Introduction
Heterobothrium Cerfontaine, Reference Cerfontaine1895 (Monogenoidea: Diclidophoridae) represents a lineage of parasitic flatworms infecting the gill lamellae primarily of tetraodontid fishes (Teleostei: Tetraodontidae), occurring within the eastern and Indo-Pacific Ocean, western Atlantic Ocean, and off southern Africa (Acosta and Smit Reference Acosta and Smit2021; Kim et al. Reference Kim, Nguyen, Truong, Dang, Vu, Chinh, Nguyen, Nguyen and Nguyen2025; Ogawa and Itoh Reference Ogawa and Itoh2022; Vidal-Martínez and Mendoza-Franco Reference Vidal-Martínez and Mendoza-Franco2008). The genus traces its origin to Goto’s (Reference Goto1894) description of Diclidophora tetrodonis Goto, Reference Goto1894, which was distinguished from other diclidophorids by the unique 180° lateral rotation of members of the anterior pair of haptoral clamps. Based in part on this feature, D. tetrodonis was transferred to Heterobothrium as Heterobothrium tetrodonis (Goto, Reference Goto1894) Cerfontaine, Reference Cerfontaine1895 by Cerfontaine (Reference Cerfontaine1895), who designated it as the type species of the genus. Currently, 21 species of Heterobothrium are listed in the World Register of Marine Species (WoRMS; www.marinespecies.org); however, in our study the genus is represented by 19 valid species (see Table 1).
List of valid species of Heterobothrium, their type hosts, type localities, and references
a The type species of Heterobothrium is Heterobothrium tetraodonis (Goto, Reference Goto1894) Cerfontaine, Reference Cerfontaine1895, which is the currently valid synonym (name) of Diclidophora tetraodonis Goto, Reference Goto1894.
Euzet and Birgi (Reference Euzet and Birgi1975) provided a revision and emended diagnosis of Heterobothrium, synonymized Tagia Sproston, 1946 and Gempylitrema Yamaguti, 1968 with Heterobothrium, and described Heterobothrium fluviatile (as H. fluviatilis) from the gills of Tetraodon lineatus Linnaeus (as Tetraodon fahaka Bennett) from the freshwaters of Chad. Following their revision, later studies recorded an unidentified species of Tagia and Heterobothrium lineatum Morsy, Saady, Abdel-Gaffar, Bashtar, Melhorn, Al Quraishy & Adel, Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012 (as H. lineatus) from T. lineatus in Egypt (Mandour et al. Reference Mandour, Ibraheem and El-Ganainy2000; Morsy et al. Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012, Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013).
In the present study and as part of a broader survey of monogenoidean parasites infecting Egyptian freshwater fishes, specimens of T. lineatus were collected from the Nile River in Qena Governorate and examined for parasitic infections. The only monogenoidean species found was initially identified as H. lineatum. The present study included an evaluation of the taxonomic status of the two species of Heterobothrium currently known to infect T. lineatus. The evolutionary relationships among the congenerics of Heterobothrium and of Heterobothrium within the Diclidophoridae were also explored.
Materials and methods
Morphological data
Specimens of the Nile puffer, Tetraodon lineatus, were periodically collected from the Nile River in Qena Governorate, Egypt (26°08′N, 32°42′E), between February 2022 and April 2024. Following collection, the fishes were immediately placed on ice and transported to the Parasitology Laboratory, Faculty of Science, Qena University, Egypt. Fish identification was performed using Bishai and Khalil (Reference Bishai and Khalil1997), while common and scientific names were verified using Froese and Pauly (Reference Froese and Pauly2025) and Fricke et al. (Reference Fricke, Eschmeyer and Van der Laan2024), respectively. External surfaces (skin and fins) of the fish were first checked for the presence of monogenoids using a dissecting microscope. Gill baskets were then surgically removed, the gill arches separated, and each gill arch immersed in normal saline solution to eliminate excess mucus. The gill arches were then placed in vials containing a hot (~65°C) 5% formalin solution for relaxation and fixation of the helminths. Worms were then picked from the sediment or removed from the gill tissue using a fine probe, transferred to labelled vials, and shipped to Idaho State University for further study.
