Restoration of the Genus Paraunisaccoides Martin, 1973 (Digenea: Haploporidae) and Description of P. elegans n. sp. and Unisaccus halongi n. sp. from Mugilid Fish in Vietnam

Abstract

We restore the genus Paraunisaccoides (Haploporidae), synonymised earlier with the genus Skrjabinolecithum. Adult worms, detected in Vietnamese mullet fish, were highly similar to trematodes described as P. lobolecithum via digestive and genital system structures and relative organ arrangement. Differences are expressed as absence and presence of pads on the hermaphrodite duct, respectively, and the disjunction of some metric parameter values, namely body, ovary and eggs. Ribosomal DNA sequences, based on the phylogenetic analysis of Haploporidae, indicates that new worms represent a sister clade to Unisaccus tonkini. Genetic divergence between new worms and Skrjabinolecithum species can be interpreted as intergeneric. Based on morphological and molecular data, we recognise Paraunisaccoides as a valid genus within Waretrematinae and worms from Vietnam as a new species of this genus, P. elegans n. sp. Other worms detected in Vietnamese mugilids are morphologically similar to representatives of Paraunisaccoides и Skrjabinolecithum. However, molecular-based phylogenetic analysis showed that these trematodes are closely related to Unisaccus tonkini; the genetic divergence between them is at the interspecific level, despite considerable differences in vitellarium structure as intergeneric character. Accepting the priority of molecular results, we include these new worms into the genus Unisaccus as new species, Unisaccus halongi n. sp.

1. Introduction

Species of Haploporidae Nicoll, 1914, as well as other Haploporoidea, parasitising a wide range of definitive hosts, marine, freshwater, and eurihaline fish species, are cosmopolitan. Species of different subfamilies (except the cosmopolitan Megasoleninae Manter, 1935) are somehow related to certain geographical territories, infecting mainly fish species of Mugilidae Jarocki, 1822 and, rarely, Cyprinidae Rafinesque, 1815. Representatives of three subfamilies, Haploporinae, Waretrematinae Srivastava, 1937 and Pseudohaploporinae Atopkin, Besprozvannykh, Ha, Nguyen, Bguyen & Chalenko, 2018, of the eight known at present, have mainly been detected in the Indo-West Pacific region [1,2,3,4,5,6,7,8,9,10,11]. Most of the trematodes have been extracted from Mugilidae fish species.

We obtained specimens of two hypothetic species of Haploporidae in mullet fish from coastal waters of Halong Bay, Vietnam. In the present study, we performed morphological and molecular analyses to validate these species and to reconstruct the phylogenetic relationships of the family Haploporidae with new data.

2. Materials and Methods

2.1. Collection of Trematodes

Adult worms were collected from intestines of mullet fish (Mugilidae) in coastal waters near Cat Ba Island, Ha Long Bay, Vietnam (20°84′ N, 106°59′ E). Worms were rinsed in saline, previously defined under a microscope using temporal slides preparation technique, killed in hot distilled water and preserved in 70% ethanol. After fixation, they were replaced in 96% ethanol. Whole mounts were made by staining specimens with alum carmine, dehydrating them in graded ethanol series and clearing in clove oil, followed by mounting the specimens in Canada balsam under a coverslip on a glass slide. All measurements are given in micrometres.

2.2. DNA Extraction, Amplification and Sequencing

Three and four adult specimens of Unisaccus halongi n. sp. and Paraunisaccoides elegans n. sp., respectively, from 96% ethanol were used for molecular analysis (

Table 1. List of taxa used for molecular analysis.

