Molecular and morphological evidence for three species of Diplostomum (Digenea: Diplostomidae), parasites of fishes and fish-eating birds in Spain.

BACKGROUND: Recent molecular studies have revealed high species diversity of Diplostomum in central and northern Europe. However, our knowledge of the distribution of Diplostomum spp. in the southern distributional range in Europe of the snail intermediate hosts (Lymnaea stagnalis and Radix spp.) is rather limited. This study aims to fill this gap in our knowledge using molecular and morphological evidence.
METHODS: Nineteen fish species and six fish-eating bird species were sampled opportunistically in three regions (Catalonia, Extremadura and Aragon) in Spain. All isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the barcode region of the cox1 mitochondrial gene and complete sequences of the ribosomal ITS1-5.8S-ITS2 gene cluster were used for molecular identification of the isolates.
RESULTS: Integrated morphological and molecular analyses demonstrated the presence of three species among the larval and adult isolates of Diplostomum spp. sampled in Spain: Diplostomum spathaceum (in fish and birds), D. pseudospathaceum (in birds) and Diplostomum sp. (in fish) referred to as Clade Q sensu Georgieva et al. (Int J Parasitol, 43:57-72, 2013). We detected ten cox1 haplotypes among the isolates of D. spathaceum with only one haplotype shared with adult isolates from central and northern Europe. No specific geographic pattern of the distribution of the novel haplotypes was found.
CONCLUSION: This first molecular exploration of the diversity of Diplostomum spp. in southern Europe indicates much lower species richness compared with the northern regions of Europe.

Background

Diplostomum von Nordmann, 1832 is a relatively large genus of widely distributed digeneans with three-host life-cycles involving lymnaeid snails and fish as intermediate hosts and fish-eating birds (predominantly gulls) as definitive hosts. There are 41 nominal species described within the Palaearctic, mainly from Europe (see [1] for details). However, treatment of the data on the geographic and host ranges of Diplostomum spp. have long been hindered by taxonomic and identification problems concerning all life-cycle stages.

The use of molecular markers has proved to be valuable and more efficient than experimental approaches in elucidating parasite life-cycles by linking larvae with adults, e.g. [1-5]. The mitochondrial cytochrome c oxidase subunit 1 (cox1) barcode region was found to be suitable for this goal as well as for the identification and recognition of cryptic species diversity within Diplostomum [1,6,7].

Recent molecular studies linking cox1 and ITS1-5.8S-ITS2 sequences for larval and adult isolates, which were identified based on parasite morphology, have revealed high species diversity of Diplostomum in central and northern Europe [1,7]. However, our knowledge of the distribution of Diplostomum spp. in the southern distributional range in Europe of the snail intermediate hosts (Lymnaea stagnalis and Radix spp.) is rather limited. Virtually no data exist for infections with Diplostomum spp. in the intermediate and definitive hosts in southern Europe. In Spain, two species have been recorded in populations of the gull definitive hosts. Diplostomum spathaceum was reported in four out of 324 yellow-legged gulls referred to as “Larus cachinnans” [8] and “Larus michahellis” [9] in Galicia and D. pseudospathaceum was recorded in one of 122 “L. cachinnans” from Medes Islands [10,11]. Similarly, there is a lack of data from the intermediate fish hosts; only unidentified metacercariae of Diplostomum sp. were reported in Anguilla anguilla in the Rivers Ulla and Tea in Galicia [12].

In this study, we used the molecular framework and the recently generated genetic datasets for Nearctic and Palaearctic species of the genus [1,6,13] to investigate species diversity of Diplostomum in birds and fishes sampled opportunistically in three regions in the northern and southern Spain. We provide the first molecular evidence associated with descriptions of the hologenophores sensu Pleijel et al. [14] for three species of Diplostomum.

Methods

Sample collection and processing

An opportunistic sampling strategy was adopted for this study, which was focused on examination of a diverse array of hosts rather than large samples of a single host species. Table 1 provides a list of the fish hosts and localities in different regions in Spain. Fish were obtained in collaboration with the regional governments of Extremadura, Aragón and Catalunya. A total of 230 fish belonging to 19 species and 10 families was examined in 2012 for the presence of eye dwelling metacercariae. The samples of Pseudochondrostoma willkommi and Salmo trutta collected in Villafranco del Guadiana and Jerte were obtained from aquiculture centres of the regional government of Extremadura whereas the remaining fish species/samples were collected in rivers. The largest number of individuals and species was collected in the Ebro Delta. The aquaculture system in Villafranco del Guadiana Aquaculture Centre comprises a central octagonal pool (depth 1 m; surface c.100 m2) surrounded by a group of pentagonal pools (depth <1 m; surface c.100 m2) (Figure 1A). The central pool is used for culturing mature breeders of P. willkommi of different ages whereas the peripheral pools are used for fish fry for up to two seasons; the latter are transferred to the central pool after reaching maturity. All pools are covered with nets to decrease predation by fish-eating birds and have an open water circulation system with a steady flow of 20 L/min. Although efforts are made to keep the water quality within the accepted ranges, the degree of eutrophication is high. Pools have been completely dried on various occasions but soon afterwards were repopulated by freshwater snails.

Table 1

Summary data for the fish species examined/infected with spp.

