The Genus Gyrodactylus von Nordman, 1832 (Monopisthocotyla: Gyrodactylidae) from Freshwater Fishes in Bulgaria: A Museum-Based Revision

Abstract

The species composition and host–parasite associations of Gyrodactylus parasitising freshwater fishes in Bulgaria were re-examined based on the revision of museum specimens. Revised data are provided for 28 species. There are 22 species confirmed as occurring in Bulgaria based on morphological examination (G. aphyae, G. cyprini, G. fossilis, G. gobii, G. gracilihamatus, G. katharineri, G. laevis, G. latus, G. leucisci, G. luciopercae, G. macrocornis, G. macronychus, G. malmbergi, G. markakulensis, G. medius, G. prostae, G. rhodei, G. shulmani, G. sprostonae, G. stankovici, G. truttae and G. vimbi). New records for the country are reported for G. cobitis, G. dykovae, G. gobiensis and G. papernai. The data about the occurrence of nine species could not be verified. The most species-rich region is the Danube Drainage (21 species), followed by the East-Aegean Sea Drainage (12 species). The smaller drainages (Black Sea Drainage—nine species; West-Aegean Sea Drainage—four species) are less studied. G. prostae (four host species from eight localities) and G. sprostonae (four host species, four localities) are revealed as the most frequent species; these euryxenous parasites infect a broad range of host species, often fishes of economic importance. Fish species of less commercial value are less studied.

1. Introduction

The parasitic platyhelminths of the genus Gyrodactylus von Nordman, 1832 (Monopisthocotyla: Gyrodactylidae) are widespread ectoparasites, infecting mainly the gills, fins and skin of teleost fishes [1,2,3] and, rarely, amphibians [2]. In some cases, they have a severe pathogenic effect on the fish host, which affects negatively fisheries and aquacultures as well as natural fish populations [4,5]. Globally, more than 500 species of Gyrodactylus have been described [2] and the expected number may exceed 20,000 [6]. The characters traditionally used for the identification of gyrodactylids are associated with the morphology of the haptoral hard parts [1,7]. The majority of the published data on the diversity of Gyrodactylus spp. from Bulgaria are based on these morphological characteristics [8].

The earliest records of Gyrodactylus in Bulgaria dated from the late 1950s, based on a small number of samples from various parts of the country, e.g., the river Danube, fishponds near Sofia and Yambol, as well as from fishes kept in the Varna Aquarium [9]. Kakacheva-Avramova [10] published her first records of Gyrodactylus from freshwater fishes in Bulgaria, which included samples from the river Maritsa and its tributaries in the Upper Thracian Plain and adjacent mountain slopes. In the span of the next three decades (1965–1999), Kakacheva-Avramova and her collaborators examined the helminth fauna of fishes in Bulgarian freshwater bodies, recording 30 species of Gyrodactylus from a total of 32 fish species [11,12,13,14,15,16,17,18,19]. The map of the localities sampled by Kakacheva-Avramova and her team is presented in the Supplementary Materials (Figure S1). Additional data on Gyrodactylus from freshwater fishes were presented by Grupcheva & Nedeva [20], Margaritov [21,22,23,24], Margaritov & Van Than [25], Margaritov & Kiritsis [26] and Kirin et al. [27]. Two recent papers were added by the present authors [28,29]. The summarised primary data are included in the Supplementary Materials (Table S1).

The total number of recorded Gyrodactylus spp. from freshwater fishes from Bulgaria is 36. They are isolated from 37 host species (Table S1). An additional five records are identified at the generic level only, as “Gyrodactylus sp.” [10,11,13,18]. The majority of the reviewed articles contain data on the host species, site of infection, prevalence and intensity of infection. Metric data of the body parts are sometimes given, as well as taxonomic drawings of the haptoral hard parts. Even though these records provide information for the sizes of the various haptoral hard parts, the lack of detailed morphological descriptions and taxonomic illustrations makes their credibility uncertain. Dedicated studies on freshwater Gyrodactylus spp. from Bulgaria have not been carried out during recent decades. Studying the literature, we observed that the published data are provided with scarce morphological information which is often not enough to confirm the identifications. Our hypothesis is that the data on the species composition are not complete and, therefore, need to be updated and verified. As a next step in the study of the diversity of Gyrodactylus spp. in Bulgaria, we re-examine the available specimens used in the works of previous researchers.

