Parasitising Oreochromis Niloticus Niloticus (L.) and O

DOI: 10.2478/s11686-011-0005-2 © W. Stefan´ski Institute of Parasitology, PAS Acta Parasitologica, 2011, 56(1), 20–33; ISSN 1230-2821 Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea) parasitising Oreochromis niloticus niloticus (L.) and O. mossambicus (Peters) (Cichlidae)

Adriana García-Vásquez1,4*, Haakon Hansen2, Kevin W. Christison3, James E. Bron4 and Andrew P. Shinn4 1Centro de Investigación en Alimentación y Desarrollo, CIAD, A.C. Unidad Mazatlán en Acuicultura y Manejo Ambiental. Avenida Sábalo-Cerritos s/n. Estero del Yugo, C.P. 82000, Mazatlán, Sinaloa, Mexico; 2National Veterinary Institute, Section for Parasitology, P.O. Box 750 Sentrum, Oslo, Norway; 3Department of Biodiversity and Conservation Biology (Zoology), University of the Western Cape, Private bag X17, Bellville 7535, South Africa; 4Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK

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

Keywords Monogenea, Gyrodactylus, G. hildae, G. ulinganisus, G. yacatli, taxonomy, cichlid, tilapia, Oreochromis n. niloticus, O. mossambicus

Introduction species, O. mossambicus and O. n. niloticus are the most widely and intensively cultured with 45,130 and 1,629,203 t In freshwater, the global production of cultured tilapia now respectively being produced in Asia, 130 t and 202,623 t exceeds 2 million tonnes (t) per annum, second only to carp across Africa, and, 21 t and 79,379 t throughout the Americas production (FAO 2006). Ten tilapia species, belonging to sev- (FAO Fishstat Plus, 2006). Oreochromis n. niloticus has be- eral different genera, are cultured in a variety of freshwater come one of the favoured species for culture in Asian aqua- and brackish environments worldwide: Oreochromis ander- culture because it is easy to breed, tolerates a wide range of sonii (Castelnau) (three spotted tilapia), O. aureus (Stein- environmental conditions, has versatile dietary preferences dachner) (blue tilapia), O. macrochir (Boulenger) (longfin and is highly marketable and affordable (Pullin 1985, Shelton tilapia), O. mossambicus (Peters) (Mozambique tilapia), O. nilo- 2002). ticus niloticus (L.) (Nile tilapia), O. spilurus spilurus (Gün- Under intensive culture conditions early phase survival is ther) (sabaki tilapia), Sarotherodon galilaeus galilaeus (L.) decreased by a broad spectrum of diseases, notably those (mango tilapia), S. melanotheron melanotheron Rüppell caused by Ichthyophthirius multifiliis Fouquet, 1876, Tri- (blackchin tilapia), Tilapia rendalli (Boulenger) (redbreast chodina Ehrenberg, 1838 and Ichthyobodo necatrix (Hen- tilapia), and, T. zillii (Gervais) (redbelly tilapia). Of these neguy, 1883) Pinto, 1928 (see the reviews of El-Sayed 2006,

*Corresponding author: [email protected] Gyrodactylids infecting Oreochromis spp. 21