Fish species	Fish family	Locality	Date of collection	No. examined (infected)	Total length (range, mm)
*Carassius auratus (L.)	Cyprinidae	Ebro Deltaa	18.ii.2012	2	121 − 248
*Cyprinus carpio L.	Cyprinidae			13 (1)	290 − 379
*Silurus glanis L.	Siluridae			2	440 − 460
*Pseudorasbora parva (Temminck & Schlegel)	Cyprinidae			15	45 − 103
*Lepomis gibbosus (L.)	Centrarchidae			1	52
Liza ramada (Risso) juv.	Mugilidae			10	90 − 183
*Misgurnus anguillicaudatus (Cantor)	Cobitidae			15 (1)	50 − 128
Anguilla anguilla (L.)	Anguillidae	Ebro Deltaa	17.v.2012	5	158 − 255
Atherina boyeri Risso	Atherinidae			10	34 − 44
*Cyprinus carpio L.	Cyprinidae			1	192
*Gambusia holbrooki Girard	Poeciliidae			18	24 − 50
Liza ramada (Risso) juv.	Mugilidae			1	58
*Lepomis gibbosus (L.)	Centrarchidae			14	43 − 65
*Misgurnus anguillicaudatus (Cantor)	Cobitidae			16 (2)	52 − 122
Pomatoschistus microps (Krøyer)	Gobiidae			1	32
*Pseudorasbora parva (Temminck & Schlegel)	Cyprinidae			27	49 − 79
*Silurus glanis L.	Siluridae			1 (1)	409
Tropidophoxinellus alburnoides (Steindachner)	Cyprinidae	River Albarragenab	21.ii.2012	4	57 − 89
Tropidophoxinellus alburnoides (Steindachner)	Cyprinidae	River Luorianillab	06.vi.2012	8	55 − 75
Pseudochondrostoma willkommii (Steindachner)	Cyprinidae	Villafranco del Guadianab	06.iii.2012	10 (10)	235 − 262
Salmo trutta L.	Salmonidae	Jertec	07.iii.2012	3	262 − 291
Parachondrostoma miegii (Steindachner)	Cyprinidae	River Piedrad	24.ix.2012	5	139 − 177
Oncorhynchus mykiss (Walbaum)	Salmonidae			2	170 − 195
Squalius pyrenaicus (Günther)	Cyprinidae			10	84 − 135
Salmo trutta L.	Salmonidae	Lake Espejod	24.ix.2012	2	490 − 497
Luciobarbus graellsii (Steindachner)	Cyprinidae			3	236 − 405
Oncorhynchus mykiss (Walbaum)	Salmonidae			1	441
Salmo trutta L.	Salmonidae	River Aragond	25.ix.2012	12	70 − 188
Salmo trutta L.	Salmonidae	River Arae	25.ix.2012	12	68 − 146
Gobio lozanoi Doadrio & Madeira	Cyprinidae	River Cincae	25.ix.2012	1	53
*Gambusia holbrooki Girard	Poeciliidae			5	21 − 29

*Invasive species are marked with a star; aTarragona; bBadajoz; cCaceres; dZaragoza; eHuesca.

Figure 1

Focus of infection with at the Aquaculture Centre in Villafranco del Guadiana. A, Pool system for culturing Pseudochondrostoma willkommi at the Aquaculture Centre in Villafranco del Guadiana; B, P. willkommii infected with large numbers of lens-dwelling metacercariae of Diplostomum spathaceum; C, Eye of P. willkommii with lens capsule close to rupture due to the large numbers of metacercariae of D. spathaceum.

A total of 31 fish eating birds were obtained from bird recovery centres in Catalunya (Spain) in 2012 in order to obtain adult specimens of Diplostomum (Table 2). Six species of birds of four families were examined: (i) Laridae [Larus ridibundus L., Larus argentatus michahellis Naumann]; (ii) Sternidae [Sterna sandvicensis Latham]; (iii) Ardeidae [Ardea cinerea L. and Ixobrychus minutus (L.)]; (iv) Phalacrocoracidae [Phalacrocorax aristotelis (L.)]. The largest number of birds was obtained from the Ebro Delta.

Table 2

Summary data for the bird species examined/ infected with spp.

Bird species	Collection site	No. examined (infected)
Larus argentatus michahellis Naumann	Ebro Delta (Tarragona)	6 (2)
Larus argentatus michahellis Naumann	Barcelona	2
Larus argentatus michahellis Naumann	Alella (Barcelona)	1
Larus argentatus michahellis Naumann	Sabadell (Barcelona)	1
Larus argentatus michahellis Naumann	Empuria Brava (Girona)	1
Larus argentatus michahellis Naumann	Figueres (Girona)	1
Larus argentatus michahellis Naumann	Roses (Girona)	2
Larus argentatus michahellis Naumann	Tarragona	1
Larus argentatus michahellis Naumann	Cambrils (Tarragona)	1
Larus ridibundus L.	Ebro Delta (Tarragona)	5 (3)
Larus ridibundus L.	Cunit (Tarragona)	1 (1)
Sterna sandvicensis (Latham)	Roda de Bará (Tarragona)	1
Phalacrocorax aristotelis (L.)	Tarragona	1
Ardea cinerea L.	Ebro Delta (Tarragona)	5
Ixobrychus minutus (L.)	Ebro Delta (Tarragona)	2

All metacercariae were dissected out from fresh fish, fixed in hot saline solution and preserved in molecular biology grade ethanol whereas all adult worms were collected from birds found dead and frozen until necropsy; these were also preserved in molecular grade ethanol. The morphology of the larval and adult stages of Diplostomum spp. was studied on live and fixed material from series of photomicrographs made for each isolate with a digital camera of an Olympus BX51 microscope prior to sequencing; measurements were taken from the digital images with the aid of Quick Photo Camera 2.3 image analysis software. The structure of the secondary excretory system was reconstructed from serial microphotographs and the number of excretory concretions was counted.

All measurements in the descriptions and tables are in micrometres and are presented as the range followed by the mean in parentheses.

Sequence generation

Total genomic DNA was isolated from single ethanol-fixed adult individuals using the Chelex method (see [15] for details). Partial fragments of the barcode region of the cox1 mitochondrial gene [16] were obtained by polymerase chain reaction (PCR) amplifications using Ready-To-Go PCR beads (GE Healthcare, UK) and the diplostomid-specific PCR primers Plat-diploCOX1F (5′-CGT TTR AAT TAT ACG GAT CC-3′) and Plat-diploCOX1R (5′-AGC ATA GTA ATM GCA GCA GC-3′) designed by Moszczynska et al. [16] (see [1] for details). PCR amplifications of the ITS1-5.8S-ITS2 gene cluster were performed as above using the primers D1 (forward: 5′-AGG AAT TCC TGG TAA GTG CAA G-3′) and D2 (reverse: 5′-CGT TAC TGA GGG AAT CCT GGT-3′) and thermocycling conditions of Galazzo et al. [17].