Preparation, illustration, and measurement of formalin-fixed specimens followed the methodology described by Kritsky (Reference Kritsky2018). Illustrations were prepared with aid of a camera lucida or microprojector. All measurements were recorded in micrometres (μm) as straight-line distances between extreme points and were presented as the range, followed by the mean and number of structures measured (n) in parentheses; body length included the haptor (see Table 2). Voucher specimens of the helminths were deposited in the National Museum of Natural History (USNM), Smithsonian Institution, Washington, D.C., and the University of Nebraska State Museum, Harold W. Manter Laboratory (HWML), Lincoln, Nebraska, U.S.A., as indicated in the ensuing species account.
Comparative measurements (μm) of Heterobothrium fluviatile in the present study with that previously described
Molecular data
Total genomic DNA was extracted from a single helminth preserved in 95% ethanol using the universal DNA Extraction Kit (Quick-DNATM Fungal/Bacterial Miniprep Kit; Zymo Research, Irvine, California) according to the manufacturer’s manuals. The primers ZX-1 (forward: 5′-ACC CGC TGA ATT TAA GCA TAT-3′) (Bray et al. Reference Bray, Waeschenbach, Cribb, Weedall, Dyal and Littlewood2009) and 1500R (reverse; 5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003) were used to amplify partial fragments of the 28S rDNA gene (domains D1–D3). Procedures for PCR amplification, gel electrophoresis, purification, and sequencing were carried out according to the protocol of Mansour and Kritsky (Reference Mansour and Kritsky2024).
The current large subunit (LSU) sequence was modified using GeneStudioTM Professional Edition V. 2.2.0.0. LSU sequences of diclidophorids and the outgroup taxa, species of Kannaphallus Unnithan, 1957 (Heteraxinidae) and Pauciconfibuloides de Aguiar, Domingues, Silva, Ceccarelli, Adriano & Soares, Reference de Aguiar, Domingues, Silva, Ceccarelli, Adriano and Soares2022, and Microcotyle Van Beneden & Hess, 1863 (both Microcotylidae) were retrieved from the GenBank database (Table 3) and aligned using MUSCLE implemented in MEGA11 V. 11.0.13 (Tamura et al. Reference Tamura, Stecher and Kumar2021) with the default settings. Phylogenetic analyses were run under Maximum Likelihood (ML) and Bayesian Inference (BI). The best model of GTR+G+I was used to construct ML trees using MEGA11 V. 11.0.13. The robustness of the resulting tree was tested using 500 bootstrap replicates. BI analysis was performed with MrBayes V. 3.2.6 123 (Ronquist et al. Reference Ronquist, Teslenko, Van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012); the best nucleotide substitution model was chosen using MrModeltest V. 2.3 (Nylander Reference Nylander2004) and the best estimators for the dataset were indicated by the Akaike Information Criterion (AIC). Posterior probability distributions for BI were generated using the Markov Chain Monte Carlo (MCMC) method with 2 chains being run simultaneously for a minimum of 100,000 generations and then increased until the standard deviation of split frequencies was <0.01. The first 3,000 samples were discarded as ‘burn-in’. Genetic divergence was calculated using an uncorrected p-distances model in MEGA11 V. 11.0.13 (Table 4).
Partial 28S rDNA sequences of diclidophorid taxa included in this study, with outgroup taxa
a This sequence is not found in its referenced source, Oliva et al. (Reference Oliva, Sepulveda and González2014), as indicated in GenBank.
b,c,e Registered in GenBank as Heterobothrium sp.
d Heterobothrium indicum Verma, Reference Verma2022, transferred herein to Upenicola indicus (Verma, Reference Verma2022) n. comb.
f Registered in GenBank as Amblyrhynchotes honckenii (Bloch).
g Registered in GenBank as Neoheterobothrium sp. n.
h Registered in GenBank as Alectis indica (Cuvier).
i Registered in GenBank as Microcotyle erythrinii.
j Registered in GenBank as Cheilodactylus variegatus (Valenciennes).
