Parasitology International 68 (2019) 31–39 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint A new species of Haliotrema (Monogenea: Ancyrocephalidae (sensu lato) T Bychowsky & Nagibina, 1968) from holocentrids off Langkawi Island, Malaysia with notes on the phylogeny of related Haliotrema species ⁎ Soo O.Y.M.a,b, a UCSI University KL, No.1, Jalan Menara Gading, Taman Connaught 56000 Cheras, Kuala Lumpur, Malaysia b Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia ARTICLE INFO ABSTRACT Keywords: Haliotrema susanae sp. nov. is described from the gills of the pinecone soldierfish, Myripristis murdjan off Monogenea Langkawi Island, Malaysia. This species is differentiated from other Haliotrema species especially those from Holocentrids holocentrids in having a male copulatory organ with bract-like extensions at the initial of the copulatory tube, Haliotrema grooved dorsal anchors and ventral anchors with longer shafts. The maximum likelihood (ML) analysis based on 28S rDNA partial 28S rDNA sequences of H. susanae sp. nov. and 47 closely related monogeneans showed that H. susanae Malaysia sp. nov. is recovered within a monophyletic clade consisting of only species from the genus Haliotrema. It is also observed that H. susanae sp. nov. forms a clade with H. cromileptis and H. epinepheli which coincides with a similar grouping by Young based on solely morphological characteristics. The morphological and molecular results validate the identity of H. susanae sp. nov. as belonging to the genus Haliotrema. 1. Introduction rubrum were collected in the coastal waters off the island of Langkawi (6°21′N, 99°46′E) in March 2011. Monogeneans were collected from The family Holocentridae (squirrelfish and soldierfish) consists of excised gills of freshly killed or frozen fish and prepared for morpho- 84 species globally with 13 species recorded in Malaysia from three logical investigation. Briefly, the monogeneans were collected from the genera (Sargocentron, Myripristis and Ostichthys)[1]. To date, species of gills, pipetted onto clean glass slides, covered with cover slips and four genera of monogeneans have been reported from holocentrids; cleared in modified ammonium-picrate-glycerin [2]. These ammonium- Benedenia Diesing, 1858, Enoplocotyle Tagliani, 1912, Pseudohalio- picrate-glycerin specimens were later washed and dehydrated through trematoides Yamaguti, 1953 (taxonomic status is incertae sedis) and a graded ethanol series and mounted in Canada balsam without staining Haliotrema Johnston & Tiegs, 1922, with the largest number of de- [2] to study the sclerotized hard parts. To study the soft anatomical scribed species coming from the latter genus (Table 1). During a field structures, some specimens were immediately fixed after collection, in trip in 2011, three species of holocentrid fish (Myripristis murdjan For- AFA (acetic acid-formalin-alcohol) onto glass slides, then stained in sskål, 1775, M. hexagona Lacépède, 1802 and Sargocentron rubrum Gomori's triple stain and mounted in Canada balsam after dehydration Forsskål, 1775) were examined for monogeneans. A new monogenean in increasing ethanol series [3]. Stained and unstained specimens were species was discovered from M. murdjan and its taxonomy is described studied under bright-field and phase contrast microscopes. Images of herein. This new species is not only the first described Haliotrema the hard and soft anatomical structures of the new species were cap- species from Malaysia but is the first study on holocentrids from Ma- tured using a Leica digital camera and an image analysis software laysian waters. Molecular information from the new species is further (QWin Plus). The hard and soft parts were illustrated using a digitizing analysed with molecular information from related Haliotrema species to tablet (WACOM) and Adobe Illustrator software (Fig. 1). The specimens confirm its validity. were measured as indicated in Fig. 1 and measurements given in mi- crometres as an average with the ranges in parentheses. Type-speci- 2. Materials and methods mens of the new species were deposited at the Zoological Reference Collection, Lee Kong Chian Natural History Museum, National Uni- The fish hosts, Myripristis murdjan, M. hexagona and Sargocentron versity of Singapore, Singapore (ZRC) and Natural History Museum ⁎ Corresponding author at: UCSI University KL, No.1, Jalan Menara Gading, Taman Connaught 56000 Cheras, Kuala Lumpur, Malaysia. E-mail address: [email protected]. https://doi.org/10.1016/j.parint.2018.09.003 Received 2 March 2018; Received in revised form 15 August 2018; Accepted 17 September 2018 Available online 20 September 2018 1383-5769/ © 2018 Elsevier B.V. All rights reserved. O.Y.M. Soo