PCR amplicons were purified using a QIAquick PCR purification kit (Qiagen Ltd, UK) and sequenced directly from both strands using the PCR primers (cox1) and the primers from [18]: BD1 (forward: 5′-GTC GTA ACA AGG TTT CCG TA-3′) and BD2: (reverse: 5′-TAT GCT TAA ATT CAG CGG GT-3′) (ITS1-5.8S-ITS2) with ABI BigDye chemistry (ABI Perkin-Elmer, UK), alcohol-precipitated, and run on an ABI Prism 3130 x 1 automated sequencer. Contiguous sequences were assembled with MEGA v5 [19] and submitted to GenBank (details and accession numbers are shown in Table 3).

Table 3

Summary data for the isolates of spp. from fishes and birds collected in Spain and used for generation of the 1 and ITS1-5.8S-ITS2 sequences

Species	Life-cycle stage a	Isolate	Haplotype	Host	Locality	GenBank accession numbers
						cox 1	ITS1-5.8S-ITS2
Diplostomum sp. (Clade Q)	M	CCED	−	Cyprinus carpio	Ebro Delta	KP025770	KP025788
Diplostomum pseudospathaceum	A	LRED1	−	Larus ridibundus	Ebro Delta	KP025771	JX986854b
Diplostomum spathaceum	A	LCED1	1	Larus argentatus michahellis	Ebro Delta	KP025772	–
Diplostomum spathaceum	A	LCED2	6	Larus argentatus michahellis	Ebro Delta	KP025773	–
Diplostomum spathaceum	A	LCED3	4	Larus argentatus michahellis	Ebro Delta	KP025774	–
Diplostomum spathaceum	A	LRC	3	Larus ridibundus	Cunit	KP025775	KP025789
Diplostomum spathaceum	A	LRED2	10	Larus ridibundus	Ebro Delta	KP025776	–
Diplostomum spathaceum	A	LRED3	8	Larus ridibundus	Ebro Delta	KP025777	–
Diplostomum spathaceum	M	MAED1	4	Misgurnus anguillicaudatus	Ebro Delta	KP025778	KP025790
Diplostomum spathaceum	M	MAED2	2	Misgurnus anguillicaudatus	Ebro Delta	KP025779	KP025791
Diplostomum spathaceum	M	PWVG1	5	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025780	–
Diplostomum spathaceum	M	PWVG2	4	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025781	KP025792
Diplostomum spathaceum	M	PWVG3	7	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025782	KP025793
Diplostomum spathaceum	M	PWVG4	1	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025783	–
Diplostomum spathaceum	M	PWVG5	9	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025784	–
Diplostomum spathaceum	M	PWVG6	3	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025785	–
Diplostomum spathaceum	M	PWVG7	7	Pseudochondrostoma willkommii	Villafranco del Guadiana	KP025786	–
Diplostomum spathaceum	M	SGED	6	Silurus glanis	Ebro Delta	KP025787	–

aM, metacercaria, A, adult; bITS sequence identical with JX986854 of Georgieva et al. [1].

Alignments and data analysis

The newly-generated and published sequences were aligned together with MUSCLE implemented in MEGA v5; cox1 sequences were aligned with reference to the amino acid translation, using the echinoderm and flatworm mitochondrial code [20]. The cox1 alignment (410 nt; 46 sequences) comprised the 18 newly-generated (Table 3) and 28 published sequences, the latter including 1 − 5 representative sequences per species/lineage identified in previous studies in Europe [1,13]; see Table 4 for details. The ITS1-5.8S-ITS2 alignment (997 nt; 35 sequences) comprised seven new sequences for Spanish isolates sub-sampled within the cox1-derived clades and 29 published sequences, representative for the species/lineages sequenced in Europe [1,13] and Canada [6,17] (for details see Table 4). Sequences for Tylodelphys clavata were used as outgroups.