The aim of the present study is to revise the data on the taxonomic identification, host range and geographical distribution of gyrodactylids from freshwater fishes in Bulgaria. For this purpose, we examined the museum specimens of the genus Gyrodactylus preserved in the Helminthological Collection of Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences (IBER-BAS). We also examined new specimens, which were collected by the senior author in 2017–2023.

2. Materials and Methods

The morphological data presented for each species are based on examination of specimens from the Helminthological Collection of the IBER-BAS. For some species, specimens collected by the senior author (to be deposited in the same collection) were also examined. The deposited material consisted of 296 microscopic slides containing 325 specimens of Gyrodactylus collected between 1962 and 1996. It included specimens examined by D. Kakacheva-Avramova and I. Nedeva (known also as I. Nedeva-Menkova or I. Menkova). The following locations were examined [8]:

Danube Drainage

Rivers: Danube and the basins of its tributaries—Archar, Barziya, Bebresh, Beli Vit, Berkovska, Bogovina, Buchinska, Cherni Vit, Chuprenska, Iskretska, Kalnik, Kesekievska, Koznitsa, Varteshnitsa (Leva), Lopushnitsa, Malki Iskar, Manina, Mirkovska, Nishava, Novachenska, Ogosta, Palakariya, Rositsa, Shiposhnitsa, Shumska (Varbnishka), Vadima, Vit, Vodomerka, Yantra and Zheleznitsa.

Reservoirs: Bratushkovo, Iskar and Maslovo.

Fish Farms: Bezden and Chelopechene.

Black Sea Drainage

Rivers: Dokuzak, Eleshnitsa, Glavnitsa (Annadere), Golyama Kamchiya, Karaagach, Izvorska, Kamchiya, Kotleshnitsa, Luda Kamchiya, Sukhoyka, Rezovska, Ticha, Veleka and Zlatina.

Reservoirs: Komarevo (Hrabrovski) and Tsonevo (Georgi Traykov).

Fish Farm: Kotel.

West-Aegean Sea Drainage

Rivers: Banderitsa, Belishka, Blagoevgradska Birstritsa, Demyanitsa, Gradevska, Iliyna, Mesta, Rilska, Struma, Strumeshnitsa, Sushichka and Yazo.

Reservoir: Dospat and Pchelina.

Fish Farm: Blagoevgrad.

East-Aegean Sea Drainage

Rivers: Asenitsa, Bedechka, Byala, Devinska, Harmanliyska, Leshnitsa, Maritsa, Mugla, Sarneshka, Shirokolashka, Stryama, Studena, Sushitsa, Tazha, Topolnitsa, Trigradska, Tundzha, Tvardishka, Vacha and Varbitsa (Syutliyka).

Reservoirs: Azmaka, Izvora and Zhrebchevo.

Fish Farms: Malo Konare, Plovdiv and Yambol.

According to Kakacheva-Avramova [8], the majority of the collected gyrodactylid specimens were examined fresh on site. Part of the specimens were mounted in glycerine–gelatine medium for microscopy examination. Selected specimens were mounted in ammonium picrate.

An additional 30 specimens of 6 species were collected by the senior author in 2017–2022 and mounted in glycerine–gelatine according to the methodology described by Gussev [30].

The nomenclature of fish species follows FishBase [31].

In the present study, we examined the microscopic slides under an Olympus BX-51 light microscope fitted with a drawing tube. The haptoral measurements were taken where possible as previously described [28].

The following metric data were collected:

Hamuli—total length (TL), shaft length (S), root length (R), point length (P);

Ventral bar—length (VBl), width (VBw); length of ventral bar membrane (VBml);

Dorsal bar—length (DBl), width (DBw);

Marginal hooks—total length (MHl), sickle length (MHs), toe basal width (MHt), aperture distance of sickle (MHad).