Species	N	Definitive Host	Authors	Accession Number in the NCBI
				28S	ITS1-5.8S-ITS2
Haploporidae
Waretrematinae
Paraunisaccoides elegans n. sp.	4/2	Planiliza subviridis	Present study	KY501639–KY501641	KY501642–KY501644
Unisaccus halongi n. sp.	3/3	Crenimugil seheli	Present study	OK644190-OK644192	OK644196-OK644198
Unisaccus tonkini	1/1	Moolgarda cunnesius	[6]	MF176843	MF176838
Skrjabinolecithum pyriforme	1/1	Planiliza haematocheila	[5]	HE806359	LN864990
Skrjabinolecithum spinosum	1/1	Planiliza haematocheila	[6]	MF176834	MF176831
Skrjabinolecithum. spasskii	3/3	Planiliza haematocheila	[21]	LN614538, HG530210, HG530207	LK022754, HE806371, HG530228
Parasaccocoelium mugili	1/1	Planiliza haematocheila	[22]	MW813991
Parasaccocoelium armatum	-/2	Mugil cephalus	[11]	-	MT298950–MT298951
Parasaccocoelium haematochelium	-/2	Liza haematocheila	[4]	-	HF548466–HF548467
Parasaccocoelium polyovum	-/2	Liza haematocheila	[4]	-	HF548477–HF548478
Elonginurus mugilus	1/1	Mugil cephalus	[9]	MH763766	MH763761
Carassotrema koreanum	3/3	Carassius gibelio	[9]	MH763763–MH763765	MH763758–MH763760
Carassotrema sp.	1/1		unpublished	MH285255
Spiritestis herveyensis	1/1	Moolgarda seheli	[2]	KC206500
Capitimitta costata	1/1	Selenotoca multifasciata	[2]	KC206497
Capitimitta darwinensis	1/1	Selenotoca multifasciata	[2]	KC206498
Pseudohaploporinae
Parahaploporus elegantus	10	Moolgarda seheli	[10]	MN639712-MN639721
Pseudohaploporus vietnamensis	6/8	Osteomugil engeli	[8]	MF774420–MF774421, MF774423–MF774426	MF774427–MF774429; MF774436–MF774440
Pseudohaploporus vietnamensis	1/1	Moolgarda seheli	[8]	MF774422	MF774431
Pseudohaploporus planilizum	3/3	Planiliza subviridis	[8]	MF774417–MF774419	MF774433–MF774435
Pseudohaploporus pusitestis	2/2	Moolgarda seheli	[10]	MH986037, MH986038	MF774430, MF774432
Haploporinae
Saccocoelium brayi	1/1	Liza saliens	[23]	FJ211234	FJ211244
Saccocoelium cephali	1/1	Mugil cephalus	[23]	FJ211233	FJ211243
Saccocoelium obesum	2/2	Liza ramada	[23]	FJ211259–FJ211260	FJ211265–FJ211266
Saccocoelium tensum	2/2	Liza ramada	[23]	FJ211257–FJ211258	FJ211263–FJ211264
Dicrogaster contracta	1/1	Liza aurata	[23]	FJ211261	FJ211267
Dicrogaster perpusilla	1/1	Liza ramada	[23]	FJ211238	FJ211248
Lecithobotrys putrescen	1/1	Liza saliens	[23]	FJ211236	FJ211246
Litosaccus_brisbanensis	1/1	Mugil cephalus	[3]	KM253765
Haploporus benedeni	1/1	Liza ramado	[23]	FJ211237	FJ211247
Ragaia lizae	1/1	Liza aurata	[23]	FJ211235	FJ211245
Forticulcitinae
Forticulcita isabelae	1/1	Mugil curema	[24]	MT957787	MT957640
Forticulcita macropharyngis	1/1	Mugil curema	[25]	MW796513	MW796548
Forticulcita minuta	1/1	Mugil cephalus	[24]	MT957822	MT957660
Forticulcita venezuelensis	1/1	Mugil curema	[25]	MW796522	MW796549
Forticulcita gibsoni	1/1	Mugil cephalus	[24]	FJ211239	FJ211249
F. apiensis	1/1	Mugil cephalus	[26]	KP761087
F. platana	1/1	Mugil liza	[26]	KP761086
Xiha fastigata	1/1	Mugil cephalus	[26]	KP761088
Overstreetoides_pacificus	2/2	Mugil curema	[24]	MT957753–MT957754	MT957629–MT957630
Ekuarhuni papillatum	2/2	Mugil sp.	[24]	MT957687–MT957688	MT957587–MT957588
Ekuarhuni mexicanum	2/2	Mugil sp.	[24]	MW796487–MW796488	MW796524–MW796525
Chalcinotrematinae
Saccocoelioides sp.	1/1	Unidentified molly (Poecilidae)	[27]	EF032696	-
Saccocoelioides beauforti	1/1	Mugil cephalus	[28]	MG925104	MG925103
Saccocoelioides elongatus	1/1	Prochilodus lineatus	[28]	MG925108	MG925107
Saccocoelioides magnus	1/1	Cyphocharax voga	[28]	MG925112	MG925111
Saccocoelioides nanii	1/1	Prochilodus lineatus	[28]	MG925114	MG925113
Saccocoelioides orosiensis	1/1	Poecilia gillii	[28]	MG925118	MG925117
Saccocoelioides tkachi	1/1	Astyanax aeneus	[28]	MG925122	MG925121
Intromugil mugilicolus	1/1	Mugil cephalus	[29]	KC430096
Intromugil alachuaensis	1/1	Mugil cephalus	[29]	KC430095
Hapladeninae
Hapladena acanthuri	1/1	Acanthurus chirurgus	[30]	MH244119
Hapladena cf. varia	1/1	Acanthurus chirurgus	[30]	MH244120
Megasoleninae
Megasolena hysterospina	1/1	Archosargus rhomboidalis	[30]	MH244121
Megasolena sp. m MA-2018	1/1	Holacanthus ciliaris	[30]	MH244122
Cadenatelinae
Cadenatella americana	1/1	Kyphosus sectatrix	[30]	MH244117
Cadenatella floridae	1/1	Kyphosus incisor	[30]	MH244118
Atractotrematidae
Isorchis anomalus	1/1	Chanos chanos	[7]	KU873018
Isorchis currani	1/1	Selenotoca multifasciata	[31]	KU873017
Isorchis megas	1/1	Selenotoca multifasciata	[7]	KU873015
Brachycladiidae
Brachycladium goliath	1/1	Balaenoptera acutorostrata	[32]	KR703279
Monorchiidae
Hurleytrematoides chaetodoni	1/1	Chaetodon striatus	[30]	MH244116