Table 4

Summary data for the isolates of spp. retrieved from GenBank

Trematode species	Isolate	Life-cycle stage a	Host species	Locality	Accession No. ( cox 1)	Accession No. (ITS1-5.8S-ITS2)	Reference
‘Diplostomum baeri’ 1	STR3	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986862	JX986837	Georgieva et al. [1]
‘Diplostomum baeri’ 1	STL1	M	Salmo trutta fario	Germany: River Lenne	JX986863	-	Georgieva et al. [1]
‘Diplostomum baeri’ 1	STR4	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986864		Georgieva et al. [1]
‘Diplostomum baeri’ 1	STL2	M	Salmo trutta fario	Germany: River Lenne	JX986865	-	Georgieva et al. [1]
‘Diplostomum baeri’ 1	STR7	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986869	-	Georgieva et al. [1]
Diplostomum baeri	PF5D3	M	Perca fluviatilis	Germany: Lake Constance	JQ639195	-	Behrmann-Godel [13]
Diplostomum baeri	PF15D9	M	Perca fluviatilis	Germany: Lake Constance	JQ639193	-	Behrmann-Godel [13]
Diplostomum baeri	PF15D4	M	Perca fluviatilis	Germany: Lake Constance	JQ639187	-	Behrmann-Godel [13]
Diplostomum baeri	PF8D7	M	Perca fluviatilis	Germany: Lake Constance	JQ639191	-	Behrmann-Godel [13]
Diplostomum baeri	PF6D3	M	Perca fluviatilis	Germany: Lake Constance	JQ639189	-	Behrmann-Godel [13]
Diplostomum baeri	–	A	Larus delawarensis (exp.)b	Canada	-	AY123042	Galazzo et al. [17]
Diplostomum huronense	–	A	Larus delawarensis (exp.)b	Canada	-	AY123044	Galazzo et al. [17]
Diplostomum huronense	D.LL.IVT.Cc.3 F.1	M	Catostomus commersoni	Canada	-	GQ292513	Locke et al. [6]
Diplostomum indistinctum	D.RL.D.Cc.1.2	M	Catostomus commersoni	Canada		GQ292508	Locke et al. [6]
‘Diplostomum mergi’ 1	RAH1	C	Radix auricularia	Germany: Hengsteysee	JX986873	JX986838	Georgieva et al. [1]
‘Diplostomum mergi’ 2	RAH2	C	Radix auricularia	Germany: Hengsteysee	JX986874	-	Georgieva et al. [1]
‘Diplostomum mergi’ 2	RAH3	C	Radix auricularia	Germany: Hengsteysee	JX986875	JX986839	Georgieva et al. [1]
‘Diplostomum mergi’ 2	RAH4	C	Radix auricularia	Germany: Hengsteysee	JX986876	-	Georgieva et al. [1]
‘Diplostomum mergi’ 3	GGR2	M	Gobio gobio	Germany: River Ruhr (Henne)	JX986877	JX986840	Georgieva et al. [1]
‘Diplostomum mergi’ 3	STR10	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986878	JX986841	Georgieva et al. [1]
‘Diplostomum mergi’ 3	STR11	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986879	-	Georgieva et al. [1]
‘Diplostomum mergi’ 3	STR12	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986880	-	Georgieva et al. [1]
‘Diplostomum mergi’ 3	GGR3	M	Gobio gobio	Germany: River Ruhr (Henne)	-	JX986842	Georgieva et al. [1]
‘Diplostomum mergi’ 3	GGR4	M	Gobio gobio	Germany: River Ruhr (Henne)	-	JX986843	Georgieva et al. [1]
‘Diplostomum mergi’ 3	STR15	M	Salmo trutta fario	Germany: River Ruhr (Henne)	JX986886	-	Georgieva et al. [1]
Diplostomum mergi	RR45	M	Rutilus rutilus	Germany: Lake Constance	JQ639178	-	Behrmann-Godel [13]
Diplostomum mergi	RR43	M	Rutilus rutilus	Germany: Lake Constance	JQ639177	-	Behrmann-Godel [13]
Diplostomum mergi	RA97	C	Radix auricularia	Germany: Lake Constance	JQ639179	JQ665458	Behrmann-Godel [13]
Diplostomum paracaudum	CL100	M	Coregonus lavaretus	Germany: Lake Constance	-	JQ665457	Behrmann-Godel [13]
Diplostomum pseudospathaceum	LCT3	A	Larus cachinnans	Czech Republic: near Tovačov	JX986896	JX986849	Georgieva et al. [1]
Diplostomum pseudospathaceum	LSB2	C	Lymnaea stagnalis	Germany: Baldeneysee	-	JX986850	Georgieva et al. [1]
Diplostomum pseudospathaceum	LSH1	C	Lymnaea stagnalis	Germany: Harkortsee	-	JX986851	Georgieva et al. [1]
Diplostomum pseudospathaceum	GAH6	M	Gasterosteus aculeatus	Germany: Hengsteysee	-	JX986852	Georgieva et al. [1]
Diplostomum pseudospathaceum	LAG2	A	Larus argentatus	Poland: near Gdańsk	JX986904	JX986853	Georgieva et al. [1]
Diplostomum pseudospathaceum	LCT4	A	Larus cachinnans	Czech Republic: near Tovačov	JX986905	JX986854	Georgieva et al. [1]
Diplostomum pseudospathaceum	GC87	M	Gymnocephalus cernuus	Germany: Lake Constance	-	JQ665456	Behrmann-Godel [13]
Diplostomum spathaceum	LCT1	A	Larus cachinnans	Czech Republic: near Tovačov	JX986887	JX986844	Georgieva et al. [1]
Diplostomum spathaceum	RAH6	C	Radix auricularia	Germany: Hengsteysee		JX986846	Georgieva et al. [1]
Diplostomum spathaceum	RAH5	C	Radix auricularia	Germany: Hengsteysee		JX986845	Georgieva et al. [1]
Diplostomum spathaceum	LAG1	A	Larus argentatus	Poland: near Gdańsk	JX986892	JX986847	Georgieva et al. [1]
Diplostomum spathaceum	LCT2	A	Larus cachinnans	Czech Republic: near Tovačov	JX986895	JX986848	Georgieva et al. [1]
Diplostomum sp. 1 SAL-2008	D.IN.SSO.Ld.2 F.6	A	Larus delawarensis (exp.)b	Canada	-	GQ292519	Locke et al. [6]
Diplostomum sp. 2 SAL-2008	D.BR.S.B.20.1	M	Pimephales notatus	Canada	-	GQ292505	Locke et al. [6]
Diplostomum sp. 3 SAL-2008	D.RL.B08.Ms.1 F.1	M	Micropterus salmoides	Canada	-	GQ292511	Locke et al. [6]
Diplostomum sp. 4 SAL-2008	D.IN.SSO.Ld.2 F.10	A	Larus delawarensis	Canada	-	GQ292520	Locke et al. [6]
Tylodelphys clavata	PFL1	M	Perca fluviatilis	Germany: River Lippe	JX986909	-	Georgieva et al. [1]
Tylodelphys clavata	CL91	M	Coregonus lavaretus	Germany: Lake Constance	-	JQ665459	Behrmann-Godel [13]

aC, cercaria, M, metacercaria; A, adult; braised in experimental infection.

