Myxosporea: Multivalvulida) Including Seventeen New Host Records for Kudoa Thalassomi

Folia Parasitologica 58[1]: 1–16, 2011 © Institute of Parasitology, Biology Centre ASCR ISSN 0015-5683 (print), ISSN 1803-6465 (online) http://www.paru.cas.cz/folia/

Low host specificity in the Kudoidae (Myxosporea: Multivalvulida) including seventeen new host records for Kudoa thalassomi

Mieke A.A. Burger1,2 and Robert D. Adlard1,2

1 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia; 2 Biodiversity Program, Queensland Museum, South Brisbane, Queensland 4101, Australia

Abstract: Kudoid parasites are known to infect a large variety of fish. A significant proportion of Kudoa species have relatively low host specificity, with a single species able to infect multiple host species representing various host families even from different host orders. Since DNA sequences have been associated with myxosporean species characterisations, it has become far easier to determine host range of new species and validate host records from earlier descriptions. This study investigated the host specificity of a kudoid parasite, Kudoa thalassomi Adlard, Bryant, Whipps et Kent, 2005, from the Great Barrier Reef in Australia using DNA sequence analysis and morphology. The results revealed the host specificity to be broad, withK. thalassomi identified in 18 different fish species representing six different fish families. This study also compares current genetic information from different host isolates of Kudoa Meglitsch, 1947 to their host ranges recorded in existing literature. From this analysis, only half of the Kudoa species with multiple host records (27 Kudoa species) have half or more isolates that are genetically characterised, and thus specifically identified with a high confidence, from their known hosts. Only five kudoid species have genetically characterised isolates from all of their recorded hosts. Keywords: Myxosporea, Kudoidae, host specificity, Kudoa thalassomi, genetic variation

Parasites from the monotypic family Kudoidae Meg- fish families from three fish orders (Egusa and Shiomitsu litsch, 1960 are microscopic, spore-forming organisms 1983, Sugiyama et al. 1999, Diamant et al. 2005), and found commonly in marine fish. Some of these parasites the cosmopolitan K. thyrsites (Gilchrist, 1924) has been are of significant concern to marine aquaculture through recorded from 18 different fish families representing nine their production of post-mortem myoliquefaction, fish orders (Whipps and Kent 2006). Recent trends to encephalomyelitis, and obvious cysts in the musculature incorporate genetic data in species descriptions facilitates (Egusa 1986a, Alvarez-Pellitero and Sitja-Bobadilla the unambiguous identification of infections of described 1993, Moran et al. 1999, Grossel et al. 2003). However, kudoid species occurring in new hosts, regardless of some kudoid species seem to cause relatively little effect phenotypic plasticity, and equally, it facilitates the in some hosts while causing significant issues in other recognition of novelty in typically character-poor, hosts, e.g. Kudoa amamiensis Egusa et Nakajima, 1980 in morphologically similar, species. This study compares pomacentrid fish (Egusa and Nakajima 1980, Burger et al. the host range of kudoid species determined by molecular 2008) compared to Seriola spp. diagnosis with those that were determined prior to, or Clearly, the presence of kudoid parasites can impact without the aid of DNA sequences, and reports 17 new on aquaculture production, with the host specificity of host records for Kudoa thalassomi Adlard, Bryant, such parasites being a key element required to determine Whipps et Kent, 2005. the actual level of threat. Host species-specific kudoids that occur in non-commercial fish obviously pose little Materials and Methods threat, while kudoids with broader specificity may Sample collection. Fish from 214 species of 41 fish families transfer to cultured stock through the presence of locally were collected (during 2005–2008) from Heron Island (23°27′S, occurring reservoir hosts. Most myxosporeans have been 151°55′E) and Lizard Island (14°39′S, 145°7′E) on the Great Barrier Reef, Queensland, Australia. Fish were caught using reported from a single host species or host genus (Mo- line, spear, or localised sprays of clove oil anaesthetic and eutha- ran et al. 1999) but many kudoid species tend to show nised by prolonged immersion in clove oil. Identification of fish lower host specificity. For example, Kudoa iwatai Egusa hosts conforms to currently valid names and common names et Shiomitsu, 1983 has been recorded from 13 different given in FishBase (http://www.fishbase.org/).

Address for correspondence: R. Adlard, Biodiversity Program, Queensland Museum, P.O. Box 3300, South Brisbane, Queensland 4101, Australia. Phone: +617 3840 7723; Fax: +617 3846 1226; E-mail: [email protected]

1 The fish were examined for kudoid infection in the somatic Bleeker, Chrysiptera cyanea (Quoy et Gaimard), Dascyllus muscle by taking a sub-sample of fish tissue (approximately aruanus (Linnaeus), and Neoglyphidodon melas (Cuvier) 20 × 10 × 5 mm) that was placed on a glass plate (76 × 50 mm), (Pomacentridae); Scarus flavipectoralis Schultz (Scaridae); moistened with vertebrate saline (0.85% salt), and shredded to and Cephalopholis boenak (Bloch) (Serranidae). The spore a pulp using scalpel blades. A second glass plate was placed suspensions were concentrated by centrifugation for 6 min on top and the liquid squeezed out into a 2-ml centrifuge tube. at 4750 g and the supernatant discarded. The DNA from the An aliquot (~15 µl) of the suspension from the bottom of the pellet was extracted using a QIAgen DNeasy Kit (QIAGEN tube was examined for kudoid spores under an Olympus BH2 Inc., Valencia, California) according to the manufacturer’s microscope at ×400 magnification. When kudoid spores were instructions. Small subunit (SSU, 18S) rDNA was amplified detected, sub-samples of skeletal muscle were preserved in 10% using PCR primers KudMB1f 5’- GCC ATG GAT AAC TGT neutral buffered formalin for histological analysis and the re- GGT AAA TCT AGA GC -3’ (Burger et al. 2008) and universal mainder of the fish and the saline preparation were preserved 18R 5’- CTA CGG AAA CCT TGT TAC G -3’ (Whipps et al. by freezing for morphological analysis and DNA extraction 2003). Large subunit (LSU, 28S) rDNA was amplified using of spores. Where material was limited, priority went to frozen PCR primer combination Kt28S1f 5’- CAA GAC TAC CTG samples from which both morphological information and DNA CTG AAC -3’ and 28S1R 5’- GTG TTT CAA GAC GGG TCG could be recovered. -3’ (Whipps et al. 2004). PCR reactions and cycling parameters Histology. Sections (5 µm thick) were cut from selected 10% were as per Burger et al. (2008). PCR products were cloned formalin-fixed samples using standard histological techniques. using TOPO TA Cloning® Kit for Sequencing (Invitrogen, Serial sections were stained alternatively with haematoxylin and Carlsbad, CA, USA) as per the manufacturer’s instructions eosin or with Giemsa and eosin, and coverslips applied using and DNA sequence data were generated as described in Burger DePeX (BDH, England). Digital, light photomicrographs of et al. (2008). One clone of LSU was sequenced for each host, the sections were taken at ×400 magnification with an Olym- while two clones were sequenced from the LSU PCR product pus BH2 microscope using a Nikon Digital Sight DS-L1 (Nikon from Amphiprion akindynos and two clones from the second Corporation, Japan) camera. Chaetodon unimaculatus individual. Morphometric analysis. Spore suspensions in saline were SSU rDNA and LSU rDNA sequence data were edited using prepared as described above from two selected fish from two BioEdit (Hall 1999) then aligned with other kudoid sequences different families: Abudefduf bengalensis (Bloch) (Pomacen- available from GenBank using ClustalW (Thompson et al. 1994) tridae) from Heron Island and Thalassoma lunare (Linnaeus) and subsequent neighbour joining distance analyses using the (Labridae) from Lizard Island. Photomicrographs were taken of p-distance model were conducted using PAUP* 4.0b10 (Swof- the spores at ×400 magnification under phase contrast lighting ford 2002). The SSU Kudoa rosenbuschi (Gelormini, 1943) se- conditions. Spore measurements were taken from the micro- quence from Abollo et al. (2005) and the Kudoa sp. sequences graphs using the measuring tool in Nikon NIS Elements imaging from Pascual and Abollo (2008) were considered as sequences software (Nikon, Tokyo). At least 30 spores from each morpho- of Kudoa alliaria Shulman et Kovaleva, 1979 as discussed in type in apical view were used to represent the measurements Burger and Adlard (2010). Unicapsula sp., a multivalvulidan taken (side view measurements were taken from the A. benga- in the family Trilosporidae, was used as an outgroup because it lensis sample only). represents the closest known relative external to the Kudoidae. The morphological data were subjected to a principal The bootstrap confidence values were generated using 1000 component analysis using the software PAST version 1.82b bootstrap replicates. Pairwise differences were also analysed (Hammer et al. 2001). Scatter plots with 95% confidence in PAUP* 4.0b10 to determine total nucleotide distance and ellipses were generated using variant-covariant matrices. All percentage differences. other statistical analysis was conducted using Stata™ 10.0 Host specificity. A survey of the literature was conducted (StataCorp 2008). The raw data of each measurement were to compare host ranges amongst the Kudoidae Meglitsch, 1960 tested for normal distribution using the Shapiro-Wilk test with (amended by Whipps et al. 2004). All records were compared a 95% confidence level. Each normally distributed dataset was to availability of genetic data correlating to Kudoa species with tested individually for significant difference between the means multiple hosts. While every effort has been made to include from two host isolates first by Bartlett’s test for equal variance all Kudoa species with multiple hosts and to include all their (one-way analysis of variance), followed by two-sample t-test host records, it is possible that some in peripheral literature (for either equal or unequal variance depending on the outcome may have been overlooked. The authors welcome any further of the Bartlett’s test). contributions. DNA analysis. DNA was extracted from Kudoa spore suspensions that had been examined under the microscope Results during sample collection. Kudoids were sampled from 18 fish species in six different host families: Cheilodipterus macrodon Identity of new Kudoa isolates (Lacépède) (Apogonidae); Chaetodon baronessa Cuvier, three New isolates of Kudoa were collected from the somatic C. unimaculatus Bloch individuals, C. vagabundus Linnaeus, Chelmon rostratus (Linnaeus), and Heniochus monoceros muscle from 18 species of six different fish families on Cuvier (Chaetodontidae); Thalassoma lunare and T. lutescens the Great Barrier Reef, Queensland, Australia. The Ku- (Lay et Bennett) (Labridae); Abudefduf bengalensis, A. whitleyi doa samples from these fish had spores with 6 or 7 polar Allen et Robertson, two Amblyglyphidodon curacao (Bloch) capsules and spore valves within a single plasmodium individuals, Amphiprion akindynos Allen, A. melanopus (Figs. 1, 2). As such, from microscopic observation all