N—number of sequences of 28S rDNA/ITS rDNA.

). Total DNA was extracted from flukes using a “hot shot” technique [12].

28S ribosomal DNA (rDNA) 1200 base pairs (bp) in lenght was amplified by a polymerase chain reaction (PCR) method using the Q5 HF polymerase (New England Biolabs, Ipswich, MA, USA) and the primers 28S-A (5′-TCGATTCGAGCGTGAWTACCCGC-3′) [13] and 1500R (5′-GCTATCCTGAGGGAAACTTCG-3′) [14] with an annealing temperature of 55 °C. The ribosomal ITS1-5.8S-ITS2 fragment 1500 bp in lenght was amplified with the primers ITSF (5′-CGCCCGTCGCTACTACCGATTG-3′) [3] and S4R (5′-TATGCTTAAATTCAGCGGGT-3′) [15] with an annealing temperature of 54 °C. Negative and positive controls using both primer pairs were included. PCR parameters began with a 1 min denaturation at 98 °C, followed by 35 cycles of 10 s at 98 °C, 5 s at 54/55 °C and 30 s at 72 °C, and concluded with a 2 min extension at 72 °C.

PCR products were directly sequenced using an ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) as recommended by the manufacturer. The internal sequencing primers for 28S rDNA are described in [14], for ITS they are described in [16]. PCR product sequences were analysed using an ABI 3500 genetic analyser at the FSC of Biodiversity FEB RAS. Sequences were submitted to the GenBank database (Table 1).

2.3. Alignments and Phylogenetic Analysis

Ribosomal DNA sequences were assembled using the SeqScape v. 2.6 software provided by Applied Biosystems. Alignments and estimations of the number of variable sites and sequence differences were performed using the MEGA v. 7.1 software [17]. The values for genetic p-distances were calculated for the 28S ribosomal DNA fragment. Phylogenetic relationships were obtained using a concatenated data set of partial sequences of the 28S rRNA gene and ITS1-5.8S-ITS2 rDNA. Phylogenetic analysis was performed using the Bayesian algorithm in the MrBayes v. 3.2.6 software [18]. The best nucleotide substitution model, a transversional model with estimates of invariant sites and gamma-distributed among-site variation TVM + G+I [19], was estimated using the jModeltest v. 2.1.5 software [20] for partial 28S rDNA and combined ITS1-5.8S-ITS2-28S rDNA sequence data sets. Bayesian analysis was performed using 10,000,000 generations with two independent runs. Summary parameters and the phylogenetic tree were calculated with a burn-in of 3,000,000 generations. The significance of phylogenetic relationships was estimated using posterior probabilities [18]. Sequences of ITS1-5.8S-ITS2 rDNA and 28S rDNA of Brachycladium goliath (van Beneden, 1858) from GenBank were used as the outgroup. The authors of these and other sequences from GenBank [3,4,5,6,7,8,9,10,11,21,22,23,24,25,26,27,28,29,30,31] and their accession numbers are given in Table 1.