Distance-based [neighbour-joining (NJ)] and model-based [maximum likelihood (ML) and Bayesian inference (BI)] algorithms were used for tree reconstruction. Prior to analyses the best-fit nucleotide substitution models were selected in jModelTest 2.1.1 [21,22] using the Akaike Information Criterion (AIC). These were the Hasegawa-Kishino-Yano model including estimates of invariant sites and among-site rate heterogeneity (HKY + I + G) for the cox1 dataset and the Hasegawa-Kishino-Yano model including estimates of among-site rate heterogeneity (HKY + G) for the ITS dataset. ML analyses were performed in PhyML 3.0 [23] with a non-parametric bootstrap validation based on 1,000 replicates. BI analyses were carried out in MrBayes 3.2 [24] using Markov Chain Monte Carlo (MCMC) searches on two simultaneous runs of four chains during 107 generations, sampling trees every 103 generations. The first 25% of the sampled trees were discarded as “burn-in” for each data set and the consensus tree topology and the nodal support were estimated from the remaining samples as posterior probability values [25]. Distance matrices (p-distance model, i.e. the percentage of pairwise character differences with pairwise deletion of gaps) were also calculated and explored with MEGA v5.

Results

Diplostomum spp. infections in fish and birds

Of the 230 fish of 19 species studied, only 15 were infected with Diplostomum spp.: one Cyprinus carpio (Cyprinidae), one Silurus glanis (Siluridae), three Misgurnus anguillicaudatus (Cobitidae) and ten Pseudochondrostoma willkommii (Cyprinidae). All infected fishes were collected in the Ebro Delta (Tarragona, Spain) with the exception of P. willkomii originating from the aquaculture centre of Villafranco del Guadiana (Badajoz, Spain) (Table 1). It is worth noting that infections with metacercariae of Diplostomum spp. were detected in some (C. carpio and M. anguillicaudatus) and not in other relatively well-sampled species (Pseudorasbora parva, Gambusia holbrooki and Lepomis gibbosus) in the Ebro Delta but also in one of the three S. glanis sampled in this locality. All infections with Diplostomum spp. in the fish from Ebro Delta were of low intensity (1 to 4 metacercariae).

All P. willkommii (n = 10) examined from the aquaculture centre in Villafranco de Guadiana were infected with 95–139 metacercariae. Due to the high parasite load, infections were detectable by visual examination especially in older mature fish (Figure 1B,C). The overall prevalence of infection is estimated as 60–65% with a trend of increase with fish age: 0–25% in fish during the first year; 25–50% during the second year; 50–75% during the third year; up to 90% during the fourth year.

A total of 31 fish-eating birds belonging to six species was examined (Table 2). Of these, only six gulls were infected with Diplostomum spp.: two Larus argentatus michahellis and three L. ridibundus originating from Ebro Delta (Tarragona) and one L. ridibundus from Cunit (Tarragona). Representative adult specimens of the two Diplostomum spp. identified in the material from gulls based on morphology, i.e. D. spathaceum and D. pseudospathaceum, and all metacercariae recovered from fish were selected for sequencing.

Molecular identification

Partial cox1 sequences were obtained for seven adult isolates collected from two gull hosts (Larus ridibundus and L. cachinnans) and 11 metacercarial isolates collected from the lenses of four fish hosts (Cyprinus carpio, Misgurnus anguillicaudatus, Pseudochondrostoma willkommii and Silurus glanis). Similar to a previous study on Diplostomum spp. in Europe [1], phylogenetic analyses of the cox1 dataset (410 nt) recovered eight species/lineages comprising D. spathaceum, D. pseudospathaceum, D. spathaceum/parviventosum referred to as Clade Q sensu Georgieva et al. [1], ‘D. mergi’ complex (including three putative species) and ‘D. baeri’ complex (representing two sibling species) (Figure 2). The analyses provided robust evidence that most of the isolates are conspecific with D. spathaceum sensu Georgieva et al. [1] (Figure 2). These represented five adult isolates ex L. ridibundus and L. argentatus michahellis from Ebro Delta, one adult isolate ex L. ridibundus from Cunit, seven metacercarial isolates ex P. willkommii from Villafranco del Guadiana, two metacercarial isolates ex M. anguillicaudatus and a single isolate ex S. glanis, the last two fish species both collected from Ebro Delta.

Figure 2

Neighbour-joining (NJ) phylogram reconstructed using the newly-generated and retrieved from GenBank 1 sequences for spp. Nodal support from Maximum Likelihood (ML) and Bayesian Inference (BI) analyses indicated as NJ/ML/BI. Outgroup: Tylodelphys clavata. The scale-bar indicates the expected number of substitutions per site. Isolates from Spain are coded as in Table 3; stars indicate adult isolates from gulls.

The intraspecific divergence within the D. spathaceum clade ranged between 0 and 1.5%, i.e. within the known range of intraspecific variation for Diplostomum spp. [1]. The material collected in Spain was represented by a total of 10 haplotypes (Table 3) with only one haplotype shared with adult isolates from central and northern Europe (haplotype 2, isolate ex M. anguillicaudatus and JX986892). There was no specific geographic pattern of the distribution of the novel haplotypes. Thus isolates from Pseudochondrostoma willkommii from the population of Villafranco del Guadiana were represented by six haplotypes with only one shared and there were shared haplotypes among isolates from geographically distant host samples, e.g. among larval isolates from Villafranco del Guadiana and adult isolates from Ebro Delta and Cunit (haplotypes 1, 3 and 4) (see Table 3 for details).

Numerous attempts were made to obtain sequences for isolates of adult D. pseudospathaceum identified based on morphology but only one was successful; this may be due to the fact that the infected birds were collected long after their death. The sequence for the single isolate ex Larus ridibundus clustered within the strongly supported clade (Figure 2) representing sequences for adult isolates of D. pseudospathaceum identified based on morphology [1]. The Spanish isolate was represented by a unique haplotype which differed by 1.2-1.7% from the remaining three haplotypes within the D. pseudospathaceum clade.

Finally, a sequence from a single metacercaria ex Cyprinus carpio from the Ebro Delta clustered together with sequences for one cercarial isolate ex Radix auricularia (RA97) and two metacercarial isolates ex R. rutilus (RR43 and RR45) from Lake Constance, all reported as D. spathaceum [13] but labelled as D. mergi in GenBank (see Clade Q in Figure 2).