2 Burger, Adlard: Low host specificity in the Kudoidae isolates were morphologically consistent with the original description of Kudoa thalassomi Adlard, Bryant, Whipps –

et Kent, 2005. 1.6 2.123 1.721 1.371 width S ide P C (1.6–2.3) (1.3–2.1) The genetic sequence data produced from the 18 iso- 2.01, 0.19 (1.25–1.513) (2.039–2.237) lates in this study possess the highest genetic affinity with (1.645–1.842) K. thalassomi ex Thalassoma lunare (minimum 99.7% affinity in SSU (range of 0–10 nucleotides difference) and – 2.5 4.921 3.684 3.235 98.5% in LSU (range of 1–10 nucleotides difference)) (see length S ide P C (3.9–4.6) (2.0–3.1) Kudoa thalassomi isolates 4.28, 0.18 (4.737–5.000) Figs. 3, 4). The neighbour joining trees (Figs. 3, 4) show (3.553–3.861) (3.026–3.553) no correlation between host relatedness and sequence affinity between host isolates. Interestingly, 4 nucleotides Side view measurements (μm) – difference (0.6%) was found in LSU between the type 6.2 6.645 6.535 5.425 L ength (6.7–7.8) (5.3–7.3) sequence of Kudoa thalassomi and the Kudoa sequence 7.26, 0.34 (5.0–6.184) (6.316–6.711) from the same host, T. lunare from Lizard Island. (6.184–7.105) To examine the breadth of morphological variation, – – – two of these Kudoa isolates were subjected to more de- – width 2.1, 0.13 (1.7–2.4) (1.9–2.3) (2.0–2.2) (2.0–2.3) 2.12, 0.11 2.12, 0.07 tailed morphological analysis. The first was an isolate 2.13, 0.06 A pical P C from the type host of Kudoa thalassomi, Thalassoma lunare (Labridae), while the second was an isolate recov- – – – ered from Abudefduf bengalensis representing a different – length (3.1–4.1) (3.2–4.0) (3.3–4.3) (3.2–4.2) 3.60, 0.26 3.56, 0.18 3.88, 0.23 3.63, 0.28 and more taxonomically distant host family (Pomacen- A pical P C tridae) of coral reef associated fishes. All measurements conformed to a normal distribution using the Shapiro-

Wilk test (p-value >0.05). A principal components analy- 3.6 3.987 3.881 2.959 radius S uture (4.3–5.1) (4.5–5.2) (4.1–5.2) (4.6–5.8) (3.0–4.4) 4.57, 0.21 4.81, 0.17 4.80, 0.24 sis comparing the apical view measurements of the 6- and 5.13, 0.26 (3.421–4.408) 7-polar capsule morphotypes from these two host isolates (3.619–4.094) (2.632–3.158) (Fig. 5) shows overlap between all the morphotypes, but – – – reveals significant differences between the two isolates – (6.2–7.5) (5.5–7.5) (6.7–7.9) (6.1–7.7) 6.84, 0.30 6.56, 0.35 7.37, 0.33 6.76, 0.39

and between the two different morphotypes. This is es- width Valve pecially obvious between the two 7-polar capsule morphotypes (Fig. 5). The two sample t-test identified signifi- Apical view measurements (μm) – – – 9.37 cant differences (p-value <0.05) in the means between the 8.14 6.794 11.70, 0.6 11.70, T hickness 11.52, 0.51 11.52, (10.4–12.6) 7-polar capsule morphotype from the different hosts in (10.5–13.5) (7.632–8.684) (6.184–7.632) width, valve width, and apical polar capsule length. For (8.553–10.789) the 6-polar capsule morphotype, both the spore width and the valve width have significantly different means. 11 8.623 7.555 Width

Compared to other species that infect somatic muscle 10.658 (9.1–13.0) 13.67, 0.59 13.16, 0.59 14.27, 0.47 13.48, 0.69 (12.4–14.9) (12.3–14.9) (12.9–15.0) (12.3–14.9) (8.026–8.947) (6.842–8.158)

and are dominated by 6-polar capsule spores (see (9.474–11.842) Table 1), the two new Kudoa isolates are closest in size, 6 6 6 6 6 6 7 7 No. but generally slightly larger than those recorded from P C s K. thalassomi described by Adlard et al. (2005). The exception being for the side view polar capsule length, where the K. thalassomi from Adlard et al. (2005) is larger (mean 4.9 µm cf. 4.3 µm) than that from Abudefduf A dlard et al. (2005) and with three other species containing six polar capsules, known to infect skeletal muscle. P C = capsule. Host bengalensis from this study. There is minimal overlap in the ranges for the thickness, length, and side view polar capsule width between the new isolates and K. thalassomi. Thalassoma lunare Abudefduf bengalensis Grammatorcynus Grammatorcynus bicarinatus Neothunnus macropterus Scomberomorus commerson Thalassoma lunare The new Kudoa isolates are easily distinguished from K. grammatorcyni Adlard, Bryant, Whipps et Kent, 2005 and K. scomberomori Adlard, Bryant, Whipps et Kent, 2005 and they are generally larger than K. neothunni (Arai et Matsumoto, 1953), but there is a small overlap in size ranges in width, length, and side view polar capsule width. Kudoa neothunni has only been recorded from yellowfin able 1. Morphometric comparison (mean, standard deviation and range in parentheses) between the 6- and 7-polar capsule morphotypes of the two new K. thalassomi A dlard et al. 2005 with the original measurements from K. thalassomi T his study tuna, Thunnus albacares (Bonnaterre) from Japan and is T Kudoa species S tudy K. grammatorcyni A dlard et al. 2005 K. neothunni A rai and Matsumoto 1953 K. scomberomori A dlard et al. 2005

3 a species that causes liquefaction of the somatic muscle (Arai and Matsumoto 1953). From here on, the new Kudoa isolates will be referred to as K. thalassomi due to their high genetic affinity with this species, similar geographic locality (on the Great Barrier Reef) to where the original type specimen was collected, and their clear distinction in both genetics and morphology to other superficially similar kudoids described to date.

Kudoa thalassomi Adlard, Bryant, Whipps et Kent, 2005

T y p e h o s t : Thalassoma lunare (Linnaeus), moon wrasse (Teleostei, Labridae). T ype locality: Swain Reefs (21°36.20′S, 152°22.26′E), Great Barrier Reef, Queensland, Australia. S ite of infection: Somatic muscle, pseudocysts not evident macroscopically. New material for Kudoa thalassomi : See Figs. 1 and 2. N o t e s : Plasmodia microscopic, polysporic. No membrane or inflammatory response evident. Spores of uniform development, extra-sporogonic stage not observed. Dominant spore morphotype has six polar capsules and spore valves, minor spore morphotype has seven (Fig. 2). Radially symmetrical in apical view. Polar capsules convergent, pyriform. In side view, spores are pyramidal. Spore measurements are shown Fig. 1. Giemsa and eosin-stained histological section through in Table 1. a skeletal muscle fibre infected with Kudoa thalassomi. Both O ther hosts: Cheilodipterus macrodon (Lacépède) – large 6- and 7-polar capsule morphotypes are visible. toothed cardinalfish (Apogonidae); Chaetodon baronessa Cuvier – eastern triangular butterflyfish, Chaetodon unimaculatus Bloch – teardrop butterflyfish,Chaetodon vagabundus numbers HM022110–HM022116 for SSU and HM022117– Linnaeus – vagabond butterflyfish, Chelmon rostratus (Lin- HM022139 for LSU (see Figs. 4 and 5 for specific isolate naeus) – copperband butterflyfish, Heniochus monoceros numbers). Cuvier – masked bannerfish (Chaetodontidae); Thalassoma lutescens (Lay et Bennett) – yellow-brown wrasse (Labri- Kudoa species with multiple hosts dae); Abudefduf bengalensis (Bloch) – Bengal sergeant, Of the 84 Kudoa species described to date, 27 of them Abudefduf whitleyi Allen et Robertson – Whitley’s sergeant, Amblyglyphidodon curacao (Bloch) – staghorn damselfish, have been associated with more than one host with a me- Amphiprion akindynos Allen – barrier reef anemone fish, dian of four hosts for Kudoa species with more than one Amphiprion melanopus Bleeker – fire clownfish, Chrysip- host. Eight of these 27 Kudoa species only have two tera cyanea (Quoy et Gaimard) – sapphire devil, Dascyllus known hosts (K. azoni Aseeva, 2004, K. camarguensis aruanus (Linnaeus) – whitetail dascyllus, Neoglyphidodon Pampoulie, Marques, Rosecchi, Crivelli et Bouchereau, melas (Cuvier) – bowtie damselfish (Pomacentridae);Scarus 1999, K. lethrini Burger, Cribb et Adlard, 2007, K. me- flavipectoralis Schultz – yellowfin parrotfish (Scaridae); Ce- gacapsula Yokoyama et Itoh, 2005, K. miniauriculata phalopholis boenak (Bloch) – chocolate hind (Serranidae). Whitaker, Kent et Sakanari, 1996, K. shiomitsui Egusa O t h e r l o c a l i t i e s : Heron Island (23°27′S, 151°55′E) and et Shiomitsu, 1983, K. trifolia Holzer, Blasco-Costa, Lizard Island (14°39′S, 145°27′E) on the Great Barrier Reef, Sarabeev, Ovcharenko et Balbuena, 2006, and K. unicap- Queensland, Australia. sula Yurakhno, Ovcharenko, Holzer, Sarabeev et Balbue- New material: Giemsa and eosin-stained histological sec- na, 2007) while K. thyrsites has the highest number of host tion vouchers deposited in the collections of the Queensland species recorded at 38, followed by K. nova Naidjenova, Museum, Brisbane, Australia: ex Neoglyphidodon melas, 1975 with 20, K. iwatai with 19, and now K. thalassomi G465422; ex Amphiprion akindynos, G465423; ex Dascyl- lus aruanus, G465424; ex Abudefduf sexfasciatus, G465425. with 18. At higher taxonomic levels, 12 of the 27 species Haematoxylin and eosin-stained histological section vouch- are restricted to a single host family and 20 of the 27 are ers: ex Neoglyphidodon melas, G465426; ex Amphiprion restricted to a single fish order. Fig. 6 shows the frequen- akindynos, G465427; ex Dascyllus aruanus G465428; cy distribution of host species range in the Kudoidae. It ex Abudefduf sexfasciatus, G465429. GenBank sequence shows a positively skewed proportion where most species

4 Burger, Adlard: Low host specificity in the Kudoidae

Fig. 2. Phase contrast photomicrographs of spores from Kudoa thalassomi ex Abudefduf bengalensis. A – 6-polar capsule morphotype; B – 7-polar capsule morphotype; C – side view of spore.

(57 of 84) have only been recorded from a single host and Kudoa camarguensis Pampoulie, Marques, Rosecchi, eight species have been recorded from only two hosts. Crivelli et Bouchereau, 1999 Following are data from the literature on the 27 species Total number of hosts: 2. Perciformes: Gobiidae – Pomatoschis- of kudoids that have been recorded from multiple hosts. tus microps (type host), P. minutus. Those hosts with corresponding rDNA sequence of their Reference: Pampoulie et al. 1999. Kudoa isolates are highlighted in bold. Kudoa ciliatae Lom, Rohde et Dyková, 1992 Kudoa alliaria Kovaleva, Shulman et Yakovlev, 1979 Total number of hosts: 3.Perciformes: Sillaginidae – Sillago Total number of hosts: 6. Gadiformes: Gadidae – Micromesis- analis, S. ciliata (type host), S. maculata. tius australis (type host). Merlucciidae – Macruronus mag- References: Lom et al. 1992, Hallett et al. 1997, Burger et al. ellanicus, Merluccius australis, Me. hubbsi. Perciformes: 2007. Nototheniidae – Patagonotothen ramsayi (Notothenia ramsayi in Kovaleva et al. 1979), Notothenia conina. Kudoa clupeidae (Hahn, 1917) References: Kovaleva et al. 1979, Abollo et al. 2005, Whipps Total number of hosts: 11. Clupeiformes: Clupeidae – Alosa aes- and Diggles 2006, Pascual and Abollo 2008, Burger and Ad- tivalis (Pomolobus aestivalis in Hahn 1917), A. mediocris, lard 2010. A. pseudoharengus (Pomolobus pseudoharengus in Hahn Kudoa amamiensis Egusa et Nakajima, 1980 1917), Brevoortia tyrannus, Clupea harengus (type host). Perciformes: Labridae – Tautogolabrus adspersus. Pomato- Total number of hosts: 11. Perciformes: Carangidae – Caranx midae – Pomatomus saltatrix (syn. Temnodon saltator in Ba- sexfasciatus, Seriola dumerili, S. quinqueradiata (type lozet 1929, 1930). Scombridae – Thunnus thynnus. Sparidae host). Pempheridae – Pempheris ypsilychnus. Pomacentri- – Stenotomus chrysops. Zoarcidae – Macrozoarces america- dae – Abudefduf bengalensis, A. sexfasciatus, A. vaigiensis, nus. Pleuronectiformes: Pleuronectidae – Platichthys flesus. A. whitleyi, Chromis chrysura (syn. Chromis isharai in Egu- References: Hahn 1917, Balozet 1929, 1930, Nigrelli 1946, sa and Nakajima 1980), Chromis notata, Chrysiptera cyanea. Meglitsch 1947, Dollfus 1955, Sindermann 1961, Kovaleva References: Egusa and Nakajima 1980, Hervio et al. 1997, Sugi- et al. 1979, Pellizzato and Canzonier 1985, Shulman 1988, yama et al. 1999, Whipps et al. 2003, Burger et al. 2008. Reimschuessel et al. 2003, Webb et al. 2005.