Pairwise estimates of evolutionary divergence between sequences from species of Diclidophoridae and outgroup taxa based on the alignments of the 28S rDNA gene
Results
During the present investigation, the gill lamellae of seven specimens of the Nile puffer were examined for monogenoidean parasites. Six (86%) of the fish examined were found infected with a species of Heterobothrium. Other species of Monogenoidea were not encountered.
Morphology
Taxonomic position
Class Monogenoidea Bychowsky, 1937
Subclass Heteronchoinea Boeger & Kritsky, 2001
Infrasubclass Oligonchoinea Bychowsky, 1937
Order Mazocraeidea Bychowsky, 1937
Diclidophoridae Cerfontaine, Reference Cerfontaine1895
Heterobothrium Cerfontaine, Reference Cerfontaine1895
Heterobothrium fluviatile Euzet & Birgi, Reference Euzet and Birgi1975
(Figure 1)
Heterobothrium fluviatile Euzet & Birgi, Reference Euzet and Birgi1975. (a) Whole mount (ventral view, composite). (b) Male copulatory organ. (c) Spine of male copulatory organ (lateral view). (d) Egg. (e) Haptoral clamp of anterior-most pair (rotated 180° along longitudinal axis from normal attitude, ventral view). (f) Haptoral clamp of posterior three clamp rows (ventral view).
Syns. Heterobothrium fluviatilis Euzet & Birgi, Reference Euzet and Birgi1975; Heterobothrium lineatus Morsy, Saady, Abdel-Gaffar, Bashtar, Melhorn, Al Quraishy & Adel, Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012; Heterobothrium lineatum Morsy, Saady, Abdel-Gaffar, Bashtar, Melhorn, Al Quraishy & Adel, Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012; Tagia sp. of Mandour et al. (Reference Mandour, Ibraheem and El-Ganainy2000).
Taxonomic summary
Type host: Nile puffer, Tetraodon fahaka Bennett (now Tetraodon lineatus Linnaeus), Tetraodontidae.
Type locality: Chari River, N’Djamena, Chad, Africa.
Other published records: T. lineatus (as T. fahaka): Nile River off El-Minia Governorate, Egypt (as Tagia sp.) (Mandour et al. Reference Mandour, Ibraheem and El-Ganainy2000). T. lineatus: Nile River off Qena Governorate, Egypt (Morsy et al. Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012) and Nile River off Helwan Region, Egypt (Morsy et al. Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013) (both as Heterobothrium lineatus).
Current records: T. lineatus: Nile River off Qena Governorate, Egypt (26°08′N, 32°42′E), 25 February 2022 (2 examined, 1 infected), 12 September 2022 (2 examined, both infected), 1 November 2022 (1 examined, infected), 10 October 2023 (1 examined, infected), 3 April 2024 (1 examined, infected).
Site of infection: Gill lamellae.
Prevalence: 86% (6 of 7 Nile puffers infected).
Intensity: 1–4 worm(s) per infected fish.
Specimens studied: 14 voucher specimens, USNM 1762784–1762793, HWML 218128.
Molecular sequence: Partial 28S rDNA sequence deposited in GenBank under accession number PX601193.
Comparative description
Body dorsoventrally flattened. Body proper tapering anteriorly from level of gonads, with nearly parallel margins posteriorly. Peduncle short to absent; haptor appearing as extension of trunk (Figure 1a). Prohaptoral suckers semi-circular in dorsoventral view, lying within posterolateral walls of subterminal cephalic cavity; each sucker with thick muscular lips opening anteroventrally. Tegument smooth. Mouth within subterminal cephalic cavity, flanked by prohaptoral suckers; pharynx large, subovate, immediately posterior to mouth, posteromedial to prohaptoral suckers; oesophagus short to absent, lacking diverticula, bifurcating anterodorsal to genital pore. Intestinal ceca not observed, obscured by vitellarium.