Shoemaker et al. 2006). In addition, several outbreaks of gy- rDNA ITS1 and 2 from the same samples confirmed that each rodactylosis resulting in the mortality of juvenile pond-reared of these groups represented a separate species, but also iden- O. n. niloticus have been reported by several authors (Fryer tified a fifth species of Gyrodactylus parasitising South Af- and Iles 1972, Roberts and Sommerville 1980, García- rican O. mossambicus. The current paper formally describes Vásquez et al. 2007) and tilapia producers (V. Vidal-Martínez, the three discrete forms identified by morphometric and mo- CINVESTAV, Mexico, pers. comm., Prof. El-Naggar from Man- lecular methods in García-Vásquez et al. (2010) as three new soura University, Egypt, pers. comm., Mr W. Turner Nam Sai species, namely G. hildae sp. nov. from O. n. niloticus from farms, Thailand, pers. comm., Mr N. Froyman, Israel, pers. Ethiopia; G. ulinganisus sp. nov. from O. mossambicus from comm.), raising the question of whether infections are due to South Africa and G. yacatli sp. nov. from O. n. niloticus from a single species or to a range of species. Mexico. Prior to the current study, six species of Gyrodactylus von Nordmann, 1832 were known from tilapiine fish – G. cichlidarum Paperna, 1968; G. nyanzae Paperna, 1973; G. niloticus Materials and methods Cone, Arthur et Bondad-Reantaso, 1995; G. shariffi Cone, Arthur et Bondad-Reantaso, 1995; G. ergensi Přikrylová, Sample collection and preparation Matĕjusová, Musilová et Gelnar, 2009; and, G. aegypticus El- Naggar et El-Tantawy, 2003. Of these, G. cichlidarum was de- Samples of gyrodactylid-infected yolk-sac fry (2 weeks post- scribed from the mango tilapia S. g. galilaeus, its type host hatch) and juvenile (2–3cm) O. n. niloticus and O. mossam- and also from Ghanaian populations of T. zillii, Hemichromis bicus were collected from Mexico (samples comprising cul- fasciatus (Peters) (banded jewelfish) and Hemichromis bi- tured stock sourced from CINVESTAV, a government funded maculatus (Gill) (jewelfish). A recent study by the current au- research facility in Merida, and from a farm in Tabasco), thors (García-Vásquez et al. 2007), also documented the South Africa (cultured stock held at the Aquaculture division, occurrence of G. cichlidarum on an aquarium-reared stock of University of Stellenbosch, Welgevalen, Stellenbosch) and O. n. niloticus in the UK and on fish from a farm in Mexico. Ethiopia (wild fish from Baro Lake, Gambela) and were fixed This latter study synonymised G. niloticus with G. cichli- in 95% ethanol. Gyrodactylids were removed using mounted darum based on identical morphology. Subsequently, García- triangular surgical needles (size 16, Barber of Sheffield, UK) Vásquez et al. (2010) sampled gyrodactylid material from the and were processed individually. Haptors were excised using same farm in the Philippines (Bureau of Fisheries and Aquatic a scalpel and subjected to a partial proteolytic digestion to re- Resources National Freshwater Fish Hatchery, Muñoz, Nueva move the tissue enclosing the haptoral armature using Harris Ecija Province) as that investigated by Cone et al. (1995) in and Cable’s (2000) proteinase K-based method. Once the tis- the description of G. niloticus and the gyrodactylid material sues had been removed, digestion was arrested by the addi- was found to have identical internal transcribed spacers 1 and tion of a 50:50 formaldehyde:glycerine solution and spe- 2 (ITS1 and 2) and 5.8S gene sequences, and hook morphol- cimens were then coverslipped and sealed with nail varnish. ogy to that of G. cichlidarum. Of the remaining species, The excised bodies were fixed in 95% ethanol and stored in in- G. nyanzae was first documented from Oreochromis variabilis dividually labelled Eppendorf tubes for subsequent molecular (Boulenger) (Victoria tilapia) (Paperna 1973); G. shariffi from analyses. a farmed population of O. n. niloticus reared in the Philippines (Cone et al. 1995), G. ergensi from a Senegalese populations Morphometric analysis of O. n. niloticus and S. g. galilaeus (see Přikrylová et al. 2009), and G. aegypticus from an Egyptian population of red- The digested haptoral hard parts were studied on an Olympus belly tilapia, T. zillii (El-Naggar and El-Tantawy 2003). Given BH2 compound microscope using an oil immersion ×100 ob- the lack of a description for G. aegypticus, this species was jective lens and a JVC KY-F30B 3CCD camera fitted via a regarded as a nomen nudum (see Harris et al. 2008). camera mount with a ×2.5 top lens. Measurements were made The Gyrodactylus-associated mortality of juvenile, pond- on the images of the attachment hooks using the Point-R mor- reared O. n. niloticus across several continents raised the ques- phometric analysis platform (ver. 1.0 © University of Stirling, tion of whether infections were due to single of infections 2003) running on Zeiss KS300 ver. 3.0 image analysis soft- either G. cichlidarum or G. shariffi, or were due a combination ware (Carl Zeiss Vision GmbH, München, Germany, 1997). A of both or other undescribed species of Gyrodactylus. The total of 25 point-to-point measurements detailed by Shinn et study of García-Vásquez et al. (2010) investigated the extent al. (2004) were made on each specimen (see Table I). Type of morphological and molecular variation in specimens of Gy- specimens were deposited in the Institute of Parasitology, Bi- rodactylus from 29 populations of both cultured and wild ology Centre of the Academy of Sciences of the Czech Re- stocks of O. n. niloticus and O. mossambicus sampled from public, České Budějovice, Czech Republic and in the Parasitic 15 countries. Four discrete groupings representing G. cichli- Worms Division, The Natural History Museum, London, UK. darum, G. shariffi and two previously unknown species were The holotype of Gyrodactylus amphiliusi Paperna, 1973 from identified from the morphometric analyses. Sequencing of the Amphilius atesuensis Boulenger (accession no. MRAC- 22 Adriana García-Vásquez et al.