All measurements are in micrometres unless otherwise specified. For identification of the species, we used the monographs of Malmberg [1] and Ergens et al. [3], as well as the original descriptions. The presented scale bars are 20 μm unless otherwise stated. All taxonomic drawings of haptor hard parts are original and based on specimens from the Helminthological Collection of IBER-BAS.

In the descriptions, based on the total length of the hamuli, the species are marked as follows: species with extra-large hamuli (over 100 μm long), species with large hamuli (70–100 μm long), species with medium-sized hamuli (40–70 μm long) and species with small hamuli (shorter than 40 μm).

3. Results

3.1. Review of Species

Gyrodactylus aphyae Malmberg, 1957

Records: Kakacheva-Avramova [11] (1); Kakacheva-Avramova [13] (2); present study (3).

Host: Phoxinus phoxinus (L.) (1–3).

Locality: Rivers—Nishava (1, 3), Stryama (2), and Sukhoyka (2).

Site of infection: Fins (1–3).

Specimens studied: 10, including 5 specimens from Nishava River near the town of Godech, three slides (No. 3548, one slide, 28 April 1965—1 specimen; No. 3557, two slides, 28 April 1965—1 and 3 specimens); 2 specimens from Stryama River near the village of Bogdan (No. 4501, two slides each with 1 specimen, 15 October 1968); and 3 specimens from Sukhoyka River near the town of Kotel (No. 4564, three slides each with 1 specimen, 27 April 1969).

Morphology: Hamuli robust, medium-sized (Figure 1). Point straight. Root slightly curving inwards. Ventral bar slightly trapezoid-shaped, with oval distal ends. Proximal ends with prominent processes. Ventral bar with central ridge along the length. Ventral bar membrane not observed. Dorsal bar mostly straight, thin. Marginal hooks small, with characteristic curve of the blade, toe pointed. For metrical data, see

Table 1. Metrical data of Gyrodactylus aphyae from Phoxinus phoxinus from various localities.

Source	Malmberg [32]									Ergens et al. [3]	Kakacheva-Avramova [11]		Present Study
Locality	Nämdö (Sweden)			Aneboda (Sweden)			Jämtland (Sweden)			Palaearctic	Nishava River		Nishava River			Stryama River			Sukhoyka River
	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	Range	n	Range	Mean	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	52–63	58	10	56–67	60	10	59–64	62	10	53–70	56–64	3	58–68	63.3	4	60	-	1	60–63	61.5	3
S	-	-	-	-	-	-	-	-	-	37–52	44–46	3	44–47	45.5	5	43–45	44	2	45	45	3
P	23–35	28	10	28–29	29	10	31–32	32	10	24–34	32–34	3	28–32	31.5	5	30–31	30.5	2	30–31	30.3	3
R	12–20	16	10	16–20	18	10	18–19	19	10	15–23	-	-	12–18	17.3	4	18	-	1	15–19	17	3
Ventral bar:
VBl	23–27	25	10	24–29	26	10	29–31	29	10	22–33	30	3	25–30	29	5	28	-	1	22–30	25.3	3
VBw	8–10	9	10	7–9	8	10	10	10	10	5–9	11	3	6–7	6.8	5	6	-	1	5–6	5.5	2
VBml	9–15	12	10	13–14	13	10	15–16	14	10	11–18	-	-	-	-	-	-	-	-	-	-	-
Dorsal bar:
DBl	18–28	24	10	20–21	20	10	23–24	24	10	16–28	-	-	15–21	17	4	-	-	-	15	15	2
DBw	1–2	2	10	2–3	3	10	2–3	2	10	2–3	-	-	2–3	2.3	4	-	-	-	1–2	1.3	3
Marginal hooks:
MHl	5–6	5	10	5	5	10	5	5	10	5–6	5–6	3	5–6	5.6	5	5–6	5.5	2	5	5	3
MHs	1–2	1	10	1	1	10	1	1	10	-	-	-	3	3	5	3	3	2	3	3	3
MHt	3–4	4	10	4–5	4	10	4–5	4	10	-	-	-	4	4	4	4	4	2	-	-	-
MHad	-	-	-	-	-	-	-	-	-	-	-	-	2–3	2.6	5	3	3	2	3	3	3

.

