 1292 - 93.25 99.28 94.33 (4) oaȱa¢ 1233 1220 1230 - 95.31 96.69 (5) oaȱa 835 822 833 832 - 95.55 (6) aaȱbęȱ 1238 1225 1235 1239 832 -
Molecular phylogenetic analyses consistently carolinusǰȱȱ£ȱǻȱřǯǼǯȱȱ place Microgemma tilanpasiri n. sp. with other divergence matrices also showed M. carolinus to ȱȱȱȱMicrogemma within the ȱȱȱǰȱȱȱȱȱǰȱ ¢ȱǰȱȱȱ- ȱȱȱ¢ȱȱşşǯŘřƖȱȱ ology used, being most closely related to M. ŗŘşśȱȱȱȱȱȱȱǻȱŘǼǯ carolinusȱȱȱȱǰȱTrachinotus Bull. Eur. Ass. Fish Pathol., 35(3) 2015, 117
Potaspora morhaphis EU534408
Kabatana takedai AF356222 1.0 EF202572 1.0 Kabatana newberryi
Kabatana sp. EU682928
66 Microgemma tilanpasiri n. sp
Microgemma carolinus JQ085991 1.0 C Microgemma vivaresi AJ252952 1.0 63 B Microgemma tincae AY651319 Tetramicridae AF364303 1.0 Tetramicra brevifilum 1.0 A Microgemma caulleryi AY033054 Spraguea gastrophysus GQ868443 98 AF033197 Spragueidae 0.01 Spraguea lophii 87 Spraguea americanus AY465876
Figure 3.ȱ¢ȱȱ¢ȱȱ ȱȱȱȱ ȱȱȱȱ ȱȱȱǯȱMicrogemma tilanpasiriȱǯȱǯȱȱ¢ȱȱȱȱȱȱȱȱ ȱȱǰȱȱȱ¢ȱȱȱȱǰȱ ȱȱǯ
a¡ooȱa¢ Type material: semi-thin sections have been Phylum: Microsporidia Balbiani, 1882 lodged in the collection ȱȱ ȱȱ¡- Class: Marinosporidia Vossbrinck and Debrun- ȱ¢ǰȱȱ ǰȱ¢ȱȱ ner-Vossbrinck, 2005 Iceland, under the accession numbers 2013:Mi- ¢DZȱȱĴ ȱȱĴ ǰȱ Mt-1-3. 1980 Genus: MicrogemmaȱȱȱĴ ǰȱŗşŞŜ Discussion Species: Microgemma tilanpasiri n. sp. ȱȱȱęȱȱȱMicrogemma in- Type host: Trypauchen vagina (Bloch and Schnei- ȱęȱȱȱ ǰȱȱȱęȱȱ der, 1801), (Gobiidae: Amblyopinae). ȱMicrogemmaȱȱȱȱ Ȭęǯȱ Type locality: Bagan Sungai Buloh, Kuala Se- M. ovoideaȱȱȱȱȱȱȱ langor, Peninsular Malaysia. ęȱȱǰȱȱȱȱęȱ ȱ ȱȱDZȱ ȱȱ¢ȱȱŝȬşȱȱȱȱȱ ¢¢DZȱęȱȱȱȱȱ¢ȱ ęȱǻàȱȱǯǰȱŗşşŜǼǯȱM. carolinus is ȱȱȱęȱȱtilan-pasir (sand eel). ǁşşƖȱȱȱȱȱǰȱȱȱȱ 118, Bull. Eur. Ass. Fish Pathol., 35(3) 2015