Haptor subrectangular in outline, with lateral sides curled ventrally, armed with four bilateral pairs of closed diclidophorid-like clamps (pairs numbered 1 to 4 from posterior to anterior end of haptor), each with similar sclerite structure; one or both members of pair of small nondescript sclerites, apparently representing anchors, located between members of clamp pair 1, often absent or missing one member of the pair. Clamps sessile, asymmetrical. Each clamp having dorsal rod-shaped midsclerite with trough-like longitudinal groove, truncate proximal end, small T-shaped distal end; paired anterolateral sclerites fused distally, having proximal end of dextral sclerite curved ventrally toward midregion of clamp and proximal end of sinistral sclerite forming frame of pad; two dorsolateral sclerites, each comprised of two subunits united by connecting tissue. Dextral dorsolateral sclerite with truncate slightly expanded proximal end; proximal end of sinistral dorsolateral sclerite flattened, with groove extending across flattened portion often appearing to form two separate subsections (Figure 1f). Clamp of pair 4 with similar sclerites but rotated 180° along longitudinal axis (Figure 1e).
Genital pore midventral, lying on body midline at level of oesophageal bifurcation; genital atrium inapparent. MCO circular in outline in dorsoventral view, apparently eversible, armed with 6–10 (usually 7) small, recurved spines lying internal to outer muscular layer (Figures 1b, 1c). Prostates, prostatic reservoir not observed; seminal vesicle small, spherical, situated dorsoposterior to MCO. Vas deferens sinuous along body midline; origin of vas deferens from testicular field not observed. Testicular field comprising 6 to 8 (usually 6) subspherical to ovate testes.
Germarium intercecal, shaped as an interrogation mark, comprising proximal germinal bulb from which main portion of germarium originates. Oviduct arising from distal end of germarium on right side of intercecal space, extending posteriorly toward body midline, forming small posterior loop prior to uniting with genitointestinal canal, uterus, vitelline reservoir. Oötype not observed. Genitointestinal canal apparently uniting with dextral intestinal cecum. Uterus delicate, extending anteriorly ventral to germarium and apparently vas deferens; not observed distally at level of common genital pore. Vitelline reservoir Y shaped, with elongate proximal branch bifurcating into two short distal arms uniting with vitellarium. Vaginal pore, vagina, seminal receptacle not observed. Vitellarium dense throughout trunk, extending into haptoral region between bilateral clamp rows as scattered follicles. Egg fusiform, with long filamentous posterior filament, lacking anterior filament (Figure 1d).
Measurements
See Table 2.
Remarks
Euzet and Birgi (Reference Euzet and Birgi1975) described Heterobothrium fluviatile (as H. fluviatilis) from the gills of Tetraodon fahaka (now T. lineatus) in Chad. In the apparent absence of the original description of H. fluviatile, Morsy et al. (Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012), who described H. lineatum (as H. lineatus), along with Mandour et al. (Reference Mandour, Ibraheem and El-Ganainy2000) and Morsy et al. (Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013), offered independent characterizations of specimens identified as either Tagia sp. or H. lineatus from locations along the Nile River in Egypt. Although Morsy et al. (Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012) indicated in the taxonomic summary for H. lineatum that the type specimens were deposited in the museum of the Department of Zoology, South Valley University (currently Qena University), no documentation nor specimens could be located that would indicate they were ever submitted to or received by the department. Further, neither Mandour et al. (Reference Mandour, Ibraheem and El-Ganainy2000) nor Morsy et al. (Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013) provided any indication concerning the fate of their respective voucher specimens, and no response was received from the Muséum National d’Histoire Naturelle, Paris, France, concerning a formal request for a loan of Euzet and Birgi’s type specimens. Nonetheless, comparison of the present monogenoidean specimens, also from T. lineatus in the Nile River in Egypt, with the original description of H. fluviatile and with the individual accounts of Mandour et al. (Reference Mandour, Ibraheem and El-Ganainy2000) and Morsy et al. (Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012, Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013), clearly supports the present proposal of conspecificity of both H. lineatum and Tagia sp. of Mandour et al. (Reference Mandour, Ibraheem and El-Ganainy2000) with H. fluviatile. Moreover, the shared host specificity to T. lineatus and the distribution of the host across northern African freshwater systems provided added support of conspecificity. Thus, H. lineatum Morsy, Saady, Abdel-Gaffar, Bashtar, Melhorn, Al Quraishy & Adel, Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012 is herein placed as a junior subjective synonym of H. fluviatile Euzet & Birgi, Reference Euzet and Birgi1975.