3. Results

3.1. Diagnosis of the Genus Paraunisaccoides

Family. Haploporidae Nicoll, 1914; Subfamily.

Waretrematinae Srivastava, 1937; Genus.

Paraunisaccoides Martin, 1973.

Body oval, fusiform or elongate. Eye-spot pigment dispersed. Oral sucker subterminal. Ventral sucker larger than oral sucker in anterior half of body. Prepharynx long, reach level of ventral sucker or posterior to ventral sucker. Pharynx round or transversely oval. Oesophagus short poorly defined. Caecum saccular. Testis single, in posterior end of body. Hermaphroditic sac oval with muscular sphincter at anterior end. External seminal vesicle sac-shaped or another form depending of fullness of sexual products. Internal seminal vesicle elongated. Hermaphroditic ducts with or without pads. Genital pore anterior to ventral sucker. Ovary round or transversal oval, immediately anterior to testis. Uterus short, between anterior margin of testis and hermaphroditic sac. Metraterm short, thin-walled. Eggs unnumerous, unembryonated, operculated. Mehlis’gland sinistrally to ovary. Vitellarium, consists from elongate thin of follicles. Vitelline fields can reach level of posterior edge of ventral sucker and to posterior end of body. Excretory bladder I-shaped with or without muscular sphincter. Type species: Paraunisaccoides lobolecithum Martin, 1973.

3.2. Paraunisaccoides elegans n. sp.

3.2.1. Taxonomic Summary

Host. Planiliza subviridis (Valenciennes, 1836).

Locality. Coastal water of Cat Ba Island, Ha Long Bay, northern Vietnam (20°84′ N, 106°59′ E).

Site. Intestine.

Prevalence. 3 of 5 specimens infected.

Intensity. 1–12 worms.

Type-deposition: Type No. 182-Tr, paratype No. 183-187-Tr. This material is held in the collection of the Zoological Museum (Institute of Biology and Soil Sciences, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia); e-mail: [email protected]. Deposited: 29 July 2020.

Etymology: species name associated with trematode body form.

3.2.2. Morphology

Based on 6 adult worms (Figure 1,

Table 2. Measurements (µm) of adult worms of new species Paraunisaccus elegans n. sp. and Unisaccus halongi n. sp. and known species Skrjabinolecithum lobolecithum and S. indicum.

	Paraunisaccoides elegans n. sp.			Skrjabinolecithumlobolecitum (Martin, 1973)	S. vitellosum (Martin, 1973)	Unisacus halongi n. sp.			S. indicum (Zhukov, 1972)
	Holotype	Range	Mean			Holotype	Range	Mean
Body length	1217	1124–1232	1170	1850; 2070	510–790	920	920–1390	1170	1000–1200
Body width	354	323–370	347	266; 406	140–300	420	390–500	430	280–370
Body length/width%	29.1	26.0–32.9	29.7	-		45.7	31.7–45.7	36.8	-
Forebody length	501	424–508	469	-		0.347	347–396	364	-
Body/forebody length%	41,2	37.5–44.0	40.1	-		32.2	28.2–37.7	31.1	-
Oral sucker length	96	77–104	92	96; 112	50–80	96	92–127	104	83–120
Oral sucker width	96	92–112	100	100; 112	59–90	131	116–154	139	110–120
Ventral sucker length	123	112–139	125	93; 143	56–74	127	96–142	120	120–150
Ventral sucker width	131	127–135	129	78; 156	56–74	127	112–150	124	140–170
Ventral/oral sucker length ratio	1:1.28	1:1.17–1.71	1:1.36	-		1:1.38	1: 0.91–1.48	1:1.15	-
Ventral/oral sucker width ratio	1:1.36	1:1.13–1.47	1:1.29	-		1:1.09	1: 0.73–1.09	1:1.06	-
Prepharynx length	539	377–566	483	426; 684	90–109	227	227–354	306	120–170
Pharynx length	77	77–116	93	109; 112	44–80	77	77–116	95	110–140
Pharynx width	85	85–123	100	131; 137	59–110	89	89–135	117	80–110
Oesophagus length	15	0–154	-	112; 249	60	58	23–58	49	-
Ovary length	62	46–77	71	109; 140	31–62	69	69–116	96	62–83
Ovary width	62	54–77	64	100; 109	31–62	69	69–116	97	62–83
Testis length	177	162–219	206	239; 345	75–165	150	150–270	180	210–300
Testis width	193	154–235	198	168; 202	50–90	193	139–227	184	120–140
Hermaphroditic sac length	254	181–270	220	258; 286	90	177	154–231	186	-
Hermaphroditic sac width	96	100–123	114	96; 118	50	104	104–173	138	-
Eggs length	62–65	62–65	-	71–93	59–65	62–73	62–73	-	71–79
Eggs width	50–54	50–54	-	56–59	42	42–50	42–50	-	39–43