ȱȱȱȱȱ£ȱȱ ȱȱȱȱ¢ȱȱęȱǻȱ ¢ȱȱŞȬşȱȱȱȱȱęȱǻȱ and Dyková, 2005). T. vagina are omnivorous et al., 2012). ęǰȱ¢ȱ¢ȱȱȱȱȱ stray close to their burrows (Murdy, 2006), so ȱȱ¢ȱ ȱȱȱȱȱȱ it is possible that either unaided direct trans- ȱȱȱ ȱ¢ǰȱT. vagina, was ȱȱȱȱȱȱ ŗŖŖƖȱǻśȦśȱęǼǯȱ ǰȱȱęȱȱ¢ǰ ȱȱȱȱȱȱȱ- T. nigrimarginatus,ȱȱȱȱȬ¢ȱ mission in M. tilanpasiri. The ęȱȱ¢ǰ (Amblyopinae), and caught at the same location T. nigrimarginatus, lives in the same habitat and ȱȱȱȱǰȱ ȱȱ- is also ȱȱȱȱȱȱȱ ed with MicrogemmaȱǻŖȦŗśȱęǼǯȱȱęȱ ȱȱǻǰȱŗşşŜǼǰȱǰȱ suggest that M. tilanpasiriȱǯȱǯȱȱȱęȱ it would almost certainly be exposed to the ȱ¢ȱȱȱ¢ȱ¢ȱȱęȱ ȱȱȱȱ¡ȱ¢ȱT. (Trypauchen spp.) or possibly only T. vagina. vaginaǰȱȱȱȱȱȱ¢ȱȱ ȱǰȱȱȱȱȱȱMicrogemma, M. by M. tilanpasiri. ovoideaǰȱȱȱȱȱȱȱȱ ȱęȱǻȱȱǯǰȱŘŖŗŘǼǯȱM. ovoidea has been Although M. tilanpasiri has a high similarity ȱȱȱȱȱęȱȱ ȱȱȱȱMicrogemma with respect ȱȱȱȱȱǰȱȱ ȱȱȱǰȱȱȱȱęȱȱ ȱȱȱǰȱȱȱȱȱ ȱȱȱȱęǰȱȱȱ ȱȱȱęȱȱȱȱǻȱ ȱȱȱ¢ȱȱȱȱȱ- ǭȱǰȱŗşŞŜDzȱàȱȱǯǰȱŗşşŜǼǯȱ ǰȱȱ ¢ȱȱęǯȱȱȱȱ ȱȱ DNA data exist and ultrastructural studies are ȱȱȱȱȱȱȱ¢ȱȱ ȱȱȱǰȱǰȱȱȱȱ ȱȱȱȱȱȱȱ ȱ ȱȱ¢ȱ¢ȱȱȱȱǯȱ ȱȱȱȱȱȱ ȱȱȱȱȱȱȱ descriptions provide these additional regions ȱȱȱȱGlugea (Lovy et al., 2009), ȱȱǰȱȱ¢ȱ ȱĴȱ¢ȱ ȱ¡ȱȱȱȱȱȱȱ ȱȱȱǯ ȱĜȱȱȱ¢ȱ studies, so it remains unknown whether G. ȱȱȱȱȱ¢ȱ¢ȱ anomolaǰȱȱ¢ȱǰȱȱȱęȱ ȱ¡Ȭȱȱȱ species or not. The GlugeaȱǯȱȱȱT. Glugea and Microgemmaȱȱȱȱȱ nigrimarginatus in this study adds another host ěȱȱȱȱȱ¢ȱ¢ȱ ȱȱȱȱǰȱȱȱȱ ȱȱȱęǯȱ ǰȱȱȱ rDNA sequence obtained was almost identical ěǰȱȱȱǰȱ ȱȱȱ to numerous sequences in the databases. to unambiguously demonstrate this.