Molecular Phylogeny (Figures 2, 3; Tables 3, 4)
The partial 28S rDNA sequence of Heterobothrium fluviatile (1,267 nucleotides) was aligned with sequences from 18 related diclidophorid species retrieved from GenBank (Table 3). The alignment, spanning over 1,317 base pairs (bp) including gaps, resulted in a final 1,211 bp sequence (excluding gaps) for phylogenetic analysis.
The phylogenetic reconstruction, developed from both ML and BI analyses, revealed two well-supported clades (Lineages A and B) within the Diclidophoridae. Lineage A included H. fluviatile and seven other species of Heterobothrium, all of which are parasitic on the gill lamellae of marine or freshwater pufferfishes (Tetraodontidae). Lineage A was found to be the sister taxon to Lineage B, which included Heterobothrium indicum Verma, Reference Verma2022, a parasite of the goldband goatfish, Upeneus moluccensis (Bleeker), and ten other species assigned to other diclidophorid genera (Figures 2, 3). Heterobothrium fluviatile emerged as the basal taxon to the remaining seven Heterobothrium species within Lineage A, forming a monophyletic clade, containing the eight Heterobothrium spp., whereas the position of H. indicum within Lineage B provided evidence that Heterobothrium (sensu lato) is polyphyletic.
Hypothesis, inferred from Maximum Likelihood (ML) analysis, of the phylogenetic relationships based on large subunit ribosomal DNA sequences from selected diclidophorids, with two heteraxinid and four microcotylid species serving as outgroup taxa. Values near nodes represent bootstrap support, with those below 50% excluded. Heterobothrium indicum Verma, Reference Verma2022 is highlighted in grey, signifying its divergence from the other Heterobothrium species (in bold) infecting tetraodontid fishes and is herein transferred to Upenicola as Upenicola indicus (Verma, Reference Verma2022) n. comb. The sequence of the type species of the genus Heterobothrium, H. tetrodonis (Goto, Reference Goto1894) Cerfontaine, Reference Cerfontaine1895, is indicated with an asterisk (*).
Hypothesis, inferred from Bayesian Inference (BI) analysis, of the phylogenetic relationships based on large subunit ribosomal DNA sequences from selected diclidophorids, with two heteraxinid and four microcotylid species serving as outgroup taxa. Values near nodes indicate posterior probabilities, with those below 0.90 excluded. Heterobothrium indicum Verma, Reference Verma2022 is highlighted in grey, signifying its divergence from the other Heterobothrium species (in bold) parasitizing tetraodontid fishes and is herein transferred to Upenicola as Upenicola indicus (Verma, Reference Verma2022) n. comb. The sequence of the type species of the genus Heterobothrium, H. tetrodonis (Goto, Reference Goto1894) Cerfontaine, Reference Cerfontaine1895, is indicated with an asterisk (*).
Genetic divergence analysis of LSU sequences revealed divergences of 5.2% to 5.5% between H. fluviatile and the seven other Heterobothrium spp. In contrast, the genetic divergence between H. fluviatile and H. indicum was significantly higher, reaching 15.7% (Table 4).
Discussion
Heterobothrium fluviatile and H. lineatum are the only members of the genus known to infect a freshwater host. Both species parasitize the Nile puffer, Tetraodon lineatus, in rivers of northern Africa. Heterobothrium fluviatile, originally described from the Chari River in Chad, has not been reported under that name since its original description by Euzet and Birgi (Reference Euzet and Birgi1975), whereas H. lineatum is known only from Nile puffer occurring in localities along the Nile River in Egypt (Morsy et al. Reference Morsy, Saady, Abdel-Ghaffar, Bashtar, Mehlhorn, Al Quraishy and Adel2012, Reference Morsy, Khalil, Gamil, Elebiarie and Ibrahim2013). The primary objectives of the present study, therefore, included 1) clarification of the taxonomic status of the two nominal species, 2) determination of their phylogenetic relationships within the genus, and 3) evaluation of the status and phylogenetic position of Heterobothrium within the Diclidophoridae.