).

Body elongate, with narrow forebody, tegument with needle-shaped spines. Eyes-spot pigmentation dispersed in anterior half of forebody. Oral sucker subterminal. Prepharynx long, reach level of ventral sucker or slightly posterior to ventral sucker. Pharynx round or transversely oval. Oesophagus short poorly defined. Caecum saccular, in middle of posterior half of body. Ventral sucker larger than oral sucker, at beginning of middle third of body. Testis single, in posterior end of body, irregular, with anterior recess. Hermaphroditic sac oval, at level ventral sucker and pharynx. Anterior end of hermaphroditic sac with muscular sphincter. External seminal vesicle sac-shaped or another form depending of fullness of sexual products. Internal seminal vesicle elongated. Prostatic cells unnumerous. Pads at hermaphroditic duct not identified. Genital pore opening on midline of body immediately before ventral sucker. Ovary round or transversal oval, adjacent to anterior margin of testis on midline of body. Uterus short, between anterior margin of testis and hermaphroditic sac. Metraterm short, thin-walled. Eggs few, light yellow, unembryonated, operculated. Mehlis’gland sinistrally to ovary. Vitellarium, consists from elongate thin of follicles. Vitelline fields can reach level of posterior edge of ventral sucker and to posterior end of body, merge at median line of body and cover of ovary and testis. Excretory bladder I-shaped with muscular sphincter, pore terminal.

3.2.3. Molecular Data

For four specimens of P. elegans n. sp. totals of 1291 and 1604 alignable characters with three (0.23%) and nineteen (1.18%) variable sites were generated for analysis in the 28S rRNA gene and ITS1-5.8S—ITS2 rDNA fragment datasets, respectively.

3.3. Unisaccus halongi n. sp.

3.3.1. Taxonomic Summary

Host. Crenimugil seheli (Fabricius, 1775).

Locality. Coastal water of Cat Ba Island, Ha Long Bay, northern Vietnam (20°84ʹN, 106°59ʹE).

Site. Intestine.

Prevalence. 5 of 37 specimens infected.

Intensity of infection. 1–5 worms.

Type-deposition: Type No. 188-Tr, paratype No. 189-193-Tr. This material is held in the collection of the Zoological Museum (Institute of Biology and Soil Sciences, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia); e-mail: [email protected]. Deposited: 29 July 2020.

Etymology: species name is associated with location, where definitive host of these worms has been caught.

3.3.2. Morphology

Based on 6 adult worms (Figure 1, Table 2).

Body elongate, with narrow forebody, tegument with needle-shaped spines. Eyes-spot pigmentation dispersed in forebody. Oral sucker subterminal, transversaly oval. Prepharynx long, slightly does not reach of ventral sucker or reach level of anterior half of ventral sucker. Pharynx at level ventral sucker, spherical, large, slightly smaller of oral sucker. Oesophagus short, poorly defined. Caecum single, saccular, thick-walled, in middle and partly in posterior third of body. Ventral sucker larger than oral sucker, at the border of anterior and posterior third of body. Testis single, in posterior end of body, irregular. Hermaphroditic sac oval, mostly at level ventral sucker. Anterior end of hermaphroditic sac with muscular sphincter. External seminal vesicle sac-shaped. Internal seminal vesicle oval. Size of seminal vesicles depends on fullness of sexual products. Prostatic cells unnumerous. Hermaphroditic duct with pads. Genital pore opening on midline of body immediately before ventral sucker. Ovary round, adjacent to anterior margin of testis. Uterus short, between anterior margin of testis and hermaphroditic sac. Metraterm equal to length hermaphroditic duct. Eggs few, light yellow, unembryonated, operculated. Mehlis’gland sinistrally to ovary. Vitellarium, consists from large elongate, wide of follicles, in close contact with each other. Vitelline fields between ventral sucker to posterior end of body, merge at median line of body and cover of ovary and testis. Excretory bladder I-shaped, pore terminal.