Kudoa azoni Aseeva, 2004 Kudoa gunterae Burger et Adlard, 2010 Total number of hosts: 2. Scorpaeniformes: Hexagrammidae – Total number of hosts: 11. Perciformes: Apogonidae – Apogon Hexagrammos octogrammus, Pleurogrammos azonus (type properuptus. Pomacentridae – Abudefduf septemfasciatus, host). A. sordidus, A. sexfasciatus (type host), Chromis viridis, Reference: Aseeva 2004. Chrysiptera cyanea, Dascyllus aruanus, Dischistodus pseudochrysopoecilus, Neoglyphidodon melas, Plectroglyphido- Kudoa bora (Fujita, 1929) don leucozonus, Pomacentrus chrysurus. Total number of hosts: 3. Mugiliformes: Mugilidae – Mugil ca- Reference: Burger and Adlard 2010. rinatus, M. cephalus, M. japonicus (type host). Reference: Fujita 1929.

5 0 0 0 0 0.5 0.6 0.4 0.1 0.9 0.7 0.5 0.3 0.1 max % max variance 0 7 10 8 6 2 0 7 0 14 1 0 4 max nt max variance 3/1 8/5 2/1 6/1 2/1 2/2 5/3 4/1 8/3 4/2 2/2 6/6 14/13 No. species /families Kudoa thalassomi (8) Kudoa Kudoa gunterae (6) gunterae Kudoa Kudoa whippsi (4) whippsi Kudoa (18) thyrsites Kudoa Kudoa megacapsula (2) megacapsula Kudoa Kudoa shiomitsui (2) Kudoa Kudoa species (no. Kudoa sequences) Kudoa alliaria (12) Kudoa Kudoa lethrini (2) lethrini Kudoa Kudoa amamiensis (12) amamiensis Kudoa Kudoa kenti (2) kenti Kudoa Kudoa hypoepicardialis (6) hypoepicardialis Kudoa Kudoa iwatai (8) iwatai Kudoa Kudoa paraquadricornis (5) paraquadricornis Kudoa 1 EU041617 2 EU041618 5 EU041621 3 EU041619 DQ182561 4 EU041620 1 EU041612 AY197771 AY312279 2 EU041615 2 EU041613 A AY623795 3 EU041616 1 EU041614 7 AF034639 0 7 0 2 2 6 4 3 0 3 2 2 9 1 1 1 2 7 4 2 3 1 8 1 4 8 4 4 8 2 8 8 5 4 4 4 3 4 1 1 5 7 4 8 1 2 1 2 4 0 2 3 3 Y3 0 2 8 3 4 3 1 FJ792724 5 3 erluccius australis erluccius 4 Y1 Y 4 erluccius hubbsi erluccius 4 4 F0 F0 F0 B1 Y5 F0 2 1 8 Y5 U 1 Y5 Ja A 2 acruronus magellanicus acruronus magellanicus acruronus magellanicus erluccius hubbsi erluccius erluccius australis erluccius 1 3 acruronus magellanicus acruronus magellanicus 7 erluccius hubbsi erluccius acruronus magellanicus acruronus magellanicus FJ792723 2 AY302723 acruronus magellanicus acruronus magellanicus 1 acruronus magellanicus acruronus magellanicus AB263074 4 0 SA A AF034640 Au A 1 Brevoortia tyrannus Brevoortia ex 4 Or A BC A F0 1 Y DQ519388 Az A 4 BC A Or A SA A M a ex SA A 1 3 i FJ792707 AF414692 M a ex 1 r i 3 ae ae FJ792725 3 FJ792714 r a Fundulus heteroclitus i ex Fundulus heteroclitus F0 d FJ792722 i l M a ex Merluccius hubbsi Merluccius a ex M a ex M a ex M a ex a M a ex i M a ex Or A l M a ex SA A F0 lyacanthus i i i i i i i i M a ex AB188529 l i FJ792713 M a ex F0 u l e r r r i r r r r i o r l DQ439814 r d r FJ792708 p a a a a a a a a FJ792712 i i i i i i i a i n a l l l l l u l Or A l i l i l l l l l l l l LI HM022113 l l l u l l EU340236 AY152750 Sebastes paucispinus Sebastes ex FJ792709 indynos AF034638 AF378347 Kudoa a operuptus operuptus AY152748 kindynos Kudoa a 3 EU567027 r yanae FJ792710 erspicillatus AB183718 Chaetodon unimaculatus DQ519387 Chaetodon ptemfasciatus 1b EU340234 e Kudoa c Kudoa a Kudoa a Kudoa a Kudoa a Kudoa f 2 EU567026 Kudoa a xfasciatus Kudoa a Kudoa a Kudoa a rdidus Kudoa a 1c EU340235 iculata Kudoa a udefduf bengalensis udefduf e ex r o b AY302724 u AY302741 olivaceus Paralichthys ex eriola quinqueradiata eriola a 100 i pogon p canthochromis p phyraena pinguis phyraena n i 0 0 hromis viridis FJ792711 0 0 mphiprion a 0.5 100 0.6 0.4 0.1 egalensis egalensis A ex 0.9 0.7 0.5 0.3 0.1 egalensis egalensis AY172511 Latris lineata ex mphiprion a hrysiptera c hrysiptera egalensis egalensis egalensis 1a EU340233 egalensis budefduf s budefduf egalensis egalensis egalensis 4 EU567028 egalensis ischistodus p max % max variance Merluccius productus Merluccius mis ex budefduf whitleyi whitleyi budefduf p. ex Paralichthys p. ex lethostigma Beryx splendens splendens Beryx ex hyrsites budefduf s budefduf Cephalopholis boenak HI HM022110 Cephalopholis ex eoglyphidodon melas eoglyphidodon r exfasciatus 1b EU340238 exfasciatus budefduf s budefduf Sphoeroides annulatus ex Sphoeroides e hippsi o exfasciatus 1a EU340237 exfasciatus Thyrsites atun Thyrsites ex hyrsites FJ792718 a Merluccius capensis ex Merluccius capensis hyrsites Paralichthys olivaceus Paralichthys olivaceus ex hyrsites f exfasciatus 1c EU340239 exfasciatus i NS E NS scombrus ex Scomber hyrsites n Lepidopus caudatus ex Lepidopus hyrsites En A scombrus ex Scomber hyrsites Chaetodon unimaculatus LI HM022112 Chaetodon ex hyrsites ex Sardinops ocellatus hyrsites Coryphaena hippurus ex Coryphaena hyrsites ex Mondactylus argeneus ex Kudoa m n Salmo salar BC A ex hyrsites hyrsites ex Salmo hyrsites salar Or AF031412 Leptocottus armatus ex Leptocottus hyrsites hyrsites ex Merluccius productus hyrsites Merluccius productus ex hyrsites capsula ex S a Parophrys vetula ex Parophrys vetula hyrsites Pholis ornata ex hyrsites A hippsi ex Salmo salar Ch A ex hyrsites hippsi ex A i a eriola quinqueradiata quinqueradiata eriola hyrsites ex Aulorhynchus flavidus hyrsites a 0 hippsi ex A 7 10 A ex enti 8 6 2 budefduf b budefduf capsula ex S empheris ypsilychnus 0 7 0 14 g 1 0 4 Kudoa t Kudoa Kudoa s Kudoa Kudoa chaetodoni Kudoa C ex unterae a budefduf b budefduf aranx s aralichthys olivaceus D enti ex Thunnus albacares Thunnus albacares a ex akifugu rubripes akifugu rubripes e Kudoa yasunagai Kudoa budefduf b budefduf g aranx s g n budefduf b budefduf Kudoa t Kudoa budefduf b budefduf Kudoa t Kudoa budefduf b budefduf A iensis ex Kudoa t Kudoa aranx s Kudoa w Kudoa C ex unterae e e max nt max variance Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Thalassoma bifasciatum a ex Kudoa t Kudoa Kudoa t Kudoa A ex unterae Gymnocranius audleyi audleyi Gymnocranius ex lethrini Kudoa Kudoa t Kudoa m Lethrinus harak DQ519388 Lethrinus ex lethrini Kudoa Kudoa t Kudoa r Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa m Kudoa t Kudoa unterae ex A unterae N unterae ex a 99 agiotaenia Kudoa d Kudoa o Kudoa t Kudoa Kudoa w Kudoa A unterae ex 60 Kudoa w Kudoa Kudoa p Kudoa u l Kudoa k Kudoa v 72 r Kudoa w Kudoa Kudoa neurophila Kudoa 99 i iensis ex S 64 99 Kudoa Kudoa Kudoa k Kudoa v iensis ex A P itsui ex iensis ex P iensis T itsui ex 65 Kudoa m Kudoa 88 ex Cynoscion nebulosus FJ790311 ex inornata Kudoa m ulvoguttatus AY078428 puensis FJ792717 iensis ex C iensis ex A ex Neoglyphidodon melas LI HM022114 thalassomi ex Kudoa Kudoa g Kudoa Kudoa thalassomi Kudoa m m a m m Kudoa m Kudoa AM183300 aurata Liza ex a iensis ex C iensis iensis ex A 79 gnobilis 1 FJ792714 Kudoa g Kudoa exfasciatus FJ792719 gnobilis 2 FJ792716 iensis ex A a o m iensis ex A a m o iensis ex A iensis ex C 100 100 i Dascyllus aruanus Dascyllus thalassomi ex Kudoa 3/1 8/5 2/1 i m 6/1 Kudoa g Kudoa 63 2/1 2/2 5/3 4/1 8/3 4/2 Kudoa g Kudoa 2/2 m m 6/6 a m a Kudoa g Kudoa 14/13 h 89 Kudoa thalassomi Kudoa m 87 m h m m m m Kudoa c Kudoa a a a 97 LI HM022115 flavipectoralis Scarus thalassomi ex Kudoa a Kudoa monodactyli Kudoa m a a m 75 a a ex Thalassoma lunare AY302738 thalassomi ex Kudoa HI HM022116 Thalassoma lutescens thalassomi ex Kudoa LI HM022111 cyanae Chrysiptera thalassomi ex Kudoa Kudoa o Kudoa 62 ex Sillago ciliata e ex DQ519390 m m m cardialis gronovii AY302722 ex Nomeus cardialis nigritus AY302722 ex Epinephelus cardialis ex Pogonias cromis AY302722 cardialis ex Pomatomus saltatrix AY302722 cardialis ex Caranx crysos AY302722 cardialis AY302722 ex Lobotes surinamensis No. species /families m i i i m m i i AY152749 ypsilichnus Pempheris ex minithyrsites Kudoa m i sp. AY382606 Lateolabrax ex lateolabracis Kudoa m 52 a aranx i t aranx i aranx p arangoides p aranx s ep a 61 ep ep ep ep ep i l 100 o Kudoa a Kudoa o o o o 78 o 59 i Kudoa s Kudoa Kudoa s Kudoa AY302739 bicarinatus Grammatorcynus ex grammatorcyni Kudoa 100 Kudoa a Kudoa p arangoides f p p p p p y y y 62 Kudoa a Kudoa y y AY302737 commerson Scomberomorus ex scomberomori Kudoa y Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa trifolia Kudoa Scomberomorus commerson AY078429 commerson Scomberomorus ex permulticapsula Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa 100 Liza ramada AM490334 Liza ex unicapsula Kudoa 88 98 Kudoa c Kudoa 82 95 100 Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa C ornis ex la sp. AY302725 c 100 i u r 100 s d 58 p 100 87 a a c u i araquadricornis ex C araquadricornis ex C araquadricornis ex C araquadricornis ex C araquadricornis ex C n U Kudoa q Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa ex Neogobius melanostomus EF644198 a ex Kudoa thalassomi (8) Kudoa Kudoa gunterae (6) gunterae Kudoa Kudoa whippsi (4) whippsi Kudoa (18) thyrsites Kudoa Kudoa megacapsula (2) megacapsula Kudoa Kudoa shiomitsui (2) Kudoa Kudoa species (no. Kudoa sequences) v Kudoa alliaria (12) Kudoa Kudoa lethrini (2) lethrini Kudoa Kudoa amamiensis (12) amamiensis