Fish are considered one of the important sources of animal protein (de Boer
et al., 2020). Several studies have been conducted about the parasites of the Red Sea fish with digenea as the largest sector of endoparasitic species
(Cribb et al., 2001). In total, 20 specimens of
Carangoides caeruleopinnatus were examined, 13 (65%) of which were infected in the intestinal region with one acanthocolpid species having morphological features corresponding with the genus
Monostephanostomum. The parasite’s mean intensity per fish host does not exceed 5. The recovered acanthocolpid species identified as
Monostephanostomum mesospinosum inhabited
C. caeruleopinnatus for the first time.
Description (Fig 1)
Body elongated, parallel-sided over most of its length, and measured 6.102-7.923 (6.870) long × 0.201-0.243 (0.231) wide. Tegument covered with spines that increased gradually in size. Forebody contains eyespot pigment. A single uninterrupted row of enlarged 18 circum-oral spines surrounds oral sucker. The oral sucker measured 0.070-0.079 (0.075) long × 0.091-0.103 (0.098) wide. Prepharynx long, narrow, and measured 0.590-0.721 (0.611) long. Pharynx pyriform and measured 0.163-0.184 (0.172) long × 0.082-0.094 (0.086) wide. Just before ventral sucker, esophagus measured 0.078-0.098 (0.082) long and divided into two intestinal caeca. Ventral sucker has a spherical shape, pre-equatorial and measured 0.118-0.147 (0.128) long × 0.124-0.168 (0.141) wide. The sucker ratio 1:1.2-1.4. Forebody is about 7% of body length. Caeca broad, long, opened into excretory bladder forming uroproct.
Testes two, oval, tandem and separated by vitelline follicles and found in posterior half of the body. The anterior testis measured 0.267-0.351 (0. 298) long × 0.173-0.185 (0.174) wide, while the posterior one was 0.291-0.371 (0.311) long × 0.189-0.184 (0.184) wide. Post-testicular region long. Cirrus-sac long, intercaecal that extends slightly more than halfway between ovary and ventral sucker, joining metraterm slightly posterior to ventral sucker. The cirrus sac measured 0.960-1.936 (1.721) long × 0.061-0.079 (0.065) wide. Seminal vesicle tubular. Pars prostatica short, sinuous, and bordered by prostate gland cells. Ejaculatory duct long, lined with knob-like protuberances. Genital atrium located anteriorly to ventral sucker and opened with a genital pore.
Ovary rounded, pre-testicular and measured 0.115-0.124 (0.119) long × 0.113-0.125 (0.119) wide. There is a uterine seminal receptacle. Uterus pre-ovarian, intercaecal and runs dorsally to cirrus sac. Metraterm opened into genital atrium. Eggs large, operculated, and measured 0.049-0.054 (0.052) long × 0.020-0.025 (0.023) wide. Vitellaria of two lateral fields of numerous vitelline follicles that extend from base of cirrus-sac to posterior end of body, interrupted at level of ovary and testes. Excretory vesicle I-shaped and opened by a terminal excretory pore.
Remarks
At the morphological and morphometric level, the current acanthocolpid species is compatible with other
Monostephanostomum species by possessing all the species’ characteristics exceptions.
Kruse (1979) established the genus
Monostephanostomum for the digenetic trematodes belonging to the family Acanthocolpidae. Parasites within this genus are characterized by relative sizes of suckers, presence of a single row of circumoral spines, small forebody, location of ventral sucker in middle or anterior third of body, forward and then backward loop of uterus, long metraterm, oval and contiguous gonads, anterior position of genital pore to ventral sucker, and distribution of vitelline follicles, caeca opened
via uroproct, elongated cirrus sac, weekly developed pars prostatica, long ejaculatory duct and excretory vesicle I-shaped reached ovary level. The present parasite species resembles the previously recorded species of
Madhavi’s (1976) and Madhavi and Bray (2018) for
M. mesospinosum from
C. malabaricus in VSK, BOB, especially in the long slender body and presence of 18 circumoral spines in uninterrupted rows. Our specimen falls within the range of measurements for different body parts suggested by
Madhavi (1976), but with different localities and species for fish hosts. This species is distinguished from other species of genus by: smaller body size and number of circumoral spines of
M. yamaguttii (12-16),
M. manteri (16-18),
M. nolani (17-20),
M. krusei (17),
M. gazzae (23), and
M. loossi (32). These results have been obtained via comparison with other data from
Ramadan (1984) and
Bray and Cribb (2007).
There was a noticeable difference from our specimen that recorded by
Ramadan (1984) by presence of two spines rows in
M. geogianum, first ventral one with 18-20 (19), and second dorsal with 3-5 smaller spines, vitellarium is restricted to hind body and interruptions in vitelline fields at gonads level, and cirrus sac reaches only to about middle of ventral sucker to ovary distance. While, it differs from
M. manteri which has a claviform body, continuous row of circum-oral spines, oval pharynx, longer post-testicular region, vitellarium reaches into forebody, and cirrus sac overlaps ovary; this agreed with
Bray and Cribb (2007). Additionally, some differences were recorded with
M. yamaguttii by
Ramadan (1984) which has an elongate pharynx, smaller cirrus sac and vitellaria that extends anteriorly only to posterior margin of ventral sucker. Extension of vitellarium into forebody was observed in
M. nolai versus that in recovered species. Characteristic feature for ventral hiatus in circum-oral spine row was recorded in