M.T.35.712) and the holotype of G. cichlidarum (accession complete but represent the haptoral hooks only. Holotype from no. 35584) from the Museé Royal de l’Afrique Centrale O. n. niloticus. Paratypes: 3 specimens from an unspecified (MRAC), Tervuren, Belgium, the holotype and a paratype of cichlid, not confirmed as O. n. niloticus. The holotype (ac- G. niloticus (accession nos. 084007 and 084008) (syn. G. cich- cession no. 2008.12.15.9) and a paratype (accession no. lidarum) and the holotype and a paratype of G. shariffi (ac- 2008.12.15.10) are deposited in the Parasitic Worms collec- cession nos. 084009 and 084010) from the USDA U.S. Na- tion of The Natural History Museum, London. In addition, two tional Parasite Collection, Maryland, USA were also re- paratypes (accession no. M-478) are deposited in the gyro- examined for the current study. dactylid collection held at the Institute of Parasitology, Biol- ogy Centre of the Academy of Sciences of the Czech Republic, Molecular analysis České Budějovice. A species profile including taxonomic traits, host details and additional metadata is provided on the DNA was extracted from individual specimens (gyrodactylid on-line database www.gyrodb.net (Harris et al. 2008, Shinn minus its haptor) using a DNeasy® Blood and Tissue minikit et al. 2010). (Qiagen). The primer pair ITS1A (5’-GTAACAAGGTTTC- Molecular sequence data: A 1124 bp consensus sequence CGTAGGTG-3’) and ITS2 (5’-TCCTCCGCTTAGTGATA- spanning part of the SSU rDNA ITS1 (475 bp), 5.8S (157 bp), 3’) (Matějusová et al. 2001) were used to amplify (PCR) a ITS2 (424 bp) region obtained from three individuals has been fragment spanning the 3’ end of the 18S ribosomal RNA sub- deposited in GenBank (accession no. FJ231869). unit, ITS1 and 2, the 5.8S subunit and the 5’ end of the 28S Etymology: This species is named after the indefatigable subunit. The 25 µl PCR reactions contained 3 µl of DNA tem- Hilda Matthews who collected the material for this study. plate, 1 µl of each primer (10 pmol) and 20 µl of milli-Q-water and were performed with PuReTaq Ready-To-Go™ PCR Morphological description (Figs 1 and 2; Table I) beads (GE Healthcare) in 0.2 ml tubes using the following protocol: 4 min at 95°C followed by 35 cycles of 1 min at 95°C, This species is described morphologically on the basis of four 1 min at 55°C and 2 min at 72°C. The PCR products were then proteolytic enzyme digested specimens. Anterior portions of purified using a NucleoSpin® Purification Kit (Macherey- all four specimens were processed for molecular analysis, no Nagel) following the manufacturer’s recommended protocol. taxonomically informative body structures [i.e. male copula- Sequencing reactions were carried out on a MegaBace 1000 tory organ (MCO), pharynx] were retained. Hamuli measuring analysis system (GE Healthcare) using DYEnamic ET dye ter- 57.5 µm (57–59) in total length; proportionately robust with a minators. The PCR primers and the internal primers ITS1R distal width of 6.0 (5–6); proximal width from the apex of the (5’-ATTTGCGTTCGAGAGACCG-3’), ITS2R (5’-GGTAAT- ventral bar attachment point to a point posterior to the dorsal CACGCTTGAATC-3’), ITSR3A (5’-GAGCCGAGTGATC- bar attachment point 9.1 (8–10) wide; shaft 37.6 (37–38) long; CACC), and ITS2F (5’-TGGTGGATCACTCGGCTCA-3’) point 28.4 (27–30) long; hamulus aperture distance 18.7 (17– (Matějusová et al. 2001, Ziętara and Lumme 2003) were used 21) long; aperture angle 34.6° (31°–40°); hamulus root 17.9 for sequencing. Sequences were proofread and assembled in (15–20) long, comprising approximately one third of the total Vector NTI 10 (Invitrogen) and Mega 4 (Tamura et al. 2007) length of the hamulus (Figs 1a; 2a, b), ventral edge outwardly was used to calculate genetic distances. The sequences were curved. Dorsal bar straight, 22.8 (22–24) wide, 3 (2–3) long submitted to a BLASTN search (Zhang et al. 2000) with de- (Figs 1a; 2a). Ventral bar approximately triangular in shape, fault parameter settings to establish possible identity with 27.6 (25–31) wide, 21.2 (19–23) long; ventral bar processes other species. triangular, 3.5 (3–5) long, with a broad base extending the entire lateral edge of the extremity of the median ventral bar; ventral bar membrane triangular with a weak central ridge, Results 11.7 (11–13) long; distal edge thin (Figs 1a, c; 2a, c). Mar- ginal hooks 27.7 (27–28) long; shaft 22.4 (22–23) long, at- Gyrodactylus hildae sp. nov. taches at a point in line with the inner face of the sickle shaft. Synonym: G. sp. 2 in García-Vásquez et al. 2010 Marginal hook instep 0.5 (0.4–0.6) high. Sickle proper 5.5 (5– 6) long; shaft narrow, slightly angled forward, straight in its Type host: Oreochromis niloticus niloticus (L.) (Cichlidae); lower half before gently curving to the point (Figs 1d, e; 2d, wild. e). Distal width 3.6 (3–4); the point terminating before the Other host: An unspecified cichlid, not confirmed as limit of the toe; proximally 4.0 (4–4.5) wide. Toe long, trian- O. n. niloticus (Cichlidae). gular (Figs 1d; 2d) to trapezoid (Fig. 1e) in shape, 1.9 (1.5–2) Site: Fins. long; flat prominent bridge (dorsal surface of sickle foot/base), Locality: Tributary of the Baro River, Gambela, Ethiopia sloping sharply downwards to the tip of the toe. Sickle heel (8°36΄N, 40°14΄E). pronounced and marked by a flat, slightly downward sloping Type material: Four proteolytically digested, formalde- dorsal edge which then curves smoothly to the point where hyde-glycerine mounted specimens. Type specimens are not the shaft articulates with the sickle (Figs 1d, e; 2d, e). Sickle Gyrodactylids infecting Oreochromis spp. 23

Fig. 1. Light micrographs of the haptoral hard parts of Gyrodactylus hildae sp. nov. from Oreochromis niloticus niloticus (L.) collected from Ethiopia, released by proteolytic digestion: a – central hook complex, b – marginal hook, c–ventral bar, d-e –marginal hook sickle. Scale bars = 10 µm (a-c), 2 µm (d-e) aperture 5.0 (4.5–5.5) long. Filament loop 17.4 (17–18) long, G. cf. longidactylus Geets, Malmberg et Ollevier, 1988 more than half the total length of the marginal hook (Fig. 1b). (AY338449), G. cf. micropsi Gläser, 1974 (AF328868, AJ427221, AY338447-8), G. gracilihamatus Malmberg, 1964 Molecular characterisation (AF484531-2), G. rugiensoides Huyse et Volckaert, 2002 (AJ427414) and G. anguillae Ergens, 1960 (AB063291-4). The 1124 bp consensus sequence which was determined from three of the four specimens consisted of the 3’ end of the 18S Comments subunit, the ITS1, ITS2, the 5.8S gene and the 5’ end of the 28S subunit. No variation was found between sequences. Although three out of the four specimens of G. hildae sp. nov. A BLASTN (Altschul 1991, Zhang et al. 2000) search in Gen- were found on the fins of an unspecified cichlid and only one Bank (accessed June, 2008) using the entire sequence revealed on O. n. niloticus, the specimen from O. n. niloticus is desig- no close hits that could indicate conspecificity with any known nated as the holotype. The morphology of the hamuli and mar- species. When the 5.8S gene was submitted to a BLASTN ginal hooks of G. hildae sp. nov. differ from those of G. cich- search separately it was found to be identical to several gyro- lidarum, the most common species of Gyrodactylus found dactylid species, namely G. rugiensis Gläser, 1974 (AF328870, on cichlids, and the difference is most evident in the shape DQ821761-2), G. jussii Ziętara et Lumme, 2003 (AY061982), of the marginal hooks (Figs 2e-f), which are unlike those 24 Adriana García-Vásquez et al.