Kudoa Kudoa kenti (2) kenti Kudoa Kudoa hypoepicardialis (6) hypoepicardialis Kudoa Kudoa iwatai (8) iwatai Kudoa o Kudoa paraquadricornis (5) paraquadricornis Kudoa 100 Kudoa n Kudoa 97 1 EU041617 2 EU041618 5 EU041621 3 EU041619 DQ182561 4 EU041620 1 EU041612 AY197771 AY312279 2 EU041615 2 EU041613 20.0 A AY623795 3 EU041616 1 EU041614 56 7 AF034639 0 7 0 Siganus ai ex rivulatus AY514039 Dascyllus trimaculatusai ex AY514039 2 Pagrus major AY641571 ai ex 2 6 4 3 t 0 t 3 2 Apogon aureus AY514038 ai ex 2 t Sparus aurata AY514038 ai ex AY514038 variegatus Lethrinus ai ex 9 1 1 1 2 7 t 4 2 t a t 3 a 1 8 1 a 4 Lutjanus erythropterus AY644704 Lutjanus erythropterus ai ex 8 4 4 8 2 a 8 8 a a 5 4 t 4 4 w 3 w 4 1 w 1 5 7 4 8 1 a 2 1 w w w 2 4 0 2 3 3 Y3 0 2 8 3 4 3 1 FJ792724 5 3 w erluccius australis erluccius 4 Y1 Y 4 erluccius hubbsi erluccius 4 4 F0 F0 F0 B1 Y5 F0 2 1 8 Y5 U 1 Y5 Ja A 2 acruronus magellanicus acruronus magellanicus acruronus magellanicus erluccius hubbsi erluccius erluccius australis erluccius 1 3 acruronus magellanicus acruronus magellanicus 7 erluccius hubbsi erluccius acruronus magellanicus acruronus magellanicus FJ792723 2 AY302723 acruronus magellanicus acruronus magellanicus 1 acruronus magellanicus acruronus magellanicus Kudoa i Kudoa AB263074 Kudoa i Kudoa 4 0 Kudoa i Kudoa SA A AF034640 Au A 55 1 Brevoortia tyrannus Brevoortia ex 4 Or A Kudoa i Kudoa BC A Kudoa i Kudoa Kudoa i Kudoa F0 1 Y DQ519388 Az A 4 BC A Or A SA A M a ex SA A 1 3 i Kudoa i Kudoa FJ792707 AF414692 M a ex 1 r i 3 ae ae FJ792725 3 FJ792714 r a Fundulus heteroclitus i ex Fundulus heteroclitus F0 d FJ792722 i l M a ex Merluccius hubbsi Merluccius a ex M a ex M a ex M a ex a M a ex i M a ex Or A l M a ex SA A F0 lyacanthus i i i 100 i 60 i i i i M a ex AB188529 l i FJ792713 M a ex F0 u l e r r r i r r r r i o r l 99 DQ439814 r d r FJ792708 p a a a a a a a 64 a FJ792712 i i i i i i i a i n a l l l l l u l Or A l i l i l l l l l l l l LI HM022113 l l l u l l EU340236 AY152750 Sebastes paucispinus Sebastes ex FJ792709 indynos AF034638 AF378347 Kudoa a operuptus operuptus AY152748 kindynos Kudoa a 3 EU567027 r yanae FJ792710 erspicillatus AB183718 Chaetodon unimaculatus DQ519387 Chaetodon ptemfasciatus 1b EU340234 e Kudoa c Kudoa a Kudoa a Kudoa a Kudoa a Kudoa f 2 EU567026 Kudoa a xfasciatus Kudoa a Kudoa a Kudoa a rdidus Kudoa a 1c EU340235 iculata Kudoa a udefduf bengalensis udefduf e ex r o b AY302724 u AY302741 olivaceus Paralichthys ex eriola quinqueradiata eriola a 100 i pogon p canthochromis p phyraena pinguis phyraena n i hromis viridis FJ792711 mphiprion a 100 egalensis egalensis A ex egalensis egalensis AY172511 Latris lineata ex mphiprion a hrysiptera c hrysiptera egalensis egalensis egalensis 1a EU340233 egalensis budefduf s budefduf egalensis egalensis egalensis 4 EU567028 egalensis ischistodus p Merluccius productus Merluccius mis ex budefduf whitleyi whitleyi budefduf p. ex Paralichthys p. ex lethostigma Beryx splendens splendens Beryx ex hyrsites budefduf s budefduf Cephalopholis boenak HI HM022110 Cephalopholis ex eoglyphidodon melas eoglyphidodon r exfasciatus 1b EU340238 exfasciatus budefduf s budefduf Sphoeroides annulatus ex Sphoeroides e hippsi o exfasciatus 1a EU340237 exfasciatus Thyrsites atun Thyrsites ex hyrsites FJ792718 a Merluccius capensis ex Merluccius capensis hyrsites Paralichthys olivaceus Paralichthys olivaceus ex hyrsites f exfasciatus 1c EU340239 exfasciatus i NS E NS scombrus ex Scomber hyrsites n Lepidopus caudatus ex Lepidopus hyrsites En A scombrus ex Scomber hyrsites Chaetodon unimaculatus LI HM022112 Chaetodon ex hyrsites ex Sardinops ocellatus hyrsites Coryphaena hippurus ex Coryphaena hyrsites ex Mondactylus argeneus ex Kudoa m n Salmo salar BC A ex hyrsites hyrsites ex Salmo hyrsites salar Or AF031412 Leptocottus armatus ex Leptocottus hyrsites hyrsites ex Merluccius productus hyrsites Merluccius productus ex hyrsites capsula ex S a Parophrys vetula ex Parophrys vetula hyrsites Pholis ornata ex hyrsites A hippsi ex Salmo salar Ch A ex hyrsites hippsi ex A i a eriola quinqueradiata quinqueradiata eriola hyrsites ex Aulorhynchus flavidus hyrsites a hippsi ex A A ex enti budefduf b budefduf capsula ex S empheris ypsilychnus g 98 Kudoa t Kudoa Kudoa s Kudoa Kudoa chaetodoni Kudoa C ex unterae a budefduf b budefduf aranx s aralichthys olivaceus D enti ex Thunnus albacares Thunnus albacares a ex akifugu rubripes akifugu rubripes e Kudoa yasunagai Kudoa budefduf b budefduf g aranx s g n budefduf b budefduf Kudoa t Kudoa budefduf b budefduf Kudoa t Kudoa budefduf b budefduf A iensis ex Kudoa t Kudoa aranx s Kudoa w Kudoa C ex unterae e e Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Thalassoma bifasciatum a ex Kudoa t Kudoa Kudoa t Kudoa A ex unterae Gymnocranius audleyi audleyi Gymnocranius ex lethrini Kudoa Kudoa t Kudoa m Lethrinus harak DQ519388 Lethrinus ex lethrini Kudoa Kudoa t Kudoa r Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa Kudoa t Kudoa m Kudoa t Kudoa unterae ex A unterae N unterae ex a 99 agiotaenia Kudoa d Kudoa o Kudoa t Kudoa Kudoa w Kudoa A unterae ex 60 Kudoa w Kudoa Kudoa p Kudoa u l Kudoa k Kudoa v 72 r Kudoa w Kudoa Kudoa neurophila Kudoa 99 i iensis ex S 64 99 Kudoa Kudoa Kudoa k Kudoa v iensis ex A P itsui ex iensis ex P iensis T itsui ex 65 Kudoa m Kudoa 88 ex Cynoscion nebulosus FJ790311 ex inornata Kudoa m ulvoguttatus AY078428 puensis FJ792717 iensis ex C iensis ex A ex Neoglyphidodon melas LI HM022114 thalassomi ex Kudoa Kudoa g Kudoa Kudoa thalassomi Kudoa m m a m m Kudoa m Kudoa AM183300 aurata Liza ex a iensis ex C iensis iensis ex A 79 gnobilis 1 FJ792714 Kudoa g Kudoa exfasciatus FJ792719 gnobilis 2 FJ792716 iensis ex A a o m iensis ex A a m o iensis ex A iensis ex C 100 100 i Dascyllus aruanus Dascyllus thalassomi ex Kudoa i m Kudoa g Kudoa 63 Kudoa g Kudoa m m a m a Kudoa g Kudoa h 89 Kudoa thalassomi Kudoa m 87 m h m m m m Kudoa c Kudoa a a a 97 LI HM022115 flavipectoralis Scarus thalassomi ex Kudoa a Kudoa monodactyli Kudoa m a a m 75 a a ex Thalassoma lunare AY302738 thalassomi ex Kudoa HI HM022116 Thalassoma lutescens thalassomi ex Kudoa LI HM022111 cyanae Chrysiptera thalassomi ex Kudoa Kudoa o Kudoa 62 ex Sillago ciliata e ex DQ519390 m m m cardialis gronovii AY302722 ex Nomeus cardialis nigritus AY302722 ex Epinephelus cardialis ex PogoniasAY302722 cromis cardialis ex Pomatomus saltatrixAY302722 cardialis ex Caranx crysos AY302722 cardialis AY302722 ex Lobotes surinamensis m i i i m m i i AY152749 ypsilichnus Pempheris ex minithyrsites Kudoa m i sp. AY382606 Lateolabrax ex lateolabracis Kudoa m 52 a aranx i t aranx i aranx p arangoides p aranx s ep a 61 ep ep ep ep ep i l 100 o Kudoa a Kudoa o o o o 78 o 59 i Kudoa s Kudoa Kudoa s Kudoa AY302739 bicarinatus Grammatorcynus ex grammatorcyni Kudoa 100 Kudoa a Kudoa p arangoides f p p p p p y y y 62 Kudoa a Kudoa y y AY302737 commerson Scomberomorus ex scomberomori Kudoa y Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa trifolia Kudoa Scomberomorus commerson AY078429 commerson Scomberomorus ex permulticapsula Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa Kudoa a Kudoa 100 Liza ramada AM490334 Liza ex unicapsula Kudoa 88 98 Kudoa c Kudoa 82 95 100 Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa Kudoa h Kudoa h Kudoa Kudoa h Kudoa C ornis ex la sp. AY302725 c 100 i u r 100 s d 58 p 100 87 a a c u i araquadricornis ex C araquadricornis ex C araquadricornis ex C araquadricornis ex C araquadricornis ex C n U Kudoa q Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa Kudoa p Kudoa ex Neogobius melanostomus EF644198 a ex v o 100 Kudoa n Kudoa 97 20.0 56 Siganus ai ex rivulatus AY514039 Dascyllus trimaculatusai ex AY514039 Pagrus major AY641571 ai ex t t Apogon aureus AY514038 ai ex t Sparus aurata AY514038 ai ex AY514038 variegatus Lethrinus ai ex t t a t a a Lutjanus erythropterus AY644704 Lutjanus erythropterus ai ex a a a t w w w a w w w w Kudoa i Kudoa Kudoa i Kudoa Kudoa i Kudoa 55 Kudoa i Kudoa Kudoa i Kudoa Kudoa i Kudoa Kudoa i Kudoa 100 60 99 64 S ea; Kudoa I sland; isolates England; A ustralia; C olumbia – L izard from A u – British LI – 98 – G enBank accession – c hile; En Kudoa thyrsites SS U rDN A I sland; A frica. C h L ocality of new sequence L ocality key: T he number of sequences in O regon (U SA ); S N – North – A zores; B C – S outh ( C anada); species including table summarising species with sequences from multiple hosts. the analysis is shown in parentheses; the number of sequences fish come familiesmaximum variance in these nucleotides and from; percent are and shown. Bootstrap support based the on 1000 nodes. replicates shown New at in sequences bold. highlighted isolates and indicated prior to number. number. SA H I – Heron O r – A z Fig. 3. Neighbour analysis joining of distance