M. nolai,
M. krusi, and
M. roytmani, this is consistent with
Bray and Cribb (2007). In addition, it distinguished by smaller egg size from
M. yamaguttii (0.068-0.074 × 0.036-0.052),
M. geogianum (0.075-0.086 × 0.039-0.050),
M. manteri (0.080-0.094 × 0.035-0.048), and
M. krusei (0.062-0.064 × 0.039-0.044), these measurements regarding studies of
Ramadan (1984), Bray and Cribb (2002, 2007). Regarding sucker ratio by
Bray and Cribb (2007), the current species has a smaller ratio than
M. krusi (1:1.8-2.7),
M. gazzae (1:2.0-2.5) and
M. nolani (1:1.6-3.1).
Monostephanostomum species occupied many hosts belonging to different families (Lethrinidae, Arripidae, and Carangidae) in different geographical locations (Egypt, Australia and Tasmania)
(Madhavi, 1976, Kruse, 1979, Reimer, 1983, Ramadan, 1984, Bray and Cribb, 2002, Bray and Cribb, 2007, Madhavi and Bray, 2018). Our findings represent the third record for this acanthocolpid species in carangid fish.
Molecular analysis
Identification and taxonomic characterization of different parasitic taxa are primarily based on their morphological and ultrastructural features, which are considered mainly criteria for their classification
(Indaryanto et al., 2015). Molecular biological tools are recently considered sensitive and advanced tests for parasite taxonomy
(Chaudhary et al., 2016). In our molecular analyses, one
Monostephanostomum species was confirmed to belong to other Opisthorchiida taxa, especially Acanthocolpidae, based on
18S and
28S rRNA sequencing.
18S rRNA gene
PCR amplification was successfully carried out to amplify a 697 bp with 51.5% GC content and submitted to NCBI database with accession number OL770106.1. The query sequence was compared with
18S rRNA sequences of other digenean taxa available in the GenBank database (Table 1).
Phylogenetic tree was represented by two orders Opisthorchiida and Plagiorchiida within Digenea (Fig 2).
First major clade of the dendrogram was subdivided into two lineages, the first one containing taxa of Opisthorchiida and represented by Acanthocolpidae with strong support value (82), the second lineage supported taxa of two suborders of Plagiorchiida: Xiphidiata, Lepocreadiata, and remaining taxa of Acanthocolpidae. Second clade contains remaining taxa of Opisthorchiata (represented by Cryptogonimidae, Heterophyidae, Opisthordiidae), and other plagiorchiids belonging to Xiphidiata, Lepocreadiata, and Troglotremata. Among Acanthocolpidae, the maximum identity (97.99%) with the lowest divergent value was recorded between the present species and
M. nolani. Monophyly of
Monoste phanostomum species was supported sister group generally to
Stephanostomum. Query sequence is well aligned and placed within Opisthorchiida with special reference to Acanthocolpidae. Sequence representing
M. mesospinosum grouped with strong bootstrap value (100) with sequence represented by EF506762.1 for the previously deposited sequence for
M. nolani parasitizes the barcheek trevally
Carangoides plagiotaenia collected from Lizard Island, Great Barrier Reef (Australia), as expected based on sequence comparisons.
28S rRNA gene
The sequence analysis revealed a band size of 693 bp with 53.5% GC content that submitted to NCBI database with accession number OL773371.1. The query sequence was compared with
28S rRNA sequences of other related digenean available in the GenBank database (Table 2).
Phylogenetic tree was constructed with two major clades (Fig 3), the first one clustered taxa of the family Acanthocolpidae within the subclass Digenea.
Whereas, the second clade contains taxa of four orders Xiphidiata (represented by Brachycladiidae, Plagiorchiidae, Choanocotylidae, and Telorchiidae), Monorchiata (represented by Monorchiidae and Lissorchiidae), Lepocreadiata (represented by Deropristidae) and Troglotremata (represented by Troglotrematidae). Among Acanthocolpidae, the maximum identity (97.98%) with the lowest divergent value was recorded between the present digenean species and
Monostephanostomum nolani. Monophyly of
Monostephanostomum species was supported sister group generally to
Stephanostomum. Query sequence
M. mesospinosum is well aligned and placed within Opisthorchiida with special reference to Acanthocolpidae with a close relationship in a well-supported taxon (100) with the previously recorded
M. nolani (EF506763.1) parasitizes the barcheek trevally
Carangoides plagiotaenia collected from Lizard Island, Great Barrier Reef (Australia), as expected based on sequence comparisons.
A close relatedness to
M. nolani (EF506762.1 and EF506763.1) was determined based on molecular similarities of those described by
Bray and Cribb (2007) infecting the carangid, the barcheek trevally,
Carangoides plagiotaenia from Lizard Island, Great Barrier Reef. This is consistent with
Bray et al., (2009) who reported that complete SSU and partial LSU rDNA sequence data enhance significantly the chance for an accurate differentiation between plagiorchiids. Thus, the nuclear RNA analyses herein showed that the
Monostephanostomum was consistent with the morphological taxa of trematodes within the same genus. Our phylogeny assured morphological evidence for a close relation between
Stephanostomum and
Monostephanostomum to be clustered together in the well-supported clade with consideration for the number and arrangement of circum-oral spines for both genera, this agreed with previous studies by
Bray and Cribb (2002, 2007) and
Bray et al., (2007). The current dendrograms demonstrated Acanthocolpidae polyphyly, which is in agreement with
Abdel-Gaber et al., (2020) who reported the same issue due to presence of relation with taxa included within Xiphidiata and Lepocreadiata. Furthermore, the current study confirms the position of the Acanthocolpidae in the superfamily Brachycladioidea along with Brachycladiidae, as consistent with
Bray et al. (2009) and
Bakhoum et al., (2015).