Fig. 2. Drawings of Gyrodactylus hildae sp. nov. collected from Oreochromis niloticus niloticus (L.) from Ethiopia (Baro River, Gambela): a – haptoral complex, b – hamulus, c – ventral bar, d – marginal hook of G. hildae sp. nov., e – marginal hook of Gyrodactylus cichlidarum Paperna, 1968 (García-Vásquez et al. 2007), f – overlay of G. hildae sp. nov. (solid line) with G. cichlidarum (dotted line). Scale bars = 10 µm (a-b), 5 µm (c), 2 µm (d-f) of any other species of Gyrodactylus previously recorded Type material: Nine proteolytically digested, formalde- from African fish. hyde-glycerine mounted specimens were prepared for morphometric analysis. The holotype (accession no. 2008.12.15.11) Gyrodactylus ulinganisus sp. nov. and 5 paratypes (accession no. 2008.12.15.12-13 and Synonym: G. sp. 3 in García-Vásquez et al. 2010 2009.6.2.11-13) are deposited in the Parasitic Worms collection of The Natural History Museum, London. In addition, one Type host: Oreochromis mossambicus (Peters) (Cichlidae); paratype (accession no. M-479) is deposited in the gyro- cultured stock. dactylid collection held at the Institute of Parasitology, Biol- Site: Skin and fins. ogy Centre of the Academy of Sciences of the Czech Republic, Locality: Ponds in grounds close to the University of Stel- České Budějovice and a further two paratypes (accession no. lenbosch, Welgevalen, Stellenbosch, South Africa (33°57΄S, SAMCTA 29459) are lodged in the helminth collection of the 18°38΄E). South African Museum in Cape Town, South Africa. A species Gyrodactylids infecting Oreochromis spp. 25 a a a a a col- n = 4 (8–9) (5–6) (7–8) (3–4) (1–2) (5–7) (11–12) (11–12) sp. nov. (15–18) (20–21) (23–26) (22–24) (22–23) (31–33) (34–40) (39–46) (16–18) (47–49) Boulenger . 2010); Gyrodacty- et al documented from at least Cone, Arthur et Bondad-Re- Cone, n = 9 (1–4) (5–9) (7–9) (3–5) (1–3) (4–8) (1.5–3) Amphilius atesuensis Amphilius sp. nov. from O.sp. nov. n. niloticus G. shariffi O. n. niloticus from sp. 2 in García-Vásquez (1–3) (3–5) (7–9) (5–6) (1–8) (8–10) (11–13) (12–16) (17–21) (22–26) (25–31)(19–23) (22–27) (23–26) (27–28) (28–32) (27–30) (27–30) (37–38) (35–40) (31–40) (36–48) (34–47) (41–50) (15–20) (22–28) (57–59) (59–65) 8.0 ± 1.23.5 ± 0.8 7.6 ± 0.9 2.0 ± 0.4 5.3 ± 0.2 ± 0.3 11.5 9.1 ± 0.5 8.0 ± 0.5 7.9 ± 0.5 2.3 ± 0.9 2.5 ± 0.7 ± 0.3 11.6 6.0 ± 0.4 4.1 ± 0.2 3.6 ± 0.1 1.9 ± 0.9 1.8 ± 0.5 1.6 ± 0.3 5.9 ± 2.8 5.5 ± 1.3 6.2 ± 1.3 (0.8–3.0) G. et al . 2010) and G. yacatli G. cichlidarum Paperna, 1973 (holotype) from (holotype) 1973 Paperna, 5.5 8.7 3.8 2.1 7.7 11.8 14.4 ± 1.1 11.7 14.8 ± 1.2 8.5 ± 0.7 15.5 18.7 ± 1.8 23.3 ± 1.2 16.8 ± 1.1 32.8 21.2 ± 1.6 24.4 ± 1.3 24.7 ± 1.3 13.3 19.4 27.7 ± 0.5 31.3 ± 0.7 22.3 ± 1.0 21.9 28.4 ± 0.9 28.5 ± 1.0 22.7 ± 0.5 31.2 37.6 ± 0.5 37.6 ± 1.4 32.2 ± 0.7 30.3 34.6 ± 4.3 41.1 ± 2.5 37.9 ± 2.4 33.5 40.5 ± 5.7 46.7 ± 3.0 43.3 ± 3.3 12.7 17.9 ± 2.2 24.6 ± 1.7 16.9 ± 0.6 47.5 57.5 ± 1.0 61.9 ± 2.1 48.4 ± 1.1 21.6 27.6 ± 2.6 24.7 ± 2.0 20.4 ± 0.6 (L.) from Ghana; (L.) collected from Ethiopia ( sp. 3 in García-Vásquez Gyrodactylus amphiliusi Gyrodactylus G. n = 3 (2–3) (1–2) (7–8) (7–9) (2–4) (9–10) (20–22) (21–24) (22–25) (12–15) (27–28) (27–28) (35–37) (39–40) (44–45) (22–23) (58–59) 4.1 ±0.3 7.4 ± 0.7 1.6 ± 0.1 8.3 ± 0.7 2.5 ± 0.6 3.0 ± 0.4 9.9 ± 0.5 (3.9–4.5) 13.7 ± 1.2 21.4 ± 0.4 23.8 ± 1.6 27.5 ± 0.7 27.1 ± 0.2 35.8 ± 0.9 39.6 ± 0.3 44.7 ± 0.6 22.4 ± 0.7 58.4 ± 0.9 22.0 ± 2.0 G. cichlidarum ) paratype n = 4 (syn. of G. cichlidarum ) (holotype and two paratypes) from the Philippines. For each variable, mean (μm) ± standard deviation is given along with Oreochromis n niloticus Sarotherodon galilaeus galialeus Sarotherodon (1–4) (1–4) (5–9) (6–9) (2–5) (1–4) O. n. niloticus (9–23) (3–13) n = 250 holo/paratypes n = 1 (19–28) (16–31) (19–33) (22–34) (21–30) (29–39) (34–57) (38–58) (16–27) (46–66) 7.1 ± 0.7 2.2 ± 0.4 7.5 ± 0.6 2.5 ± 0.6 3.8 ± 0.4 1.9 ± 0.6 6.2 ± 1.7 13.5 ± 1.4 23.0 ± 7.5 22.8 ± 1.6 28.5 ± 1.4 26.1 ± 1.6 35.3 ± 1.7 43.6 ± 2.9 48.8 ± 3.0 21.4 ± 3.1 56.8 ± 3.1 23.0 ± 1.8 sp. nov. from sp. nov. (Peters) collected from South Africa ( (Peters) collected from South . 2010). Data are presented alongside those of those alongside presented are Data 2010). . 5.4 1.5 7.7 1.2 3.6 3.1 11.4 12.9 22.2 19.2 27.9 24.3 29.2 45.2 51.5 21.4 54.3 19.6 n = 1 et al et holotype study current Gyrodactylus niloticus et al . 2010); and, Gyrodactylus Gyrodactylus hildae Gyrodactylus Paperna, 1968 (holotype) from 6.2 3.9 2.8 11.3 11.8 14.6 34.6 12.4 14.5 19.5 25.9 37.9 25.4 10.5 28.5 24.7 53.5 20.8 n = 1 sp. García-Vásquez in 1 holotype G. amphiliusi G. cichlidarum G. cichlidarum G. niloticus G. shariffi G. hildae G. ulinganisus G. yacatli G. Paperna, 1973 Paperna, 1968 Paperna, 1968 (syn. of Cone et al. , 1995 sp. nov. sp. nov. sp. nov. mossambicus from Oreochromis Gyrodactylus cichlidarum Gyrodactylus Morphological measurements of HAD VBML VBMemL Table I. Table HPSW VBTL VBPML MHTL VBPL 13 different countries (see García-Vásquez 13 different range in parentheses Measurement from Ghana; HPL lus ulinganisus lected from Mexico ( Mexico from lected antaso, 1995 (paratype), which were collected from a population of HDSW HSL HICL HAA HPCA HIAA HRL HTL VBTW 26 Adriana García-Vásquez et al.