Table 1 Monogeneans from fish of the Family Holocentridae. Monogenean Host Localities Present status Authority Haliotrema auribaculum Zhukov, 1980 Holocentrus adscensionis Osbeck (formerly known as Holocentrus ascensionis) Gulf of Mexico Valid Zhukov, 1980 Haliotrema chelicirrus Yamaguti, 1968 Neoniphon aureolineatus Liénard (formerly known as Holocentrus scythrops), N. samara Forsskål, Sargocentron Hawaii Valid Yamaguti, 1968 punctatissimum Cuvier (formerly known as Holocentrus lacteoguttatus) Haliotrema curvicirrus Zhukov, 1980 (questionable) Holocentrus adscensionis Osbeck (formerly known as Holocentrus ascensionis) Gulf of Mexico Valid Zhukov, 1980 Haliotrema holocentri Young, 1968 Sargocentron rubrum Forsskål Heron Island, Queensland, Valid Young, 1968 Australia Haliotrema longirectocirrus Zhukov, 1980 Holocentrus adscensionis Osbeck (formerly known as Holocentrus ascensionis) Gulf of Mexico Valid Zhukov, 1980 Haliotrema myripristisi Zhukov, 1980 Myripristis jacobus Cuvier Gulf of Mexico Valid Zhukov, 1980 Haliotrema papillibaculum Zhukov, 1980 Myripristis jacobus Cuvier Gulf of Mexico Valid Zhukov, 1980 Haliotrema tenuihamus Zhukov, 1980 Myripristis jacobus Cuvier Gulf of Mexico Valid Zhukov, 1980 Benedenia hawaiiensis Yamaguti, 1968 Neoniphon aureolineatus Liénard (formerly known as Holocentrus scythrops) Hawaii Valid Yamaguti, 1968 Enoplocotyle hawaiiensis Yamaguti, 1968 Neoniphon aureolineatus Liénard (formerly known as Holocentrus scythrops) Hawaii Valid Yamaguti, 1968 Pseudohaliotrematoides incertae sedis falcatus Yamaguti, Sargocentron spiniferum Forsskål Hawaii Incertae sedis Yamaguti, 1968 1968 32 reaction (50 μl) wasand reverse performed primer in D2rDNA, (5′-TGGTCCGTGTTTCAAGAC-3′) 1.5 using mM forward [ primers MgCl C1in (5′-ACCCGCTGAATTTAAGCAT-3′) [ the PCR reactiontion kit to from QIAGEN. amplify The the75% extracted DNA partial ethanol. (5 Genomic D1-D2 μl) was DNA domainwere used was as of removed a then the from template extracted 28S the using gills DNEasy of extrac- thecarried hosts, out identified as and there preservedto weren't in enough be specimens forpers. a comm., revision. 2017). Monogeneans collectedhosts) from because the slides cannotwas be not taken out able ofHaliotrema the to banana museum (Gerasev, obtainLondon Zhukov's (NHMUK). type The specimens following (from material was holocentrid examined: paratypes, sists of copulatory tube, no accessoryservoir piece, length to 88 enter μm (76–101) initial with anterior part part of of copulatory prostatic tube.prostatic Copulatory reservoir; reservoir, organ prostatic con- duct multipleform leaves seminal prostatic prostatic vesicle, gland re- narrowscaecal to ducts branch, enter deplete copulatory ascends tube. into diagonally An in the elongated intercaecalenlarged ends, region, 46 μm distends (37–56). slightly to bent dorsal bar, 44 μmlength (28–51) wide; 46 V-shaped μm ventral bar(18–25); (41–49); with outer point root 11 13point μm μm (8–13); 13 (10–16); μm inner10 length two (11–15); μm 54 (7–12); connecting μm inner two length (48–61); bars; 59chors; ventral μm outer (50–65); two outer anchors dorsal length 47 anchorssimilar with μm (43–50); with sized inner marginal inner hooks, root rootmost length 24 rectangular; μm 22 12 μm length μm (20–28); 90 (10–14);intestine μm outer bifurcates two (68–113) root posterior pairs width to of 123anterior pharynx. μm an- Haptor region (86–181), set with 14 off 2 from body, pairs al- of pigmented eye spots. Mouth ventral; 3.1. Description of new species 3. Results robustness of the inferredBootstrap relationships. procedure with 1000 replications wasMaximum performed to likelihood assess (ML) the v7.164 analysis was [ performedupdated. with These MEGA7 DNA [ sequences(WoRMS) were [ edited andphylogenetic aligned tree with follow MAFFT those inIt the should World be Register noted ofdiscoides that Marine indianus the Species names ofmonogeneans the from species the used forPartial GenBank building ( 28S the rDNAboratories, KL) sequence using dataautomated the DNA of same sequencing the (ABI primerswas 3730 new purified used DNA using Sequencer, for species Firstviewed PCR a and Base under amplification. DNApurification a La- selected UVphoresed illuminator. kit(QIAGEN) The in remaining and a 40subjected to μlat 1.3% of 72 agarose each °C amplicon gel, for 101 stained min. min, with 50 An °C aliquot ethidium for 1 (10itial bromide min μl) denaturation and from and 72 at °C the 95 for amplicon °C 1cycler for min, was (Eppendorf followed 4 electro- Mastercycler) min, by a using followed finaleach by the extension PCR 35 following cycles primer conditions: of and an 95mentas), 1 °C in- U 200 for of μM of each deoxyribonucleotide