6 0 2.1 9.2 1.8 0.7 0.7 0.8 max % max variance 14 5 5 13 0 6 59 max nt max variance 18/6 3/1 3/2 3/1 6/2 10/1 11/10 No. species /families (12) Kudoa thalassomi (24) Kudoa Kudoa thyrsites (15) thyrsites Kudoa Kudoa whippsi (9) whippsi Kudoa Kudoa species (no. Kudoa sequences) Kudoa gunterae gunterae Kudoa Kudoa kenti (4) kenti Kudoa Kudoa amamiensis (5) Kudoa Kudoa paraquadricornis (4) paraquadricornis Kudoa FJ790312 ulvoguttatus AY302733 FJ792755 Merluccius productus AY302732 FJ792763 FJ792761 1a FJ792762 1 FJ792733 1b Caranx ignobilis 1 FJ792749 Caranx mis ex r o f i HM022120 n HM022121 HM022130 Carangoides ornis ex f Paralichthys olivaceus Ja AY924196 olivaceus Paralichthys c ornis ex Carangoides plageotaenia FJ792751 Caranx sexfasciatus FJ792752 ornis ex Caranx ignobilis ornis ex 2 FJ792750 ptemfasciatus FJ792732 i HM022117 c c c r e HM022118 i i i r r r d LI HM022127 Burger, Adlard: Low host specificity in the Kudoidae d d d HM022122 a a a a HM022139 udoa p a HM022137 HM022124 K Liza ramada AM490335 Liza Salmo salar Ch AY924190 Parophrys vetula Or AY924187 vetula Parophrys ex Cynoscion nebulosus ex inornata Kudoa Merluccius productus BC AY924188 Merluccius Merluccius productus Or AY924188 Merluccius Salmo salar Or AY924189 Pholis ornata Or AY924187 Pholis ornata Salmo salar BC AY924189 Salmo salar BC AY302734 Sillago ex udoa ciliatae ciliata FJ792731 SA AY924191 atun SA Thyrsites SA AY924193 SA ocellatus Sardinops Lepidopus caudatus Az AY924191 Lepidopus caudatus SA AY924192 SA ocellatus Sardinops SA AY924191 SA capensis Merluccius Scomber scombrus En AY924191 scombrus Scomber SA AY924194 SA capensis Merluccius Acanthochromis polyacanthus FJ792762 polyacanthus Acanthochromis Coryphaena hippurus Au AY924195 K HM022138 HM022126 Sebastes paucispinus AY302730 Sebastes ex miniauriculata Kudoa LI HM022134 Abudefduf whitleyi Abudefduf ex Apogon properuptus Apogon ex Amphiprion ex akindynos 1 Abudefduf bengalensis 1 FJ792757 Abudefduf ex udoa q u budefduf s budefduf Amphiprion ex melanopus FJ792759 Abudefduf whitleyi Abudefduf ex Amphiprion ex akindynos 2 FJ792756 budefduf sexfasciatus 2 FJ792734 budefduf budefduf sordidus budefduf FJ792735 K Abudefduf sexfasciatus sexfasciatus Abudefduf 0 2.1 9.2 1.8 0.7 0.7 0.8 udoa paraq u udoa paraq u udoa paraq u ex ex thyrsites Kudoa max % max variance ex ex paraquadricornis Kudoa K K K 100 Liza aurata AM490336 aurata Liza ex trifolia Kudoa 14 udoa whippsi 5 udoa whippsi 5 udoa whippsi 13 udoa whippsi 0 6 udoa whippsi 59 udoa whippsi udoa whippsi ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K Abudefduf bengalensisudoa whippsi Abudefduf 2 FJ792758 ex ex ex thyrsites Kudoa K K ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K K K K ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex whippsi Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K ex ex unicapsula Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa 100 Amblyglyphidodon curacao 1 LI curacao Amblyglyphidodon max nt max variance Abudefduf bengalensis HI HM022119 Abudefduf Chelmon rostratus LI HM022128 Chelmon rostratus 60 Cheilodipterus macrodon macrodon Cheilodipterus Scarus flavipectoralis LI flavipectoralis Scarus Dascyllus aruanus Dascyllus Amphiprion LI akindynos 1a Amphiprion 100 Chaetodon 3 HI HM022132 unimaculatus Chaetodon Chaetodon 2a LI unimaculatus Chaetodon Heniochus monoceros HI HM022135 Heniochus monoceros Neoglyphidodon LI HM022136 melas Amblyglyphidodon curacao 2 LI curacao Amblyglyphidodon 53 HI HM022123 whitleyi Abudefduf Amphiprion LI akindynos 1b Amphiprion Amphiprion LI melanopus Amphiprion 65 Chaetodon 1 LI HM022129 unimaculatus Chaetodon 59 Chaetodon vagabundus LI HM022133 Chaetodon Chaetodon baronessa LI Chaetodon Dischistodus pseudochrysopoecilus FJ792739 udoa g unterae ex FJ792741 chrysurus Pomacentrus udoa g unterae ex Thalassoma lutescens HI lutescens Thalassoma 88 Cephalopholis boenak HI HM022125 Cephalopholis Thalassoma lunare AY302728 2b LI HM022131 2b unimaculatus Chaetodon 61 Plectroglyphidodonudoa g unterae ex 1 FJ792742 leucozonus A udoa g unterae ex Thalassoma lunare LI Thalassoma Chysiptera udoa g unterae ex cyanae FJ792736 Chrysiptera cyanae LI cyanae Chrysiptera Dascyllusudoa g unterae ex FJ792738 aruanus Plectroglyphidodonudoa g unterae ex 2 FJ792743 leucozonus A udoa g unterae ex Neoglyphidodon udoa g unterae ex melas FJ792740 K K 90 Chromis udoa g unterae ex viridis FJ792737 ex ex gunterae Kudoa A udoa g unterae ex K K K K K K K 98 K K 100 76 61 18/6 3/1 3/2 3/1 98 6/2 10/1 11/10 100 100 No. species /families FJ792726 AY302735 Latris lineata neurophila ex Kudoa 65 a AY302736 olivaceus Paralichthys yasunagai ex Kudoa hile; En – England; – En C hile; b FJ792727 AY302729 bicarinatus Grammatorcynus ex grammatorcyni Kudoa FJ792748 Monodactylus argenteus monodactyli ex Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa Kudoa thalassomi ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa Kudoa thalassomi ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex thalassomi ex Kudoa ex ex thalassomi Kudoa h – C h (12) ex ex thalassomi Kudoa ex ex thalassomi Kudoa 100 ex ex thalassomi Kudoa ex ex thalassomi Kudoa 54 74 AY302731 100 63 98 73 T he number of sequences in the udefduf bengalensis 2 FJ792728 100 udefduf bengalensis 1 udefduf b udefduf whitleyiudefduf FJ792729 udefduf bengalensis 1 b b b Amphiprion melanopus ex 2 FJ792745 enti Amphiprion melanopus ex 1 FJ792744 enti Kudoa thalassomi (24) Kudoa Kudoa thyrsites (15) thyrsites Kudoa Kudoa whippsi (9) whippsi Kudoa Plectroglyphidodon ex enti leucozonus FJ792747 Kudoa species (no. Kudoa sequences) Kudoa gunterae gunterae Kudoa Kudoa kenti (4) kenti Kudoa Kudoa amamiensis (5) Kudoa Kudoa paraquadricornis (4) paraquadricornis Kudoa FJ417057 Thunnus albacares ex FJ792730 sexfasciatus Caranx ex udoa k udoa k udoa k K K Dischistodus perspicillatus FJ792746 Dischistodus ex kenti Kudoa K 100 Kudoa crumena Kudoa A udoa a mamiensis ex olumbia ( C anada); C olumbia A udoa a mamiensis ex K A udoa a mamiensis ex A udoa a mamiensis ex K FJ790312 K K Kudoa amamiensis Kudoa 40.0 100 ex Thalassoma bifasciatum ex ovivora Kudoa sp. AY302727 ulvoguttatus AY302733 FJ792755 Unicapsula FJ792763 FJ792761 1a FJ792762 1 FJ792733 1b Caranx ignobilis 1 FJ792749 Caranx mis ex Merluccius productus AY302732 r o f i HM022120 n HM022121 100 HM022130 Carangoides ornis ex f Paralichthys olivaceus Ja AY924196 olivaceus Paralichthys c ornis ex Carangoides plageotaenia FJ792751 Caranx sexfasciatus FJ792752 ornis ex Caranx ignobilis ornis ex 2 FJ792750 – British – B C A zores; ptemfasciatus FJ792732 i HM022117 c c c r e HM022118 i i i r r r d LI HM022127 d d d HM022122 a a a a HM022139 udoa p a HM022137 HM022124 K Liza ramada AM490335 Liza Salmo salar Ch AY924190 Parophrys vetula Or AY924187 vetula Parophrys ex Cynoscion nebulosus ex inornata Kudoa Merluccius productus BC AY924188 Merluccius Merluccius productus Or AY924188 Merluccius Salmo salar Or AY924189 Pholis ornata Or AY924187 Pholis ornata Salmo salar BC AY924189 Salmo salar BC AY302734 ex Sillago ex udoa ciliatae ciliata FJ792731 79 SA AY924191 atun SA Thyrsites SA AY924193 SA ocellatus Sardinops Lepidopus caudatus Az AY924191 Lepidopus caudatus z – A z SA AY924192 SA ocellatus Sardinops SA AY924191 SA capensis Merluccius Scomber scombrus En AY924191 scombrus Scomber SA AY924194 SA capensis Merluccius Acanthochromis polyacanthus FJ792762 polyacanthus Acanthochromis Coryphaena hippurus Au AY924195 K HM022138 HM022126 Sebastes paucispinus AY302730 Sebastes ex miniauriculata Kudoa LI HM022134 Abudefduf whitleyi Abudefduf ex Apogon properuptus Apogon ex Amphiprion ex akindynos 1 Abudefduf bengalensis 1 FJ792757 Abudefduf ex udoa q u budefduf s budefduf Amphiprion ex melanopus FJ792759 Abudefduf whitleyi Abudefduf ex Amphiprion ex akindynos 2 FJ792756 budefduf sexfasciatus 2 FJ792734 budefduf budefduf sordidus budefduf FJ792735 K Abudefduf sexfasciatus sexfasciatus Abudefduf Chromis viridis FJ792737 udoa paraq u udoa paraq u udoa paraq u ex ex thyrsites Kudoa ex ex paraquadricornis Kudoa K K K 100 Liza aurata AM490336 aurata Liza ex trifolia Kudoa udoa whippsi A ustralia; udoa whippsi udoa whippsi udoa whippsi udoa whippsi udoa whippsi udoa whippsi ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K Abudefduf bengalensisudoa whippsi Abudefduf 2 FJ792758 ex ex ex thyrsites Kudoa K K ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K K K K ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex whippsi Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa K ex ex unicapsula Kudoa ex ex thyrsites Kudoa ex ex thyrsites Kudoa 100 Amblyglyphidodon curacao 1 LI curacao Amblyglyphidodon Abudefduf bengalensis HI HM022119 Abudefduf Chelmon rostratus LI HM022128 Chelmon rostratus 60 Cheilodipterus macrodon macrodon Cheilodipterus Scarus flavipectoralis LI flavipectoralis Scarus Dascyllus aruanus Dascyllus Amphiprion LI akindynos 1a Amphiprion 100 Chaetodon 3 HI HM022132 unimaculatus Chaetodon Chaetodon 2a LI unimaculatus Chaetodon Heniochus monoceros HI HM022135 Heniochus monoceros Neoglyphidodon LI HM022136 melas Amblyglyphidodon curacao 2 LI curacao Amblyglyphidodon 53 HI HM022123 whitleyi Abudefduf Amphiprion LI akindynos 1b Amphiprion Amphiprion LI melanopus Amphiprion 65 Chaetodon 1 LI HM022129 unimaculatus Chaetodon 59 Chaetodon vagabundus LI HM022133 Chaetodon Chaetodon baronessa LI Chaetodon Dischistodus pseudochrysopoecilus FJ792739 udoa g unterae ex chrysurus FJ792741 Pomacentrus udoa g unterae ex Thalassoma lutescens HI lutescens Thalassoma 88 Cephalopholis boenak HI HM022125 Cephalopholis Thalassoma lunare AY302728 2b LI HM022131 2b unimaculatus Chaetodon 61 Plectroglyphidodonudoa g unterae ex leucozonus 1 FJ792742 A udoa g unterae ex Thalassoma lunare LI Thalassoma Chysiptera udoa g unterae ex cyanae FJ792736 Chrysiptera cyanae LI cyanae Chrysiptera Dascyllusudoa g unterae ex aruanus FJ792738 Plectroglyphidodonudoa g unterae ex leucozonus 2 FJ792743 A udoa g unterae ex Neoglyphidodon udoa g unterae ex melas FJ792740 K K 90 udoa g unterae ex 60 ex ex gunterae Kudoa A udoa g unterae ex K K K K K K u – A u K 98 K K 100 76 61 98 100 100 FJ792726 AY302735 Latris lineata neurophila ex Kudoa 65 a AY302736 olivaceus Paralichthys yasunagai ex Kudoa b FJ792727 AY302729 bicarinatus Grammatorcynus ex grammatorcyni Kudoa Monodactylus argenteus FJ792748 Monodactylus argenteus monodactyli ex Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa Kudoa thalassomi ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa Kudoa thalassomi ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex thalassomi ex Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa ex ex thalassomi Kudoa 100 ex ex thalassomi Kudoa ex ex thalassomi Kudoa 54 74 ocality key: L ocality AY302731 100 63 98 73 udefduf bengalensis 2 FJ792728 100 udefduf bengalensis 1 udefduf b udefduf whitleyiudefduf FJ792729 udefduf bengalensis 1 b b b Amphiprion ex melanopus 2 FJ792745 enti Amphiprion ex melanopus 1 FJ792744 enti Plectroglyphidodon ex enti leucozonus FJ792747 FJ417057 Thunnus albacares ex FJ792730 sexfasciatus Caranx ex A frica. udoa k udoa k udoa k K K Dischistodus perspicillatus FJ792746 Dischistodus ex kenti Kudoa K 100 Kudoa species including table summarising species with sequences from multiple hosts. Kudoa crumena Kudoa – S outh A udoa a mamiensis ex A udoa a mamiensis ex K A udoa a mamiensis ex A udoa a mamiensis ex K K K Kudoa amamiensis Kudoa 40.0 100 from ex Thalassoma bifasciatum ex ovivora Kudoa sp. AY302727 enBank accession number. number. accession G enBank Unicapsula LS U rDN A 100 79 isolates indicated prior to prior indicated isolates 60 Kudoa thyrsites Kudoa H I – Heron sland; LI L izard O r regon (U SA ); Fig. 4. Neighbour joining distance analysis of analysis is shown in sequence parentheses; new of the Locality number sample. of that fish from families clone these to sequences refers come letter from; and and number fish theindividual maximumto variancerefers inhost nucleotidesafter andNumber percentbold. arein shown.highlighted Bootstrapsequences New supportnodes. basedat on shown replicates 1000 and isolates