The evaluation of the taxonomic status of H. fluviatile and H. lineatum indicated that the two species represent subjective synonyms, with H. fluviatile having priority. In addition to the morphological similarities detected between the present specimens and the descriptions and published reports of H. fluviatile and H. lineatum (type and voucher specimens of both nominal species were not available for study), the proposed synonymy is further supported by their common host preferences and geographic distributions. Both species are parasitic on the same host species, and all geographic localities from which they have been reported are major river systems of northern Africa, in many of which the host T. lineatus is endemic (Froese and Pauly Reference Froese and Pauly2025). This current distribution of T. lineatus in northern Africa suggests that H. fluviatile may enjoy a much larger distribution than presently known.
The hypotheses derived from the ML and BI analyses were consistent, with H. fluviatile occurring within a highly supported major clade containing the seven other species of Heterobothrium (Figures 2, 3). In both analyses, H. fluviatile was shown to have a basal position relative to the other Heterobothrium species in the clade, suggesting that the invasion into freshwater by the parasite occurred early during the evolutionary development of the genus, perhaps concurrently with the invasion event of the marine ancestor of its host, T. lineatus. The river system in which the invasion occurred is unknown, but a larger effort to obtain specimens of H. fluviatile and its host from throughout northern Africa and applying a broader examination of morphological and molecular diversity of the parasite and its host may provide a better understanding of their historical zoogeography.
Heterobothrium was found to represent unnatural taxon under both ML and BI analyses (Figures 2, 3). Although 21 species are currently listed under Heterobothrium in the World Register of Marine Species (WoRMS), our integrative morphological and molecular assessment supports the recognition of only 19 valid species within Heterobothrium (sensu stricto) (Table 1), all of which parasitize tetraodontid fishes. Representing the species parasitizing a non-tetraodontid host, Verma (Reference Verma2022) described H. indicum Verma, Reference Verma2022 (as H. indicus) from the gills of the goldband goatfish, Upeneus moluccensis (Bleeker) (Mullidae) from coastal India. A genetic divergence of 15.7% separating H. indicum from all eight of the other Heterobothrium species (Table 4) and H. indicum being a member of the clade of diclidophorids serving as sister taxon to Heterobothrium (sensu stricto), support H. indicum as a member of a different diclidophorid genus. Given its host association with a mullid and its clear morphological similarity with species of Upenicola Unnithan, 1966 (Diclidophoridae), H. indicum is herein transferred to Upenicola as Upenicola indicus (Verma, Reference Verma2022) n. comb.
The literature search disclosed that the taxonomic status of Diclidophora indica Tripathi, Reference Tripathi1959, and Neodiclidophora simhai Gupta & Krishna, Reference Gupta and Krishna1980, requires added investigation regarding their validity and placement within the Diclidophoridae. Diclidophora indica was described by Tripathi (Reference Tripathi1959) from the lattice blaasop, Tetradon (sic) oblongus (Bloch) (now Takifugu oblongus (Bloch)) from the Calcutta fish market, India. Gupta and Krishna (Reference Gupta and Krishna1980) proposed Neodiclidophora Gupta & Krishna, Reference Gupta and Krishna1980, for N. simhai Gupta & Krishna, Reference Gupta and Krishna1980, collected from the same host from the Bay of Bengal near Puri, Orissa (now Odisha), India. However, the descriptions of both D. indica and N. simhai are insufficient to confidently determine their validity and generic placement. Having a common host and the specimens of both species apparently originated from the Bay of Bengal, suggesting that the two nominal species are synonyms and probable members of Heterobothrium. Because of the insufficiency of the original descriptions, however, proposals of synonymy of Neodiclidophora with Heterobothrium and of the two nominal species are withheld pending new collections of the monogenoids infecting T. oblongus in the Indo-Pacific Ocean.
Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021) reported Heterobothrium lamothei Vidal-Martínez & Mendoza-Franco, Reference Vidal-Martínez and Mendoza-Franco2008, from the gills of the silver-cheeked toadfish, Lagocephalus sceleratus (Gmelin) (Tetraodontidae) in the Gulf of Arabia off Saudi Arabia. This study is plagued with numerous contradictions and errors, such that the entire record must be approached with caution. This conclusion is based on the following: Heterobothrium lamothei was originally described by Vidal-Martínez and Mendoza-Franco (Reference Vidal-Martínez and Mendoza-Franco2008) from the checkered puffer, Spheriodes testudineus (Linnaeus), from the coastal waters off the Yucatan Peninsula. Given the relatively high host-specific associations of monogenoids and that few reports of specific species of Monogenoidea occurring naturally in both the Atlantic and Indo-Pacific oceans exist, it appears likely that the identification of the helminth as H. lamothei by Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021) is erroneous. In addition, GenBank records concerning the sequence MT322610, which was submitted by Morsy and published by Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021), significantly conflict with the published account of these authors. GenBank records indicate that MT322610, published as representing a 28S sequence from H. lamothei by Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021), was submitted to GenBank by Morsy on 11 April 2020 as being from the Nile puffer, Tetraodon lineatus (host of H. fluviatile). No mention of a species of Monogenoidea, much less one of Heterobothrium, was included in the GenBank record. However, a BLAST analysis indicated that the sequence MT322610 aligned with parasitic taxa assigned to the Diclidophoridae, with that of Heterobothrium okamotoi Ogawa, Reference Ogawa1991 (AB162155) closest but differing from Morsy’s sequence by 7.45%. That the sequence apparently originated from a heteronchoinean infecting Nile puffer indicates a possible misidentification of the parasite specimens as H. lamothei by Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021). Further, GenBank records the sequence MT322610 as 18S, whereas it was published as 28S by Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021), all adding to the unreliability of their report. Thus, H. lamothei of Morsy et al. (Reference Morsy, Al-Malki, Dajem, Hamdi, Ali and Adel2021) is herein considered a species inquirenda.
Resolution of the chaos concerning the identity of the heronchoinean infecting L. sceleratus off Saudi Arabia will require added collections and analysis of the monogenoids infecting this puffer and perhaps other species of Lagocephalus occurring in the western Indian Ocean. It should be mentioned that the only heteronchoinean previously recorded as a parasite of L. sceleratus (as L. sceleratus suezensis) is Heterobothrium tonkinensis Bychowsky, Mamaev, & Nagibina, Reference Bychowsky, YuL and Nagibina1976, from the South China Sea off Hainan, China (Bychowsky et al. Reference Bychowsky, YuL and Nagibina1976), and that a total of seven species of Lagocephalus occur in the waters off Saudi Arabia that could serve as hosts for species of Heterobothrium in the region (see Froese and Pauly Reference Froese and Pauly2025).
Acknowledgements
The authors extend their sincere gratitude to the Zoology Department, Faculty of Science, Qena University, Qena, Egypt, for invaluable support and the provision of essential facilities that enabled Ali Mansour to conduct the fish collections for this study. The authors also thank Dr. Walter Boeger, Universidade Federal do Paraná, Curitiba, Paraná, Brazil, for his valuable advice and consultation regarding the molecular analyses and recent publications related to the higher taxonomy of Monogenoidea.
Author contribution
Ali Mansour: Conceptualization, methodology, investigation, formal analysis, writing—original draft and editing, visualization. Delane C. Kritsky: Methodology, writing—review and editing, visualization, supervision.
Financial support
No funds, grants, or other support were received.
Data availability
All data generated or analyzed during this study are included in the manuscript.
Competing interests
The authors declare no competing interests.
Ethical standard
We affirm that all procedures undertaken in this research strictly adhere to the ethical standards set forth by national and institutional guidelines for the care and use of animals. Furthermore, ethical approval for the study protocol was granted by the Scientific Research Ethics Committee, Faculty of Science, Qena University, Egypt (Approval No. 004/04/25).