profile including taxonomic traits, host details and additional metadata is provided on the on-line database www.gyrodb.net (4–5) (3–4) (1–2) (3–4) (4–5) (Harris et al. 2008, Shinn et al. 2010). (17–20) Molecular sequence data: An 802 bp consensus sequence spanning part of the SSU rDNA, ITS1 (342 bp), 5.8S (157 bp) and ITS2 (303 bp) region obtained from nine individual specimens has been deposited in GenBank (accession no. FJ231870). Etymology: The hook morphology of this species closely (7–8) (4–5) (1–2) resembles that of G. cichlidarum, and the name is derived (4.5–5.5) from a word in the African Xhosa dialect which means “to resemble or imitate something else”.

Morphological description (Figs 3 and 4; Table I) (5–6) (3–4) (4–4.5) (1.5–2) Proteolytically digested specimens. Total length of hamulus (22–23) (21–24) 5.5 ± 0.1 7.7 ± 0.1 4.5 ± 0.2 4.0 ± 0.1 4.4 ± 0.2 3.3 ± 0.2 1.9 ± 0.1 1.4 ± 0.1 1.5 ± 0.2 3.6 ± 0.3 4.7 ± 0.3 3.2 ± 0.1 5.0 ± 0.2 7.1 ± 0.2 4.2 ± 0.3 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.03 (4.5–5.5) (7–7.5) (0.4–0.6) (0.1–0.3) (0.3–0.4) 61.9 (59–65); shaft 37.6 (35–40) long with flat ventral bar ar- ticulation point; point slender, 28.7 (27–30) long; root 24.4 (22–28) long, ventral edge inwardly curved; hamulus aperture 24.2 (22–26) wide, with the point arising at an angle (HAA) 4.3 3.3 1.4 2.7 3.5 0.4 of 42.4° (36°–48°) to the shaft (Figs 3a; 4a, b). Dorsal bar 15.1 22.4 ± 0.6 23.7 ± 0.7 18.0 ± 1.4 straight, 20.3 (18–24) wide; 2.1 (2–2.5) long (Fig. 4a), the union of the dorsal bar with the hamulus is prominent, the anterior edge of which is notably right angled. Ventral bar 23.8 (22–27) wide, 24.4 (23–26) long; ventral bar processes rounded and approximately triangular, 2.0 (1.5–3) long; median portion 7.6 (5–9) wide marked by a circular plaque on the poste- (7–8) (4–5) (1–2) (4–5) rior edge; ventral bar membrane square to spatulate, 14.8 (20–21) 7.3 ±0.1 7.4 ± 0.2 4.4 ± 0.2 1.7 ± 0.2 4.6 ± 0.2 0.2 ± 0.1 (7.2–7.4) (0.1–0.2) 20.8 ± 0.6 (12–16) long (Figs 3c; 4c) with medial spatulate to limoni- form-shaped ridge. Marginal hooks total length 31.3 (28–32); shaft length 23.7 (21–24), sickle proper 7.7 (7–8) long; steeply sloping, triangular toe of the sickle proper 1.4 (1–2) long, upper surface of toe weakly concave (Figs 3d; 4d, e). Base of the sickle proper is flat with the toe and heel approximately (6–8) (4–5) (1–2) (3–6) (6–9) level with a small instep, 0.3 (0.1–0.3) high. Heel rounded. (0–0.5) (16–27) 7.4 ± 0.3 4.4 ± 0.2 1.4 ± 0.1 4.7 ± 0.3 7.1 ± 0.3 0.3 ± 0.1 21.4 ± 1.2 Sickle shaft gently curves from its base to a point beyond the toe; inner curve of the sickle is open with an aperture of 7.1 (7–7.5). Sickle slender, proximally 4.4 (4–5) wide; distally 4.7 (4.5–5.5) wide (Figs 3d; 4d, e). 6.5 2.9 1.1 3.9 6.8 0.2

21.6 Molecular characterisation

The amplified nucleotide sequence of the rDNA cluster was 850 bp long and comprises the 3’ end of the 18S subunit, the ITS1, the 5.8S gene, ITS2 and the 5’ end of the 28S subunit. No variation was found between sequences obtained from the nine specimens that were analysed. A BLASTN search (Alt- 4.5 4.7 3.1 3.7 4.6 0.5 15.3 schul 1991, Zhang et al. 2000) in GenBank (accessed June, 2008) using the entire sequence revealed no close hits that could indicate conspecificity with any known species, con- HAA –HAA hamulus aperture angle, HAD – hamulus aperture distance, – HDSW hamulus distal shaft width, – HIAA hamulus inner aperture angle, – HICL hamulus inner firming that it differed from G. cichlidarum (DQ124228). When the 157 bp long 5.8S gene was submitted to a BLASTN search separately, however, this gene was found to be identical to that of G. cichlidarum. MHSL MHSiL MHSiPW MHToeL MHSiDW MHA Abbreviations: curve length, – HPCA hamulus point curve angle, – HPL hamulus point length,– – HPSW length, total hamulus hamulus proximal shaft width,– MHA –aperture, HRL hook marginal – MHI/AH hamulus root length,height, instep/arch hook marginal – HSL – – MHSL MHSiL hamulus shaft length,length, shaft hook marginal hook sickle length, marginal MHSiDW HTL – – MHSiPW width, distal sickle hook marginal hook sickle proximal width, marginal – MHTL hook total length, marginal bar membrane length, – MHToeL – hook toe length, VBML marginal – VBMemL ventral bar median length, ventral – VBPL ventral bar process length,ventral bar total width. – VBPML ventral bar process-to-mid length, – VBTL ventral bar total length, – VBTW MHI/AH Gyrodactylids infecting Oreochromis spp. 27