7 Kudoa hypoepicardialis Blaylock, Bullard et Whipps, 2004 Kudoa miniauriculata Whitaker, Kent et Sakanari, 1996 Total number of hosts: 7. Perciformes: Carangidae – Caranx Total number of hosts: 2. Scorpaeniformes: Sebastidae – Se- crysos. Lobotidae – Lobotes surinamensis. Lutjanidae – bastes elongatus, S. paucispinis (type host). Lutjanus canipechanus. Nomeidae – Nomeus gronovii (type References: Moser et al. 1976, Heckmann and Jensen 1978, host). Pomatomidae – Pomatomus saltatrix. Sciaenidae – Whitaker et al. 1996, Hervio et al. 1997, Moran et al. 1999. Pogonias cromis. Serranidae – Epinephelus nigritus. Reference: Blaylock et al. 2004. Kudoa nova Naidjenova, 1975 Total number of hosts: 20. Perciformes: Carangidae – Tra- Kudoa iwatai Egusa et Shiomitsu, 1983 churus mediterraneus ponticus, T. picturatus, T. trachurus, Total number of hosts: 19. Beloniformes: Hemiramphidae T. trecae. Gobiidae – Gobius niger, G. ophiocephalus, Knip- – Hyporhamphus gamberur. Mugiliformes: Mugilidae – owitschia longicaudata, Neogobius cephalaris, N. fluviati- Mugil cephalus. Perciformes: Apogonidae – Apogon au- lis, N. melanostomus, N. platyrostris, N. ratan, N. syrman, reus, A. fleurieu. Carangidae – Seriola quinqueradiata. Pomatoschistus marmoratus (syn. Pomatoschistus microps Chaetodontidae – Chaetodon paucifasciatus. Lethrinidae – leopardinus in Kovaleva et al. 1979), Proterorhinus mar- Lethrinus nebulosus, L. variegatus. Lutjanidae – Lutjanus moratus. Pomatomidae – Pomatomus saltatrix (type host). erythropterus. Moronidae – Dicentrarchus labrax. Scombridae – Euthunnus alleteratus, Thunnus obesus. Spari- Oplegnathidae – Oplegnathus fasciatus, O. punctatus. dae – Dentex macrophthalmus, Pagellus acarne. Pomacentridae – Dascyllus trimaculatus, Neopomacentrus References: Aldrin 1962, Naidjenova et al. 1975, Kovaleva et miryae. Priacanthidae – Priacanthus hamur. Siganidae – al. 1979, Campbell 2005, Gorchanok and Yurakhno 2005, Siganus rivulatus. Sparidae – Argyrops filamentosus, Pagrus Machkevsky and Gorchanok 2005. major (type host), Sparus aurata. References: Paperna 1982, Egusa and Shiomitsu 1983, Egusa Kudoa ovivora Swearer et Robertson, 1999 1986a, Sugiyama et al. 1999, Diamant et al. 2005, Wang et Total number of hosts: 7. Perciformes: Labridae – Halichoeres al. 2005, Holzer et al. 2006. bivittatus, H. garnoti, H. poeyi, Thalassoma bifasciatum (type host). Scaridae – Sparisoma aurofrenatum, S. radians, Kudoa kenti Burger et Adlard, 2010 S. rubripinne. Total number of hosts: 4. Perciformes: Pomacentridae – Am- References: Swearer and Robertson 1999, Whipps et al. 2004. phiprion akindynos, A. melanopus (type host), Dischistodus perspicillatus, Plectroglyphidodon leucozonus. Kudoa paraquadricornis Burger et Adlard, 2010 Reference: Burger and Adlard 2010. Total number of hosts: 4. Perciformes: Carangidae – Ca- rangoides plagiotaenia, Caranx ignobilis (type host), Kudoa lethrini Burger, Cribb et Adlard, 2007 C. papuensis, C. sexfasciatus. Total number of hosts: 2. Perciformes: Lethrinidae: Gymnocra- Reference: Burger and Adlard 2010. nius audleyi (type host), Lethrinus harak. Reference: Burger et al. 2007. Kudoa quadratum (Thélohan, 1895) Total number of hosts: 10. Perciformes: Blenniidae – Blen- Kudoa lunata Lom, Dyková et Lhotáková, 1983 nius gattorugine. Callionymidae – Callionymus lyra. Total number of hosts: 3. Pleuronectiformes: Bothidae – Ar- Carangidae – Trachurus trachurus. Labridae – Coris julis. noglossus imperialis (type host), A. laterna, A. thori. Scorpaeniformes: Cottidae – Myoxocephalus scorpius (type Reference: Lom et al. 1983. host). Siluriformes: Ariidae – Plicofollis polystaphylodon (Ariodes polystaphylodon in Shulman 1988). Syngnathi- Kudoa megacapsula Yokoyama et Itoh, 2005 formes: Syngnathidae – Entelurus aequoreus (syn. Nerophis aequorens in Shulman 1988), E. olquerus, Syngnathus acus, Total number of hosts: 2. Perciformes: Carangidae – Seriola S. tenuirostris. quinqueradiata. Sphyraenidae – Sphyraena pinguis (type References: Thélohan 1895, Parisi 1912, Meglitsch 1947, Kova- host). leva et al. 1979, Shulman 1988, Longshaw et al. 2004. References: Yokoyama and Itoh 2005, Yokoyama et al. 2006. Note: There is a possible synonym of K. megacapsula. Tetraspina Kudoa sciaenae Terán, Llicán et Luque, 1990 decapterus Xie et Chen, 1988 described from Decapterus Total number of hosts: 4. Pleuronectiformes: Sciaenidae – Para- maruadsi (Carangidae, Perciformes) has extraordinary lonchurus peruanus, Sciaena deliciosa (type host), S. fascia- morphological similarity with K. megacapsula and has all ta, Stellifer minor. the features of Kudoa (see Naidjenova and Gajevskaja 1991). References: Terán et al. 1990, Oliva et al. 1992. The genus proposed by Xie and Chen (1988) is not valid as it is based on the family Tetracapsulidae Shulman, 1959 Kudoa shiomitsui Egusa et Shiomitsu, 1983 which does not follow the International Code of Zoological Nomenclature Article 11.7.1.1, and has since been recognised Total number of hosts: 2. Pleuronectiformes: Paralichthyidae – as a synonym of Kudoa (see Lom and Dyková 2006). Until Paralichthys olivaceus. Tetraodontiformes: Tetraodontidae this species identity is confirmed, we will not officially – Takifugu rubripes (type host). associate D. maruadsi as a host of K. megacapsula. References: Egusa and Shiomitsu 1983, Ogawa and Inouye