3.3.3. Molecular Data

For three specimens of S. indicum totals of 1240 and 972 alignable characters were generated for analysis in the 28S rRNA gene and ITS1-5.8S—ITS2 rDNA fragment datasets, respectively. Sequences within both datasets were identical to each other.

4. Discussion

On the basis of the last known classification of Haploporidae, provided by Overstreet and Curran [1], Vietnamese worms belong to this family by morphological characteristics, including organ topology, single testis, identical hermaphrodite sac structure, and single caecum. The latter characteristic is representative for worms of the genera Paraunisaccoides within Haploporidae, as well as Unisaccus and Unisaccoides (Unisaccinae Martin, 1973, Haploporidae), validated by Martin [33,34]. In 2005, Paraunisaccoides and Unisaccoides were synonymised with Skrjabinolecithum Belous, 1954 (Waretrematinae), and the type genus Unisaccus of Unisaccinae was transferred to Haploporinae [1]. This has led to recognising Unisaccinae as a synonym of Haploporinae [1].

Among representatives of Haploporidae, worms ex Planiliza subviridis from Vietnam are morphologically most similar with representatives of Skrjabinolecithum that can possess double for type species S. spasskii Belous, 1954, and also S. indicum (Zukov, 1972), S. puriforme Besprozvannykh et al., 2017 and S. spinosum Besprozvannykh et al., 2018 or single for S. lobolecitum (Martin, 1973), S. vetillosum (Martin, 1973) caecum. Vietnamese flukes and S. vetillosum characterized by hiatus in metric parameters for body, ventral sucker, hermaphrodite sac etc. (Table 2). Worms from our study show highest similarity with trematodes, detected in Mugil cephalus Linnaeus, 1758 in Australia [33], denoted as Paraunisaccoides (=Skrjabinolecithum) lobolecithum. These worms differ by the presence pads on the hermaphrodite sac for worms from Martin’s study [33] and, according to the figure provided, the presence of a large space between the ventral sucker and anterior end of the vitellarium (description of vitellarium arrangement absent in [33]). Alongside this, there is a disjunction of metric parameter values, namely body length and sizes of ovary and eggs (Table 2), between worms from [33] and those from our study. Although trematode specimens from both studies were detected in mugilids from relatively close geographical regions and with similar morphology, a number of differences, reported above, and the absence of molecular data for specimens from Australia do not allow to conclude that these trematodes belong to the same species. In our opinion, it is rational to currently consider Vietnamese worms as a new species of Haploporidae. A final conclusion of the validity or conspecificity of these species can be made after obtaining molecular data for the Australian worms, reported in [33]. Phylogenetic analyses performed on the basis of 28S rDNA partial sequences 1050 bp in length and concatenated 28S rDNA and 5.8S + ITS2 rDNA (1460 bp overall length) fragments for Haploporidae confirmed the membership of considered here Vietnamese worms to Waretrematinae (Figure 2 and Figure 3). However, within this subfamily, Vietnamese worms formed a common clade with Unisaccus tonkini instead of Skrjabinolecithum species. At the same time, the genetic distance values between Vietnamese worms, Unisaccus and Skrjabinolecithum, represent an intergeneric divergence level by both markers: 7.05 ± 0.78%–12.05 ± 1.0% by 28S rDNA and 8.05 ± 0.97%–12.10 ± 1.14% by ITS2 rDNA (alignment length 379 bp). These values are compatible with those between different genera within each subfamily: 3.62 ± 0.56%–13.93 ± 1.03% and 4.21 ± 0.73%–16.38 ± 1.35% for Waretrematinae, 5.9 ± 0.62% and 8.33 ± 0.91% for Pseudohaploporinae, 6.13 ± 0.68% for 10.11 ± 1.08% for Forticulcitinae Blasco-Costa, Balbuena, Kostadinova & Olson, 2009 by 28S rDNA and ITS2 rDNA, respectively [8,23,24]. Based on this, accepting the morphological similarity of Vietnamese worms and Australian described by Martin as Paraunisaccoides lobolecithum, we conclude that synonymisation of Paraunisaccoides with Skrjabinolecithum is unreasonable. In the light of this, we restore the genus Paraunisaccoides with the type species Paraunisaccoides lobolecithum Martin, 1973 with the inclusion of the new species Paraunisaccoides elegans n. sp. from the present study.