A total of seven ITS1-5.8S-ITS2 sequences was generated after a selective sub-sampling of the Spanish isolates within the three cox1 clades of Diplostomum spp. The analysis of the ITS data (997 nt positions) resulted in molecular identification of these isolates concordant with that based on the cox1 gene trees with strong support (Figure 3). The intraspecific divergence within the D. spathaceum clade ranged between 0 and 0.4%. The five representative isolates from the cox1 dataset corresponded to four genotypes (with one genotype shared between an isolate ex M. anguillicaudatus from Ebro Delta and one ex L. ridibundus from Cunit).

Figure 3

Neighbour-joining (NJ) phylogram reconstructed using the newly-generated and retrieved from GenBank ITS1-5.8S-ITS2 sequences for spp. Nodal support from Maximum Likelihood (ML) and Bayesian Inference (BI) analyses indicated as NJ/ML/BI. Outgroup: Tylodelphys clavata. The scale-bar indicates the expected number of substitutions per site. Isolates from Spain are coded as in Table 3; stars indicate adult isolates from gulls.

The sequence from the single adult isolate identified as D. pseudospathaceum based on morphology and cox1 phylogeny was identical with six sequences of Georgieva et al. [1] based on larval and adult isolates from the Czech Republic and Germany and one sequence of Berhrmann-Godel [13]; all these sequences formed a strongly supported clade representing D. pseudospathaceum (Figure 3) which also included Diplostomum sp. 3 of Locke et al. [6] as in previous studies [1,7].

As in the cox1 solution, the sequence for the metacercarial isolate ex C. carpio clustered together with a sequence labelled in GenBank as “D. mergi” for a cercarial isolate (RA97) ex Radix auricularia from Lake Constance [13] within the Clade Q sensu Georgieva et al. [1]. The divergence between the two sequences was 0.8%.

Descriptions of the molecular voucher material

Diplostomum spathaceum (Rudolphi, 1819) (adult)

:Larus argentatus michahellis Naumann; L. ridibundus L.

: Ebro Delta, Cunit (Tarragona, Spain).

: Small intestine.

[Based on five frozen specimens (hologenophores) preserved in ethanol (molecular biology grade)]. Body 1,971 − 2,189 (2,085) long (Figure 4). Forebody oval, dorso-ventrally flattened, 782 − 1,155 long [40 − 43 (42)% of total body length], with maximum width 504 − 726 (592) at level of holdfast organ. Hindbody, elongate-oval, narrower anteriorly, 1,252 − 1,368 (1,285) long, with maximum width 387 − 575 (477) at level of anterior testis.

Figure 4

. Adult ex Larus argentatus michahellis (hologenophore). Scale-bar: 500 μm.

Oral sucker ventro-subterminal, subspherical, 71 − 93 × 70 − 92 (81 × 78). Pseudosuckers well developed, 109 − 155 × 44 − 62 (139 × 56). Ventral sucker subglobular, 65 − 95 × 80 − 99 (83 × 89), similar in size to oral sucker, located just anterior to mid-forebody. Holdfast organ large, subglobular, 150 − 236 × 202 − 288 (215 × 224), fairly close to or contiguous with ventral sucker. Prepharynx short or absent; pharynx elongate-oval, 55 − 89 × 45 − 59 (73 × 52); oesophagus indistinct; caeca narrow.

Testes 2, large, in posterior half of hindbody; anterior testis transversely elongate, asymmetrical, 171 − 203 × 154 − 224 (183 × 191); posterior testis transversely elongate, symmetrical, horseshoe-shaped, 190 − 317 × 240 − 399 (247 × 334). Seminal vesicle voluminous. Gentital pore dorso-subterminal. Ovary small, dextral, pretesticular, subglobular, 87 × 83, contiguous with anterior testis. Vitellarium follicular, follicles numerous, small, arranged in four lateral bands surrounding holdfast organ in forebody; bands reach to mid-level of holdfast organ, converge close to posterior margin of forebody, posteriorly to holdfast organ; vitelline follicles in hindbody in two wide, not well-delimited lateral bands, converging medially at level of testes, reaching fairly close to posterior extremity of body. Eggs few, 89 − 99 × 61 − 66 (95 × 63).

Diplostomum spathaceum (Rudolphi, 1819) (metacercaria)

: Pseudochondrostoma willkommii (Steindachner); Misgurnus anguillicaudatus (Cantor); Silurus glanis L.

: Villafranco del Guadiana (P. willkommii) and Ebro Delta (M. anguillicaudatus and S. glanis), Spain.

: Eye lens.

[Based on 10 metacercariae (hologenophores) fixed in hot saline solution and preserved in ethanol (molecular biology grade)]. Body elongate-oval, flattened, 277 − 453 × 198 − 295 (376 × 248); primordial hindbody 10 − 26 (16) long (Figure 5). Oral sucker elongate-oval, 40 − 57 × 36 − 41 (45 × 39). Ventral sucker transversely oval, 30 − 43 × 33 − 48 (38 × 43). Two contractile lappets (pseudosuckers) present on each side of oral sucker, 44 − 55 (48) long, with maximum width 22 − 30 (26). Prepharynx very short; pharynx elongate-oval, 29 − 43 × 19 − 26 (37 × 23); oesophagus short; caeca long, wide, reach posterior to holdfast organ. Holdfast organ large, elongate-oval, 63 − 89 × 59 − 90 (75 × 80). Reserve excretory system with numerous, relatively large excretory granules (170 − 184 in number), distributed in a median and two lateral fields.

Figure 5

Metacercariae of ex . A, Live metacercaria (hologenophore); B, Fixed metacercariae (hologenophore). Scale-bars: A, 200 μm; B, 100 μm.

Diplostomum pseudospathaceum Niewiadomska, 1984 (adult)

:Larus ridibundus L.

: Ebro Delta (Tarragona, Spain).