8 Burger, Adlard: Low host specificity in the Kudoidae

1997. Direct submission to GenBank of partial small subunit dynos, Am. melanopus, Chromis viridis, Neoglyphidodon rDNA sequence for P. olivaceus. melas, Pomacentrus chrysurus. Reference: Burger and Adlard 2010. Kudoa thalassomi Adlard, Bryant, Whipps et Kent, 2005 Total number of hosts: 18. Perciformes: See above in new mate- Kudoa yasunagai (Hsieh et Chen, 1984) rial for K. thalassomi. Total number of hosts: 7. Perciformes: Carangidae – Seriola Reference: Adlard et al. 2005. quinqueradiata. Lateolabracidae – Lateolabrax japonicus (type host). Oplegnathidae – Oplegnathus fasciatus. Sparidae Kudoa thyrsites (Gilchrist, 1924) – Pagrus major. Pleuronectiformes: Paralichthyidae – Paral- Total number of hosts: 38. Beryciformes: Berycidae – Beryx ichthys olivaceus. Siluriformes: Plotosidae – Plotosus linea- splendens. Clupeiformes: Clupeidae – Sardinella lemuru, tus (syn. Plotosus anguillaris in Cheung and Nigrelli 1990) Sardinops ocellatus, Sardinops sagax neopilchardus, Spra- Tetraodontiformes: Tetraodontidae – Takifugu rubripes. telloides robustus. Engraulidae – Engraulis australis, E. ja- References: Yasunaga et al. 1981, Hsieh and Chen 1984, Egusa ponicus. Gadiformes: Gadidae – Theragra chalcogramma. 1986b, Cheung and Nigrelli 1990, Ogawa and Inouye 1997, Merlucciidae – Merluccius capensis, M. productus. Gaste- Whipps et al. 2004. rosteiformes: Aulorhynchidae – Aulorhynchus flavidus. Per- ciformes: Coryphaenidae – Coryphaena hippurus. Gempy- Genetic intraspecific variation in kudoids among lidae – Thyrsites atun (type host). Pholidae – Pholis ornata. different host isolates Scombridae – Scomber japonicus, S. scombrus. Trichuridae – Lepidopus caudatus. Pleuronectiformes: Cynoglossidae The trimmed SSU rDNA sequence alignment con- – Pleuronectes bilineatus. Paralichthyidae – Paralichthys tained 109 Kudoa sequences from 36 species and was adspersus, P. olivaceus. Pleuronectidae – Atheresthes sto- 1539 nucleotides long. Of the sequences that spanned the mias, Eopsetta jordani, Hippoglossus stenolepis, Lepidosetta whole alignment, the sequence size varied in length from bilineata, Microstomus pacificus, Parophrys vetula, Platich- 1403 nucleotides (K. unicapsula) to 1512 (K. crumena thys stellatus. Salmoniformes: Salmonidae – Oncorhynchus Iversen et Van Meter, 1967). The trimmed LSU rDNA gorbuscha, O. kisutch, O. mykiss (syn. Salmo gairdneri in sequence alignment contained 88 Kudoa sequences from Kabata and Whitaker 1989), O. tshawytscha, Salmo salar, 20 species and was 814 nucleotides long. The sequences S. trutta. Scorpaeniformes: Cottidae – Icelinus filamentosus, Leptocottus armatus. Hexagrammidae – Ophiodon elonga- varied in length from 644 (K. ovivora) to 759 (K. kenti) tus. Zeiformes: Zeidae – Zeus capensis, Z. faber. nucleotides. References: Gilchrist 1924, Pérard 1928, Davies and Beyers From the SSU rDNA analysis, 13 of the 36 species had 1947, Meglitsch 1947, Willis 1949, Prudhomme and Pan- DNA sequences from multiple hosts (Fig. 3). From these taleon 1959, Patashnik and Groninger 1964, Priebe 1967, 13 Kudoa species, only K. lethrini (two hosts), K. mega- Kovaleva et al. 1979, Kabata and Whitaker 1981, Patashnik capsula (two hosts), K. paraquadricornis (four hosts) and et al. 1982, Harrell and Scott 1985, Kabata et al. 1986, Stehr K. shiomitsui (two hosts) have SSU sequences from all and Whitaker 1986, Kudo et al. 1987, Kabata and Whitaker their known hosts. 1989, Langdon 1991, Langdon et al. 1992, Barja and Toranzo In the SSU analysis, the numbers of different host 1993, Holliman 1994, McDonald and Margolis 1995, Castro families per Kudoa species ranged from one (K. alliaria, and Burgos 1996, Hervio et al. 1997, Shaw et al. 1997, Mo- K. gunterae, K. kenti, and K. paraquadricornis), up to ran and Kent 1999, Whipps et al. 2003, Shukhgalter 2004, 13 (K. thyrsites). Nucleotide variance between different Yokoyama et al. 2004, Yokoyama and Itoh 2005, Whipps and host isolates of the same kudoid species ranged from zero Kent 2006, Levsen et al. 2008. (K. paraquadricornis and K. shiomitsui) to 14 (0.9% in Kudoa trifolia Holzer, Blasco-Costa, Sarabeev, K. thyrsites, where the maximum distance was between Ovcharenko et Balbuena, 2006 isolates from both Beryx splendens Lowe and Parali- Total number of hosts: 2. Mugiliformes: Mugilidae – Liza au- chthys olivaceus Temminck et Schlegel compared with rata (type host), L. ramada. those from both Merluccius capensis Castelnau and Thyr- Reference: Holzer et al. 2006. sites atun (Euphrasen) nucleotides). The second largest SSU genetic variance amongst host isolates in a single Kudoa unicapsula Yurakhno, Ovcharenko, Holzer, Kudoa species was from K. thalassomi. Out of the eight Sarabeev et Balbuena, 2007 sequences from five different host families, the range of Total number of hosts: 2. Mugiliformes: Mugilidae – Liza au- nucleotide variance is between zero and 10 (0.7%), with rata, L. ramada (type host). the maximum occurring between isolates from the serra- Reference: Yurakhno et al. 2007. nid host Cephalopholis boenak and a pomacentrid host Neoglyphidodon melas. Kudoa whippsi Burger et Adlard, 2010 Analysis of LSU rDNA (Fig. 4) revealed that seven of Total number of hosts: 9. Perciformes: Apogonidae – Apogon the 18 Kudoa species have LSU rDNA sequences from properuptus. Pomacentridae – Abudefduf bengalensis, Ab. more than one host and of these seven, only K. kenti (four whitleyi, Acanthochromis polyacanthus, Amphiprion akin- hosts), K. paraquadricornis (four hosts), and K. thalas-

9 differences (0.9%), while four sequences from isolates derived from three individuals of Chaetodon unimaculatus differed by 4–11 nucleotides (0.6–1.2%) and sequences from isolates derived from each of two Thalassoma lunare (the type host) individuals showed 4 nucleotide differences (0.6%).

Discussion Kudoa thalassomi is one of four Kudoa species described to date represented by a dominant 6-polar capsule spore morphotype and that infect the somatic musculature of their host fish. One of the other three species, Kudoa neothunni infects yellowfin tuna from Japan and is distinguished from K. thalassomi by having smaller polar capsules. The other two of the three species, K. grammatorcyni and K. scomberomori, are sympatric with K. thalassomi i.e. they occur on the Great Barrier Reef, Australia, but are smaller in spore size (Table 1) and are Fig. 5. Principal component analysis with 95% ellipses of readily distinguished in genetic analyses (Figs. 3, 4). In- morphometric data from apical measurements of Kudoa terestingly, K. thalassomi is closer in genetic distance to thalassomi. Open squares – 6-polar capsule morphotype from two species that infect brain tissue, K. lethrini, a species Abudefduf bengalensis; plus signs – 7-polar capsule morphotype dominated by a 7-polar capsule morphotype also from the from Abudefduf bengalensis; filled squares – 6-polar capsule Great Barrier Reef, and K. neurophila (Grossel, Dyková, morphotype from Thalassoma lunare; crosses – 7-polar capsule Handlinger et Munday, 2003), a species dominated by morphotype from Thalassoma lunare. a 5-polar capsule morphotype from Tasmania in southern Australia (having 21 nucleotides and 1.5% difference in somi (18 hosts) have LSU sequences from all their known SSU to both species) (Grossel et al. 2003). Kudoa thalas- hosts. somi differs in SSU sequence from K. grammatorcyni and The number of different host families per Kudoa spe- K. scomberomori by 2.4% and 2.5%, respectively. What cies with LSU rDNA sequences ranged from one (K. gun- drives such a genetic relationship that places K. thalas- terae, K. kenti, and K. paraquadricornis) to 10 (K. thyr- somi closer to brain-infecting species is unclear. Kudoa sites). Nucleotide differences between isolates from lethrini and K. neurophila infect different host tissues, are different hosts ranged from zero (K. paraquadricornis) not more morphologically similar and do not infect either to 59 (9.2% in K. thyrsites, where the maximum distance genetically or ecologically similar hosts. We suspect that was between isolates from the hosts Coryphaena hippurus with more comprehensive taxon sampling the basis for Linnaeus and Paralichthys olivaceus). The second larg- such relationships will become clear. est LSU genetic variance amongst isolates from different Kudoa thalassomi also differs from related hosts in a single Kudoa species was again demonstrated muscle-infecting species, K. grammatorcyni and by K. thalassomi. Out of the 24 sequences of K. thalas- K. scomberomori, in having a low host specificity. somi from the six different host families, the nucleotide From 214 fish species the authors sampled on the Great variance ranged from one (0.1%) to 14 (2.1%), while the Barrier Reef, K. grammatorcyni has been found only in maximum difference was between isolates from a scarid Grammatorcynus bicarinatus Quoy et Gaimard (see Ad- host (Scarus flavipectoralis) with isolates from two dif- lard et al. 2005) and G. bilineatus (Rüppell) (unpublished ferent chaetodontid hosts (Chaetodon unimaculatus and data), while K. scomberomori has been found only in Chelmon rostratus) and one from a pomacentrid host Scomberomorus commerson Lacéepède (see Adlard et al. (Neoglyphidodon melas). 2005). Kudoa thalassomi has now been found in 18 host It is noteworthy that there was significant LSU se- species from six different host families (see Fig. 4 and quence variance between K. thalassomi isolates collected new material detailed in the Results). from different individuals of the same host species. For Kudoa thalassomi has now been recorded from the example, isolates from two individuals of Amblyglyph- highest number of host species for any kudoid species for idodon curacao showed 11 nucleotide differences (1.6%). which the specific identity has been confirmed through Genetic variation also occurred at other levels, e.g. two DNA sequence analysis (LSU rDNA). In comparison, individual kudoid LSU genotypes (single genotypes iso- K. thyrsites has DNA sequences matching a broader range lated and amplified using bacterial cloning) from a single of host species (14 kudoid isolates having SSU rDNA Amphiprion akindynos individual showed 6 nucleotide sequences, and 11 isolates with LSU rDNA sequences)