ȱȱ¡Ȭȱ- ians is known to take place directly between ȱȱȱ¢ȱ ȱȱ¢ȱȱ- ęȱȱȱȱǰȱȱȱȱ ¢ȱȱ¢ȱȱŖŖŗȬŗřǯ Bull. Eur. Ass. Fish Pathol., 35(3) 2015, 119
M, Hordijk W and Gascuel O (2010). References New algorithms and methods to estimate ȱǰȱ ȱǰȱ ȱ ȱȱ ěȱ maximum-likelihood phylogenies: assessing MA (2010). “Fishes of Malaysia”. Penerbit ȱȱȱ¢ȱřǯŖǯȱSystematic Universiti Malaysia Terengganu. 334 pp. Biology 59ǰȱřŖŝȮřŘŗǯ àȱ ǰȱàȱ ǰȱ ȱȱȱ¸ȱ Lom J and Dyková I (2005). Microsporidian CP (1996). Ultrastructure and development ¡ȱȱęȱȱȱ ȱǯȱ ȱMicrosporidium ovoideum (Thélohan,1895) Folia Parasitologica 52ǰȱŜşȮŞŗǯ ǰȱŗşŝŝǰȱȱȱȱȱ ȱȱȱęȱǻCepola macrophthalma L.). Lovy J, Kostka M, Dyková I, Arsenault G, European Journal of Protistology 32ǰȱśřŘȮśřŞǯȱ Pecková H, Wright GM and Speare DJ ǻŘŖŖşǼǯȱ¢¢ȱȱ¢ȱȱGlugea Canning EU and Lom J (1986). “The hertwigiȱȱ ȱȱOsmerus mordax Microsporidia of Vertebrates”. Academic ȱȱȱ ȱ ǰȱǯȱ Press, London, UK. Diseases of Aquatic Organisms 86ǰȱŘřśȮŘŚřǯ Canning EU, Feist SW, Longshaw M, Okamura B, ¢ȱȱǻŘŖŖŜǼǯȱȱȱȱȱȱęȱ Anderson CL, Tsuey TM and Curry A (2005). genus Trypauchen (Gobiidae: Amblyopinae). Microgemma vivaresi n. sp. (Microsporidia, Zootaxa 1343, 55-68. Ǽǰȱȱȱȱȱ ȱȱȱǰȱTaurulus bubalis ¢ȱȱǻŘŖŗŗǼǯȱ¢ȱȱ¢ǯȱ ǻǰȱŗŝŞŜǼȱǻ¢ǰȱĴǼǰȱ In “The Biology of Gobies” (B. G. Kapoor), ȱǰȱĴȱęǯȱJournal of Eukaryotic p. 107-118. Science Publishers. DZŗŖǯŗŘŖŗȦ Microbiology 52ǰȱŗŘřȮŗřŗǯȱ b11397-10. Casal G, Matos E, Garcia P, Al-Quraishy S Nylander JAA, Ronquist F, Huelsenbeck JP and Azevedo C (2012). Ultrastructural and and Nieves-Aldrey JL (2004). Bayesian ȱȱȱMicrogemma carolinus ¢ȱ¢ȱȱȱǯȱ ǯȱǯȱǻǼǰȱȱȱȱȱ Systematic Biology 53ǰȱŚŝȮŜŝǯȱ ęȱTrachinotus carolinus (Carangidae) in Rainboth WJ (1996). ȃȱȱęȱ Southern Brazil. Parasitology 139, 1720-1728. ęȱȱȱę¢ȱǯȱȱ Freeman MA, Kasper J and Kristmundsson ȱȱȱȄ. Rome, FAO. Á (2013). Nucleospora cyclopteri n. sp., an pp. 213. ȱȱȱ ȱ ȱ ȱȱȱ ȱǻŘŖŖŖǼǯȱȱȱȱ ęǰȱCyclopterus lumpus L., in Icelandic ȱęȱȱȱȱȱǯȱThe Raĝes waters. Parasites & Vectors 6, 49. Bulletin of Zoology 8, 569-667. Freeman MA and Kristmundsson Á (2013). Ronquist F and Huelsenbeck JP (2003). MrBayes ȱȱNucleospora cyclopteri, an řDZȱ¢ȱ¢ȱȱȱ ȱȱȱȱ mixed models. Bioinformatics 19ǰȱŗśŝŘȮŗśŝŚǯ ȱęȱǻCyclopterus lumpus L.). Bulletin of the European Association of Fish Saitou N and Nei M (1987). The Neighbour- Pathologists 33, 194-19. ȱ DZȱ ȱ ȱ ȱ ȱ reconstructing phylogenetic trees. Molecular Gill A and Mooi R (2012). Thalasseleotrididae, Biology and Evolution 4, 406-425. ȱ¢ȱȱȱȱęȱȱ New Zealand and temperate Australia, Ward RD, Zemlak TS, Innes BH, Last PR ȱȱȱęȱȱȱȱ¡ǰȱ and Hebert PDN (2005). DNA barcoding the Gobiidae (Teleostei: Acanthomorpha). Ȃȱ ȱ ǯȱ Philosophical Zootaxa 3266ǰȱŚŗȮśŘǯ Transactions of the Royal Society London B 360, 1847-1857. ȱǰȱ¢ȱ ǰȱȱǰȱȱ