: Small intestine.

[Based on a single frozen specimen (hologenophore) preserved in ethanol (molecular biology grade)]. Body 2,884 long (Figure 6). Forebody elongate-oval, narrow, dorso-ventrally flattened, tapering anteriorly, 1,075 long (37% of total body length), with maximum width at level of ventral sucker, 526. Hindbody, elongate, sub-cylindrical, narrower anterior to ovary, 1,891 long, with maximum width at level of posterior testis, 163.

Figure 6

. Adult ex Larus ridibundus (hologenophore). Scale-bar: 500 μm.

Oral sucker ventro-subterminal, subspherical, 69 × 73. Pseudosuckers well developed, 128 × 49. Ventral sucker transversely oval, 67 × 85, slightly larger than oral sucker, located just posterior to mid-forebody. Holdfast organ subglobular, 126 × 118, located well posterior to ventral sucker (at a distance >2 ventral sucker diameters). Prepharynx fairly short; pharynx elongate-oval, 53 × 35; oesophagus short; caeca narrow.

Testes 2, large, in posterior half of hindbody; anterior testis transversely elongate, asymmetrical, 132 × 75; posterior testis larger, transversely elongate, symmetrical, horseshoe-shaped, 237 × 315. Seminal vesicle voluminous. Gentital pore dorso-subterminal. Ovary small, submedian, pretesticular, subglobular, 79 × 78, nearly contiguous with anterior testis. Vitellarium follicular, follicles numerous, small, arranged in two median inter-caecal and four lateral extra-caecal bands in forebody, reaching to the posterior margin of ventral sucker anteriorly; bands, converge close to posterior margin of forebody, posteriorly to holdfast organ; vitelline follicles in hindbody in two wide, dense lateral bands, converging medially at level of gonads, reach fairly close to posterior extremity of body. Eggs few, 96 − 110 × 58 − 63.

Diplostomum sp. (metacercaria)

:Cyprinus carpio L.

: Ebro Delta (Tarragona, Spain).

: Eye lens.

[Based on a single metacercaria (hologenophore) fixed and preserved in ethanol (molecular biology grade).] Body elongate-oval, flattened, 229 × 180; primordial hindbody not evident (Figure 7). Oral sucker spherical, 29 × 29. Ventral sucker subspherical, 37 × 42. Two small contractile lappets (pseudosuckers) present on each side of oral sucker, 31 − 32 long, with maximum width 15 − 16. Prepharynx absent; pharynx subspherical, 24 × 23; oesophagus very short; caeca long, narrow, reach posterior to holdfast organ. Holdfast organ large, transversely elongate, 50 × 84. Reserve excretory system with numerous, dispersed, relatively large excretory granules (c. 215 in number).

Figure 7

sp. ex . Fixed metacercaria (hologenophore). Scale-bar: 100 μm.

Discussion

This first molecular exploration of the diversity of Diplostomum spp. in southern Europe indicates much lower species richness compared with the northern regions of Europe (3 vs 12 species). Of the six species of fish-eating birds studied in the north of Spain only two gull species were found to host adult Diplostomum spp.; however, sample sizes were rather small. The detection of metacercariae in fish also might have been influenced by the differential sample sizes. However, we found infections in an under-sampled fish host as well in some but not in other hosts with relatively large sample sizes. Notably, metacercariae of Diplostomum spp. were recovered in three out of the seven invasive fish species examined (C. carpio, M. anguillicaudatus and S. glanis; Table 1) thus indicating that these hosts may have a considerable contribution to the transmission of Diplostomum spp. in the Ebro Delta and elsewhere. M. anguillicaudatus and S. glanis are new host records for D. spathaceum.

Another important finding is the high prevalence and abundance of infection with D. spathaceum in P. willkommii, a native vulnerable species [26] with distribution restricted to the southern Iberian Peninsula in Spain and Portugal. The high levels of infections in the aquaculture centre in Villafranco de Guadiana, where mature breeders from natural populations are being added yearly to the cultured population, reveal a further threat upon this fish species in both natural and fish farming conditions. The shallow, open nature of the pools probably contributes significantly to the establishment of a focus of infection with D. spathaceum.

To the best of our knowledge, this study is the first to provide detailed morphometric data and morphological description of the isolates of Diplostomum spp. in association with the molecular data used for identification. The morphology of the adult specimens of D. spathaceum and D. pseudospathaceum used for sequence generation agrees well with the descriptions of D. spathaceum sensu stricto and D. pseudospathaceum of Niewiadomska [27], respectively. The material of D. spathaceum ex Larus spp. from Ebro Delta is characterised by lower values (outside the lower range for the material ex Larus fuscus L. and L. ridibundus from Poland studied by Niewiadomska [27] for the size of the hindbody, holdfast organ, ovary and testes (Table 5). Similarly, the specimen of D. pseudospathaceum ex L. ridibundus from Ebro Delta had smaller holdfast organ, ovary and testes and much narrower hindbody and longer pseudosuckers compared with the specimens from the same host studied in Poland (Table 5). These data indicate much higher geographic variation in the morphometric features in both Diplostomum spp.

Table 5

Comparative metrical data for adults of and .