corrected “p” distance). These substitutions can be attributed to 24 in ITS1 (12 transitions and 12 transversions) and 18 substitutions in ITS2 (9 transitions and 9 transversions), and in addition 1 indel was found. The large number of differences (42 out of 802) observed in the ITS regions suggests that these two species are different, and although no fixed threshold ex- ists for the separation of Gyrodactylus species, Ziętara and Lumme (2003) suggest that a 1% difference in ITS be used as a cut-off value to delineate species. The hamuli of G. ulinganisus sp. nov. although slightly larger than those of G. cichlidarum, have similar sized hamulus aperture angles (42.4° for G. ulinganisus sp. nov. cf. 45.0° for G. cichlidarum). Size alone, however, should not be a basis for discrimination, as the size of hooks can vary with temper- ature (Mo 1991a-c). Subtle morphological features separating the two species include the ventral bar attachment points on the hamuli, the shape of the marginal hook sickles, and features of the median portion of the ventral bar. Gyrodactylus cichlidarum also possesses a central plaque on its dorsal surface referred to as a “characteristic crescent-shaped depres- sion” in the revised description by García-Vásquez et al. (2007), which appears to differ from the more spherical to rectangular plaque observed on the ventral bar of G. ulinganisus sp. nov. (Figs 3c; 4c). This spherical feature, which probably serves as a muscle anchoring point (Shinn and Bron, unpublished data), is only visible when the dorsal surface of the ventral bar is examined and appears to vary in size and shape from individual to individual, which itself may be a consequence of the proteolytic digestion step. Given the variability of this plaque, it is not presented on the illustrations provided for this species (Fig. 4c). The ventral bar attachment points on the hamuli of G. ulinganisus sp. nov., when viewed in profile, are flatter (Figs 3a; 4a, b) than those seen for G. cichlidarum, which appear to be gently curving (see Figs 2a, b; 3a, c in Gar- cía-Vásquez et al. 2007). The marginal hook sickles can also be discriminated in that the dorsal surface (i.e. bridge) of the marginal hook sickle base or foot is longer in G. ulinganisus sp. nov. than that of G. cichlidarum which has a thicker sickle base and a toe that flattens into the bridge at a point closer to the shaft of the sickle. The shaft and point regions of the mar- Fig. 3. Light micrographs of the haptoral hard parts of Gyrodactylus ginal hook sickle of G. ulinganisus sp. nov. are not as broad as ulinganisus sp. nov. from Oreochromis niloticus niloticus (L.) and those of G. cichlidarum and follow a tighter curve. The sickle Oreochromis mossambicus (Peters) collected from South Africa (Stellenbosh, cultured stock) released by proteolytic digestion: heel of G. ulinganisus sp. nov. also appears more rhomboid in a – hamulus, b – marginal hook, c – ventral bar, d – marginal hook profile than the gently semi-circular heels of G. cichlidarum, sickle. Scale bars = 10 µm (a-b), 5 µm (c), 2 µm (d) which are wider, given that they have a thicker base to the marginal hook sickle. The relative proportion of the toe length Comments to heel length in G. ulinganisus sp. nov. is approximately 1:1 (Fig. 4d, e) as opposed to 1:2 for that of G. cichlidarum. Differences in the hook morphology of G. ulinganisus sp. nov. and G. cichlidarum are subtle and although a principal com- Gyrodactylus yacatli sp. nov. ponents analysis (PCA) of morphometric data suggested a dif- Synonym: G. sp. 1 in García-Vásquez et al. 2010 ference (see García-Vásquez et al. 2010), their clear differen- tiation from each other was confirmed by the molecular study. Host: Oreochromis niloticus niloticus (L.) (Cichlidae), cul- Altogether, there are 42 differences between the ITS1 and tured stock. ITS2 of G. ulinganisus sp. nov. and G. cichlidarum (0.049 un- Site: Gills and fins. 28 Adriana García-Vásquez et al.

Fig. 4. Drawings of the haptoral hard parts of Gyrodactylus ulinganisus sp. nov. collected from Oreochromis niloticus niloticus (L.) and Oreochromis mossambicus (Peters) from South Africa, China and Mexico: a – haptoral hard complex, b – hamulus, c – ventral bar, d – marginal hook, e – marginal hook of Gyrodactylus cichlidarum Paperna, 1968 (see García-Vásquez et al. 2007), f – overlay of G. ulinganisus sp. nov. (solid line) with G. cichlidarum (dotted line). Scale bars = 10 µm (a-b), 5 µm (c) 2 µm (d-f)

Locality: Gania de Pucté, Municipal de Chablé, Tabas- at the Institute of Parasitology, Biology Centre of the Academy co, Mexico (17°56΄N, 92°33΄W); cultured stock, Merida, Me- of Sciences of the Czech Republic, České Budějovice. A xico (21°01΄N, 89°37΄W) and Culiacan, Mexico (24°50΄N, species profile including taxonomic traits, host details and ad- 107°24΄W). ditional metadata is provided on the on-line database www.gy- Type material: Four proteolytically digested specimens. rodb.net (Harris et al. 2008, Shinn et al. 2010). Formaldehyde-glycerine preserved preparations of the haptoral Molecular sequence data: No sequences were obtained. armature of the holotype (accession no. 2008.12.15.14) and Two specimens were prepared for molecular analysis but two paratypes (accession no. 2008.12.15.15 and 2009.6.2.14) failed to amplify. are deposited in the Parasitic Worms collection of The Natural Etymology: Named after the pronounced ventral bar History Museum, London. In addition, one paratype (acces- processes using a word taken from the Nahuatl Mexican di- sion no. M-480) is deposited in the gyrodactylid collection held alect, which means “to resemble horns or tips”. Gyrodactylids infecting Oreochromis spp. 29