New trematodes ex Crenimugil seheli from Vietnam, share many morphometrical characteristics with Paraunisaccoides, and also with Skrjabinolecithum. Alongside this, Vietnamese worms differ from Paraunisaccoides lobolecithum by lesser body length along with similar body width, higher ventral sucker width, lesser prepharynx and oesophagus length and lesser hermaphroditic sac length along with its higher width (Table 2). New worms differ from Paraunisaccoides elegans n. sp., described above, by presence of pads on hermaphroditic duct, and different structure of vitellarium follicules, and also by metric parameters of body width relative to body length, forebody length and forebody lengh/overall body length ratio and by a number of other characteristics (Table 2). Among Skrjabinolecithum, S. vitellosum and S. indicum, which were firstly detected in Planiliza subviridis and Etroplus surutensis (Bloch, 1790) from Australia coastal waters and the Arabian Sea, respectively [1,35], are most similar to new trematodes ex Vietnamese Crenimugil seheli from our study. However, new worms differ from S. vitellosum by presence of pads on hermaphroditic duct, different structure of vitellarium follicules and by most of metric parameters (Table 2). New worms are similar to S. indicum by most of metric parameters, except prepharynx length (Table 2). The main morphological difference between these worms is the presence of single and double caeca, and presence and absence of pads on hermaphroditic duct, respectively. Another difference for these trematodes can be found in the vitellarium structure. The vitellarium of S. indicum represents two poorly developed follicular fields [35], whereas for Vietnamese worms, the vitellarium is well developed and represents large elongate follicules. On the basis of stated above morphometrical differences between trematodes, we consider that worms ex Crenimugil seheli from Vietnam belong to neither S. vitellosum nor S. indicum. Additional molecular data on S. vitellosum and S. indicum from type locations are needed to clarify taxonomical and phylogenetic questions of these two species and new Vietnamese trematodes.

Molecular results support the membership trematodes ex Crenimugil seheli to Waretrematinae and indicate close relationships with Unisaccus tonkini (Figure 2 and Figure 3). Alongside this, worms from our material possess both common and uncommon morphological characteristics for Unisaccus. The similarity of these worms appears in the structure of genital and digestive systems. Differences of Unisaccus spp. and worms from our study observed in following characteristics: body pyriform to fusiform vs. body elongate; eggs embryonated vs. unembryonated; subspherical follicles vs. elongate follicles. Trematodes from our material are similar in terms of the listed characteristics to Paraunisaccoides elegans n. sp., which appears as sister clade relative to the Unisaccus clade on the Bayesian tree (Figure 2 and Figure 3). Genetic differentiation between these two clades ranged from 6.53 ± 0.76%–7.05 ± 0.5% by 28S rDNA sequence data and 7.94 ± 0.95%–8.05 ± 0.67% by ITS2 rDNA sequence data. These results indicate that trematodes from the two clades represent different genera.

Within clade, the p-distance values between worms ex Crenimugil seheli and U. tonkini were 2.59 ± 0.52% and 3.54 ± 0.67% by 28S rDNA and ITS2 rDNA, respectively, which is compatible with the interspecific values for other haploporid genera by these molecular markers: 0.43 ± 0.19% to 4.85 ± 0.65% by 28S rDNA and 2.15 ± 0.56% to 5.86 ± 0.87% by 5.8S + ITS2 rDNA [4,8,23,24,36].

These data provide evidence that worms from our material represent distinct valid species. Despite the existence of morphological differences between worms ex Vietnamese Crenimugil seheli and representatives of Unisaccus, we prefer the molecular results and recognise Vietnamese worms as a new species within Unisaccus, U. halongi n. sp. However, as we said above, additional morphological and molecular data on S. vitellosum and S. indicum from type locations are needed to clarify the taxonomical status of these two species and new trematodes from our study.