Species	Dipostomum spathaceum		Dipostomum pseudospathaceum
Host	Larus fuscus L., Larus ridibundus L.	Larus argentatus michahellis Naumann; Larus ridibundus L.	Larus ridibundus L.	Larus ridibundus L.
Locality	Lake Mamry (Poland)	Ebro Delta (Spain)	Lake Mamry (Poland)	Ebro Delta (Spain)
Source	Niewiadomska [ 27 ]	Present study	Niewiadomska [ 27 ]	Present study
TL	up to 4,000	1,971 − 2,189	up to 3,600	2,884
FBL	1,110 − 1,480	782 − 1,155	1,030 − 1,720	1,075
FBW	590 − 850	504 − 726	400 − 680	526
HBL	1,560 − 2,920	1,252 − 1,368	960 − 2,190	1,891
HBW	560 − 660	387 − 575	420 − 720	163
OSL	57 − 95	71 − 93	67 − 78	69
OSW	74 − 102	70 − 92	68 − 95	73
PSL	102 − 153	109 − 155	51 − 115	128
PSW	–	44 − 62	–	49
VSL	78 − 95	65 − 95	68 − 103	67
VSW	89 − 102	80 − 99	62 − 119	85
HOL	238 − 374	150 − 236	153 − 335	126
HOW	259 − 399	202 − 288	163 − 388	118
PHL	59 − 74	55 − 89	44 − 74	53
PHW	51 − 74	45 − 59	47 − 66	35
ATL	185 − 540	171 − 203	188 − 503	132
ATW	421 − 629	154 − 224	296 − 629	75
PTL	348 − 592	190 − 317	255 − 666	237
PTW	466 − 658	240 − 399	370 − 666	315
OVL	138 − 222	87	111 − 187	79
OVW	163 − 236	83	142 − 238	78
FO/BL (%)	31 − 48	40 − 43	41 − 58	37
Egg-length	–	89 − 99	–	96 − 110
Egg-width	–	61 − 66	–	58 − 63

Abbreviations: TL total body length, FBL forebody length, FBW forebody width, HBL hindbody length, HBW hindbody width, OSL oral sucker length, OSW oral sucker width, PSL pseudosucker length, PSW pseudosucker width, VSL ventral sucker length, VSW ventral sucker width, HOL holdfast organ length, HOW holdfast organ width, PHL pharynx length, PHW pharynx width, ATL anterior testis length, ATW anterior testis width, PTL posterior testis length, PTW posterior testis width, OVL ovary length, OVW ovary width, FO/BL (%) forebody as a percentage of body length.

The dimensions of the metacercariae from the three fish hosts identified molecularly as D. spathaceum varied within the range provided by Niewiadomska [28] for the metacercariae of this species raised experimentally in C. carpio. However, the mean values for the length of body and the size of suckers were lower in the specimens obtained in Spain (Table 6). The metacercaria of Diplostomum sp. that was found to be conspecific with the isolates of Clade Q sensu Georgieva et al. [1] had distinctly smaller oral sucker and shorter holdfast organ compared with both Spanish and Polish isolates of D. spathaceum (Table 6). Finally, the metacercariae of both Diplostomum spp. examined in Spain had distinctly lower number of excretory granules in the secondary excretory system than the experimentally raised metacercariae ex C. carpio (see [28]; Table 6).

Table 6

Comparative metrical data for the metacercariae of and sp. (Clade Q)

Species	Dipostomum spathaceum				Diplostomum sp. (Clade Q)
Host	Cyprinus carpio L.		Pseudochondrostoma willkommii (Steindachner); Misgurnus anguillicaudatus (Cantor); Silurus glanis L.		Cyprinus carpio
Locality	Experimental infection		Villafranco del Guadiana and Ebro Delta (Spain)
Source	Niewiadomska [ 28 ]		Present study		Present study
	Range	Mean	Range	Mean	n =1
BL	340 − 451	398	277 − 453	376	229
BW	170 − 296	217	198 − 295	248	180
HL	–	–	10 − 26	16	0
OSL	42 − 54	48	40 − 57	45	29
OSW	42 − 52	45	36 − 41	39	29
PSL	–	–	44 − 55	48	31 − 32
PSW	–	–	22 − 30	26	15 − 16
VSL	39 − 56	46	30 − 43	38	37
VSW	42 − 59	53	33 − 48	43	42
PHL	25 − 39	31	29 − 43	37	24
PHW	12 − 25	20	19 − 26	23	23
HOL	68 − 93	77	63 − 89	75	50
HOW	62 − 102	85	59 − 90	80	84
No. of excretory granules	c. 300	–	170 − 184	178	c. 215

Abbreviations: BL body length, BW body width, HL primordial hindbody length, OSL oral sucker length, OSW oral sucker width, PSL pseudosucker length, PSW pseudosucker width, VSL ventral sucker length, VSW ventral sucker width, HOL holdfast organ length, HOW holdfast organ width, PHL pharynx length, PHW pharynx width.

Although the molecular and morphological identification of the larval and adult isolates of D. spathaceum and D. pseudospathaceum were straightforward, we failed to identify one isolate recovered in C. carpio. The analysis of both cox1 and ITS1-5.8S-ITS2 sequences placed this isolate within the Clade Q (i.e. questionable), a label used by Georgieva et al. [1] to indicate five identical ITS1 sequences from Europe: two for cercariae ex R. ovata identified as D. spathaceum and one for cercariae ex R. ovata identified as D. parviventosum by Niewiadomska & Laskowski [29] in Poland; one for a metacercaria ex R. rutilus from Finland submitted to GenBank as D. cf. parviventosum/spathaceum by Rellstab et al. [30]; and one for cercariae ex R. auricularia (isolate RA97) from Lake Constance [13]; the latter was designated as D. spathaceum but submitted to GenBank as D. mergi. Using the sequences of Behrmann-Godel [13] for both cox1 and ITS1-5.8S-ITS2, we found that this clade, incorporating our sequence for the metacercaria ex C. carpio, is strongly supported and reconstructed as sister to the species-level lineages of the ‘Diplostomum mergi’ species complex sensu Georgieva et al. [1]. Unfortunately, no identification to the species level can be attempted for the isolates within this clade since all represent larval stages for which, with the exception of the present data, no morphological evidence has been provided. The congruent morphological and molecular identification of the adult isolates of D. spathaceum achieved here, supports the suggestion of Georgieva et al. [1] that isolates in Clade Q may represent D. parviventosum. Further molecular and morphological evidence is required, preferably based on adult isolates, in order to solve the species-level identification of this clade.

Conclusion

This first molecular exploration of the diversity of Diplostomum spp. in southern Europe indicates much lower species richness compared with the northern regions of Europe.