Morphological description (Figs 5 and 6; Table I) ginal hooks 22.3 (22–24) long; shaft length 18 (17–20); sickle proper 4.5 (4–5) long; 3.3 (3–4) wide proximally, 3.2 (3–4) This species is described morphologically on the basis of one wide distally; rhomboid toe, 1.5 (1–2) long; toe region has a whole mount and three proteolytic enzyme digested speci- long bridge turning into a slender, forward sloping sickle shaft; mens. Total body length 420, width mid-body 95. Anterior inner curve to sickle follows a square to rhomboid line, point pharynx bulb 16.4 long, 21.8 wide. Posterior pharynx bulb terminating beyond the limit of the toe; sickle aperture 4.2 (4– 21.6 long, 31.5 wide. Gut not extending beyond the anterior 5) wide; sickle heel, circular to rhomboid in shape (Figs 5c-e; edge of the testes. Haptor, scallop-shaped and clearly delin- 6d, e). eated from body, length 63, width 66. No MCO visible on whole mount. Total length of proteolytically digested hamuli Comments 48.4 (47–49); shaft 32.2 (31–33) long; point 22.7 (22–23) long; hamulus root tapering, 16.9 (16–18) long, representing While the haptoral armature of G. yacatli sp. nov. resembles approximately one third of the length of the hamulus; hamu- that of the holotype of G. shariffi in both shape and approx- lus aperture angle 37.9° (34°–40°); flat, ventral edge to the imate dimensions, the two species can be separated by dif- ventral bar attachment point; dorsal bar attachment point ferences in the shape of the ventral bar membrane and the small, anterior edge more prominent than the lower (Figs 5a; marginal hook sickle. Both species possess ventral bars with 6a, b). Straight dorsal bar 2.7 long, 20.2 wide (Fig. 6a). Ven- large pronounced ventral bar processes (Figs 6c; 7b). The tral bar 20.4 (20–21) wide, 24.7 (23–26) long; prominent ven- ventral bar membrane of G. yacatli sp. nov. is, however, tral bar processes, 11.5 (11–12) long, the anterior edges of short (8.5 long), rectangular, marked by longitudinal ridges which align with the terminus of the root; centre of each ven- and its base covers one third of the median ventral bar (Fig. tral bar process marked by a circular structure; rectangular, 6c). The membrane of G. shariffi by comparison is longer ridged ventral bar membrane, 8.5 (8–9) long, base of which (14.4 long), lingulate, bears a medial spatulate ridge and its represents one third of median ventral bar width; median bar base occupies the entire posterior edge of the median ven- bears prominent, square-edged extremities (Figs 5b; 6c). Mar- tral bar (Fig. 7b). Both marginal hooks possess forward slop-

Fig. 5. Light micrographs of the haptoral hard parts of Gyrodactylus yacatli sp. nov., released by proteolytic digestion, collected from Oreochromis niloticus niloticus (L.) from Mexico (Merida and Tabasco): a – hamulus, b – ventral bar, c – marginal hook, d-e – marginal hook sickle. Scale bars = 10 µm (a), 5 µm (b-c), 2 µm (d-e) 30 Adriana García-Vásquez et al.

ing shafts, rhomboid toes and rounded heels; the heel of Ghana but these two species can also be separated on differ- G. yacatli sp. nov., however, is broader and more rounded ences in the shape of the marginal hook sickle (Fig. 7g). The (Figs 6d, e, f). Although the inner marginal hook sickle marginal hook sickles of G. amphiliusi possess broad, forward curves of both species are approximately square, that of sloping shafts, narrow toes, flat undersides to the sickles, heels G. shariffi is more angular, notably at the point where the that appear to be stepped on their dorsal surface and articulate shaft joins the base (Figs 6d, e, f; 7c). with their marginal shafts at a point that is close to the poste- The ventral bar of G. yacatli sp. nov. is also similar to that rior edge of the heel dividing the regions of the sickle base of G. amphiliusi described from Amphilius atesuensis from into 1:4–5 heel:toe (Figs 7e, f).

Fig. 6. Drawings of Gyrodactylus yacatli sp. nov. from Oreochromis niloticus niloticus (L.) collected from Mexico: a – haptoral complex, b – hamulus, c – ventral bar, d – marginal hook, e – G. shariffi Cone, Arthur et Bondad-Reantaso, 1995 marginal hook, f – overlay G. yacatli sp. nov. (solid line) with G. shariffi (dotted line). Scale bars = 10 µm (a-b), 5 µm (c), 2 µm (d-f) Gyrodactylids infecting Oreochromis spp. 31

Fig. 7. Drawings of the haptoral hard parts of Gyrodactylus shariffi Cone, Arthur et Bondad-Reantaso, 1995 from Oreochromis niloticus niloticus (L.) (a-c), and Gyrodactylus amphiliusi Paperna, 1973 from Amphilius atesuensis Boulenger from Lake Bosomtwi, Ghana (d-f): a – hamulus (re-examination), b – ventral bar (re-examination), c – marginal hook sickle (re-examination), d – haptoral complex showing hamuli, e – ventral bar, f – marginal hook sickle, g – overlay of the marginal hook sickle of Gyrodactylus yacatli sp. nov. (dotted line) with that of G. amphiliusi (solid line). Scale bars = 10 µm (a-b, d-e), 2 µm (c, f-g)