10 Burger, Adlard: Low host specificity in the Kudoidae and geographic distribution with representatives from 60 13 different host families within eight host orders (see Figs. 3, 4); however, this increased host diversity and geographic range also corresponds to a higher maximum sequence difference in K. thyrsites isolates, especially 40 in LSU rDNA which has been shown to be a superior indicator of species differentiation than SSU (see Burg-

er and Adlard 2010). In the LSU, Kudoa thalassomi has Frequency 2.1% maximum sequence variation between isolates, 20 while K. thyrsites has a 9.2% variation. The total number of K. thyrsites host species for which there are DNA sequences (14) is less than half (37%) of the total number of recorded hosts (38) for that species. This suggests that 0 the maximum sequence variation in K. thyrsites isolates 0 10 20 30 40 Number host species may be higher than that recorded to date. In comparison, there are DNA sequences (LSU) of K. thalassomi for all Fig. 6. Frequency distribution of recorded host specificity of of the currently recorded hosts for that parasite. Kudoa species. There have been many records of K. thyrsites since it was first described by Gilchrist in 1924 which, when Prior to the use of DNA, all Kudoa species with combined, report a large morphological variance for a stellate spore shape and one polar capsule larger than spores of this species. For example, the smallest average the other three were likely to be assigned to K. thyrsites, width of a K. thyrsites isolate was 12.0 µm (Gilchrist with the only recorded exceptions being K. cruciformum 1924) from Thyrsites atun compared to the largest of 16.7 (Matsumoto, 1954) and K. mirabilis Naidjenova et µm from Merluccius productus (Ayres) (see Kabata and Gajevskaja, 1991. With the benefit of DNA data it now Whitaker 1981, Whipps and Kent 2006). Smaller, but appears that K. thyrsites may represent a species complex significant morphometric differences have also been noted (Moran et al. 1999, Whipps and Kent 2006) due largely between host isolates of K. hypoepicardialis with mean to the significant genetic variance among isolates from spore width of 10.1 µm (range 9.3–11.2 µm) from Nomeus different hosts and to the broad geographic distribution grovonii (Gmelin), and 7.6 µm (range 6.5–8.4 µm) from of this species. How many species are represented by Pogonias cromis (Linnaeus) (see Blaylock et al. 2004). K. thyrsites is currently uncertain and will remain so until Our data show evidence for host-induced variation in morphological or biological characters are discovered spore valve size between K. thalassomi isolates from that map, with confidence, to genetic profiles. Abudefduf bengalensis and those from Thalassoma lunare Kudoa cruciformum and K. lateolabracis Yokoyama, (average spore width of the 6-polar capsule morphotype Whipps, Kent, Mizuno et Kawakami, 2004 are clearly was 13.7 µm (range 12.4–14.9 µm) for spores from species that are closely related morphologically but are A. bengalensis and 13.2 µm (range 12.3–14.9 µm) for considered to be distinct (Yokoyama et al. 2004). No spores from T. lunare). We suspect that a small proportion DNA data are yet available for K. cruciformum. In SSU of the morphological variation reported here and the rDNA K. lateolabracis is 23–39 nucleotides (1.9–2.0%) original description of K. thalassomi ex T. lunare (mean different to sequences from K. thyrsites (see Yokoyama spore width 10.7 µm, range 9.5–11.8 µm) may have been et al. 2004). the result of post-collection treatment of specimens (e.g. Kudoa mirabilis seems distinct from all other species number of times frozen/thawed), but clearly there is based on its morphology (it has a much more significant a degree of variation in spore dimensions displayed by difference between the sizes of the large polar capsule even the same genetic species, for example the previously compared to the three small polar capsules) and from mentioned K. hypoepicardialis isolates all have identical the distinctive pathology associated with the infection. SSU sequences (Blaylock et al. 2004) (see Fig. 3). Given Naidjenova and Gajevskaja (1991) describe macroscopic that kudoid species are now known to display a degree of cysts (3–5 mm diameter; occurring in clusters) that cause morphometric plasticity (see Blaylock et al. 2004, Whipps blisters filled with spores on the body surface and inside and Diggles 2006, Whipps and Kent 2006, Burger and the body. This pathological presentation has not been seen Adlard 2010), it is even more critical that an independent with any other stellate, unequal polar capsule kudoid. character set such as DNA sequence be employed as Since the use of DNA characterisation, it has been a datum to help establish species boundaries. Nonetheless, possible to distinguish unambiguously the 4-polar morphology still provides useful characters for defining capsule, stellate kudoids K. gunterae, K. lateolabracis, species thus we would favour a whole evidence approach K. minithyrsites Whipps, Adlard, Bryant, Lester, Findlay for identification. et Kent, 2003, and K. whippsi from K. thyrsites (see Figs.

11 3 and 4). In addition, DNA has also assisted in resolving regions of the SSU rDNA fragment at the 5’ end of the confusion with other species identities. For example, the gene. Therefore, until more sequences are generated close genetic similarity between the K. iwatai sequences for these two species we consider that they should be published by Diamant et al. (2005) and K. lutjanus from maintained. Wang et al. (2005) coupled with spore shape and tissue The examples given above demonstrate that having tropism led to the latter species being considered a junior multiple host records for a kudoid species should not, synonym of K. iwatai (see Holzer et al. 2006). Another of itself, be an indicator of broad host specificity. Unless example is the sequences of K. rosenbuschi published by such records include DNA sequences from different hosts Abollo et al. (2005) and Kudoa sp. published by Pascual such a conclusion may be unwarranted. By increasing the and Abollo (2008) which had high sequence similarity number of parasite/host isolates represented in the DNA with that of K. alliaria published by Whipps and Diggles database, the more certainty there will be in the host range (2006). The infections that these sequences represent of species in the Myxosporea. Just over half of the total have now been recognised as referable to K. alliaria (see number of Kudoa species with multiple hosts listed in the Burger and Adlard 2010) with further evidence from the results section have been sequenced from 50% or more identical pathology that they cause in the host. of their recorded hosts (14 of 27); and less than a quar- An example of a possible synonym that could be ter of the total number of species have DNA sequences determined using DNA is that of two cardiac tissue- representing their entire host range (6 of 27: K. lethrini, infecting species K. pagrusi Al Quraishy, Koura, Abdel- K. kenti, K. megacapsula, K. paraquadricornis, K. shi- Baki, Bashtar, El Deed, Al Rasheid et Abdel Ghaffar, omitsui, and K. thalassomi). Although the numbers are 2008, described from Pagrus pagrus in the Red Sea and relatively low, patterns in host specificity are slowly be- K. shiomitsui (see Egusa and Shiomitsu 1983) described coming apparent in the Kudoidae. from Takifugu rubripes from Japan. The spores are similar The specific mechanisms that drive host specificity of in size and shape and the cyst shape and location in the host kudoids are unknown. In the Bivalvulida, actinospores tissues are identical. Also, it has previously been shown of Myxobolus cerebralis Hofer, 1903 respond positively that K. iwatai has been found in both Japan and in the to signals in the mucus of a range of fish, but infection Red Sea (Diamant et al. 2005), reducing any emphasis for only progresses in susceptible hosts (Kallert et al. species distinction based on large geographic separation. 2009). Yokoyama et al. (2006) showed that Myxobolus It appears that the proposers of K. pagrusi were unaware arcticus Pugachev et Khokhlov, 1979 actinospores of the existence of K. shiomitsui, as they did not include responded quickly and non-specifically to fish mucus, but any reference to this species in their publication. Also, Thelohanellus hovorkai Akhmerov, 1960 actinospores the authors may have misidentified the fish host because showed a slower selective response to mucus from Pagrus pagrus does not occur in the Red Sea; instead it the susceptible carp host. Clearly, development in any is found in the Atlantic Ocean and the Mediterranean Sea particular host relies on a combination of cues to enhance (http://www.fishbase.org/). A SSU rDNA sequence of the the encounter of infective stages with hosts, followed by infection from the sparid fish from Al Quraishy et al. 2008 the ability of parasites to establish infections regardless of should be able to determine its relationship with reference host defensive mechanisms. to K. shiomitsui which has two SSU sequences available At higher taxonomic levels the specificity of kudoid (see Fig. 3). species does not appear to show any broad trends. The distinction between K. clupeidae from Brevoortia Taxonomic relatedness of hosts is not significantly tyrannus (Latrobe) published by Webb et al. (2005) correlated with genetic relatedness of species in the and K. funduli from Fundulus heteroclitus (Linnaeus) Myxosporea for either multivalvulids (see Burger et published by Akaishi et al. (2004) may be invalid because al. 2007) or for bivalvulids within the Ceratomyxidae SSU rDNA sequences are identical (see Fig. 3). Meglitsch (see Gunter et al. 2009), Chloromyxidae (see Holzer (1948) differentiated between these two morphologically et al. 2004), Myxidiidae (see Holzer et al. 2004, Fia- similar Kudoa species on several subtle shape and size la 2006), Myxobolidae (see Andree et al. 1999), and differences and that K. funduli was found also on the fish Sphaerosporidae (see Holzer et al. 2004). fins rather than only within somatic muscle. The lack of At a species level, the majority of kudoids have only differentiation between these two species in the SSU rDNA been reported from a single host species, but a significant analysis does not include the four indels that occur in the proportion display a broader host range. Furthermore, alignment as differences between the two sequences. The the host range of a species is often more a function of lack of informative characters is accentuated because the research effort rather than a true representation of its K. funduli sequence is much shorter (739 nucleotides long range. Only a few myxosporean genera have been studied in the alignment from this study) than that of K. clupeidae with sufficient intensity to have confidence in specificity (1394 nucleotides long in the alignment from this study) trends, with Ceratomyxa and Myxobolus showing and does not span across one of the more highly variable relatively high levels of host specificity (Molnár 1994,

12 Burger, Adlard: Low host specificity in the Kudoidae

Gunter et al. 2009). Prior to this study, K. thalassomi was Acknowledgements. The authors would like to thank: Dr. Rod reported only from Thalassoma lunare, but now from Bray, Dr. Mal Bryant, Tony Byrne, Assoc. Prof. Thomas Cribb, the data we present in this study it is obvious that the Abigail Downie, Ricky Gleeson, Dr. Nicole Gunter, Holly host specificity of K. thalassomi is broad. This is a clear Heiniger, Dr. Tim Littlewood, Marissa McNamara, Dr. Terrence demonstration that any interpretation of host specificity Miller, Dr. Matthew Nolan, Dr. Peter Olson, Chris Rohner, and Tane Sinclair-Taylor for their assistance in fish collection cannot be made from serendipitous records of presence throughout the project. We are grateful to the staff and for the fa- in a particular host, but rather, through a focused study cilities of Heron and Lizard Island Research Stations. For access using combined morphological and molecular characters to molecular analysis equipment and assistance, we would like to allow unambiguous identification of parasites from to thank Dr. Andy Barnes and Dr. Justice Baiano. For histology multiple hosts. Regardless of the current interest in assistance, we would like to thank Chris Cazier and Sue Ther- myxosporean parasites, it will likely take some years to kelsen from the Veterinary Pathology Laboratory, the University increase and validate the taxonomic dataset to a level of Queensland. We appreciate Dr. Nicole Gunter’s comments on where further trends in host specificity can be predicted the manuscript. We would also like to thank the ARC/NHMRC with any confidence for these parasites. Results from this Research Network for Parasitology for travel funds. This study study reveal that myxosporeans in general and kudoids in is a contribution funded by the Australian node of the CReefs particular, have adopted strategies that result in various global research initiative, a partnership between the Australian Biological Resource Study (ABRS) and BHP Billiton, the Great levels of host specificity. Nonetheless, identifying the host Barrier Reef Foundation, the Australian Institute of Marine Sci- range of parasites can be of enormous value for predicting ence and the Alfred P. Sloan Foundation, grant number: 209/29. the threat of introduction of a potential pathogen and for CReefs is a field program of the Census of Marine Life. managing an existing disease.

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Received 25 May 2010 Accepted 3 August 2010