Discussion counts in the literature do not specify which sub-species are being cultured. Clearly further study is required to elucidate the Gyrodactylus hildae sp. nov. collected from O. n. niloticus and primary host of G. hildae sp. nov. including a study of the pos- also from another host, not confirmed as O. n. niloticus from sible gyrodactylid fauna on all sub-species of O. niloticus with Ethiopia, is interesting. This raises the question of whether the a particular focus on the populations throughout Ethiopia and different subspecies of Oreochromis niloticus can be confidently Kenya, the latter of which has three indigenous sub-species separated on morphology alone or whether support is required (O. n. baringoensis, O. n. sugutae and O. n. vulcani). from molecular markers. The taxon O. niloticus consists of eight The finding of a separate species, G. yacatli sp. nov., sub-species: O. n. baringoensis Trewavas, O. n. cancellatus on Mexican populations of O. n. niloticus may have two ex- (Nichols), O. n. eduardianus (Boulenger), O. n. filoa Trewavas, planations, either this species has not yet been detected on O. n. niloticus, O. n. sugutae Trewavas, O. n. tana Seyoum et O. n. niloticus of African origin, or, more likely, G. yacatli sp. Kornfield and O. n. vulcani (Trewavas) (see Trewavas 1983, nov. represents an accidental infection or a host switch from Froese and Pauly 2008). Of these, at least four (O. n. cancella- a fish species inhabiting the water source feeding the farm. tus, O. n. filoa, O. n. tana and O. n. vulcani) are indigenous to A survey of the fish fauna and their parasites inhabiting the Ethiopian lakes (see Trewavas 1983); however, whether waters feeding the two Mexican farms is, therefore, warranted O. n. niloticus has also been introduced is unknown as the ac- and is in progress. 32 Adriana García-Vásquez et al.

Although G. ulinganisus sp. nov. appears to be restricted chlidarum specimens in the PCA plot (see García-Vásquez et to South Africa, it is unknown whether this species has been al. 2010). It was not possible to obtain new Gyrodactylus ma- translocated with shipments of O. mossambicus to other coun- terial from S. g. galilaeus for the current study and therefore tries such as China (Fitzsimmons 2006). With this in mind, the sequence of its ITS1 and 2 remains to be established to de- the gyrodactylid fauna of O. mossambicus from its original termine whether the holotype is identical to the “G. cichli- location, Mozambique, requires study to determine whether darum” specimens considered here or whether they represent G. ulinganisus sp. nov. also occurs there. another cryptic species. Although the attachment hooks of G. ulinganisus sp. nov. This study confirms that Nile and Mozambique tilapia har- are morphologically similar to those of G. cichlidarum, a PCA bour a number of different species of Gyrodactylus, G. cich- analysis of the morphometric data suggested that they can be lidarum being the most commonly encountered. It is clear separated (see García-Vásquez et al. 2010). This finding was from the present study that several species of Gyrodactylus supported by a parallel molecular analysis which identified 42 may be associated with the mortalities of juvenile Oreo- nucleotide differences in the ITS regions between the two chromis species that have been reported worldwide. We know species. It is possible, however, that other morphologically little regarding the host-specificity, ecology and populations cryptic species of Gyrodactylus parasitising closely related dynamics of these particular species of Gyrodactylus on cich- hosts also exist. Given that Paperna (1968) documented G. ci- lid hosts and whether different management strategies are re- chlidarum from multiple hosts of at least three different gen- quired for their effective control. Further research is therefore era (i.e. Sarotherodon, Tilapia and Hemichromis), a re-exa- required to investigate this, and until this information is avail- mination of these hosts and their gyrodactylids using molec- able, we should exercise extreme caution in the future translo- ular approaches should also be conducted. cation of commercial tilapiine species into areas where In addition, G. cichlidarum is described from O. n. niloti- cichlids are already resident. cus (reported as G. niloticus in López-Jiménez 2001), from O. mossambicus (reported as G. niloticus in Hernández-Mar- Acknowledgements. We would like to thank Dr. Rudy Jocqué from the Musée Royal de l’Afrique Centrale (MRAC) for the loan of the tínez 1992, Salgado-Maldonado et al. 2005), and, from O. au- G. cichlidarum holotype and Patricia Pilitt from the USDA U.S Na- reus (see Salgado-Maldonado et al. 2005). Gyrodactylus sp. tional Parasite Collection for the loan of G. niloticus and G. shariffi has also been reported from a range of principally Mexican type material from the Philippines. We gratefully acknowledge the cichlids including Cichlasoma geddesi (Regan) (see Vidal- contribution of Hilda Matthews, a remarkable lady, who, for two Martínez et al. 2001), Parachromis managuensis (Günther) years worked her way solo across Africa from fish market to fish (see Vidal-Martínez et al. 2001), Thorichthys aureus (Gün- landing station collecting unique fish and parasite samples for the authors to analyse. We would also like to thank Dr. Victor Vidal- ther) (see Vidal-Martínez et al. 2001), T. helleri (Steindachner) Martínez and Mrs. Clara Vivas from CINVESTAV, Merida, Mexico, (see Vidal-Martínez et al. 2001) and T. meeki Brind (see Vidal- Dr. Emma J. Fajer-Avila from CIAD, A.C. Mazatlán, Mexico, Martínez et al. 2001). Each of these latter species are worthy and Mr. Tobias Roman from Aquacorporación de Honduras and of study to determine whether they are parasitised by G. ya- Mr. Adrian Piers from the Aquaculture Division, Welgevalen, Uni- catli sp. nov. or other closely related species. versity of Stellenbosch, South Africa for the provision of infected Unfortunately, much of Paperna’s gyrodactylid collection material for study. We also thank Professor Tor Bakke (Natural His- tory Museum, University of Oslo) and the three anonymous referees was lost in a laboratory fire and cannot be re-examined. for their comments on this manuscript. This study was supported Whether G. cichlidarum displays broad host specificity re- through financial support to AGV from Mr. Jaime García and Mrs. mains to be established, however, during an outbreak of G. ci- Miriam Vásquez de García. chlidarum in an aquarium facility in Scotland, G. cichlidarum was also found to infect Oreochromis karongae (Trewavas) in the same system (unpublished data). Subsequent experimen- References tal infections of O. karongae with G. cichlidarum were found Altschul S.F. 1991. Amino acid substitution matrices from an infor- to last at least 28 days, suggesting that the parasite can com- mation theoretic perspective. Journal of Molecular Biology, plete its life-cycle on this host. More detailed studies on the 219, 555–565. DOI: 10.1016/0022-2836(91)90193-A. host-specificity of G. cichlidarum are required. Cone D.K., Arthur R., Bondad-Reantaso M.G. 1995. 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(Accepted September 21, 2010)