Remarks: This species was described as a subspecies of G. wageneri (G. wageneri aphyae Malmberg, 1957) from skin and fins of P. phoxinus from Nämdö, Archipelago of Stockholm, Sweden [32]. It is considered an oioxenous parasite of P. phoxinus, distributed throughout the geographical range of the host in the Palaearctic [3,33]. Harris et al. [2] mentioned unpublished data about the occurrence of this species on Salmo salar L. but no further details were given. In Bulgaria, it has been recorded from Nishava River near the town of Godech [11] and the rivers Tazha, Byala, Stryama, Yantra, Kotleshnitsa and Sukhoyka [13]. The metrical characteristics of the studied specimens (Table 1) correspond well to the original description [32]. The observed minor differences are within the known limits of variation of the haptoral hard parts of G. aphyae [3,32].

In the present study, we found one specimen of G. aphyae and two specimens of G. laevis from P. phoxinus from Nishava River (mounted on the same slide—No. 3557) that were identified as G. macronychus.

Gyrodactylus cf. cernuae Malmberg, 1957

Record: Kakacheva-Avramova [15].

Host: Gymnocephalus schraetser (L.).

Locality: River—Bulgarian section of the Danube near the village of Dolno Linevo (Montana Province).

Site of infection: Gills.

Remarks: Two specimens labelled as G. cernuae were found in the collection (Coll. Nos. 5704, two slides, one specimen on each slide, sampled in 1973). However, they did not correspond to the morphology of the species because their hamuli were 50 μm long, while the hamuli of G. cernuae were described as being 56–62 μm long [32]. Ergens et al. [3] reported a wider range of the length of hamuli (51–70 μm) in this species (

Table 2. Metrical data of Gyrodactylus cernuae from G. cernua and G. cernuae sensu Kakacheva-Avramova [15] from G. schraetser.

Source	Malmberg [32]		Ergens et al. [3]	Present Study
Host	G. cernua		G. cernua	G. schraetser
Locality	Mälaren		Palaearctic	Danube (Dolno Linevo)
	Range	n	Range	Range	n
Hamuli:
TL	56–62	2	51–70	50	2
S	-	-	39–53	38–40	2
P	25–29	2	21–30	18–23	2
R	19–20	2	17–28	13–14	2
Ventral bar:
VBl	6–9	2	5–8	-	-
VBw	24–16	2	23–28	-	-
VBml	13–14	2	12–17	-	-
Dorsal bar:
DBl	22–24	2	20–24	-	-
DBw	1–2	2	1–3	-	-
Marginal hooks:
MHl	6–7	2	5–7	5–6	2
MHs	-	-	-	2	1
MHt	4	2	-	5	1
MHad	-	-	-	3	1

). The available specimens had no distinct ventral and dorsal bars (Figure 2) and, therefore, they did not allow reliable identification at the species level. This species was also reported from the Danube form Gymnocephalus cernua (L.) by Margaritov [8]. Based on the available slides, the occurrence of G. cernuae in the Bulgarian sector of the Danube can be accepted only conditionally because the quality of the mounted specimens did not allow observation of the diagnostic characters of the species.

Gyrodactylus cobitis Bychowsky, 1933

Record: Present study.

Host: Cobitis sp.

Locality: Byala Reka River (Byalgradets Village, Haskovo Province, 41.4240 N, 25.9160 E).

Site of infection: Fins.

Specimens studied: One (to be deposited, 5 July 2017).

Morphology: Hamuli medium-sized, thick, robust. Point straight. Hamulus angular at the point of connection between shaft and root. Root distal ends flat, wide. Ventral bar with oval lateral sides, mid-section with a median ridge and numerous small vertical ridges. Ventral bar membrane almost hemispherical with ridges extending from the point of connection to the ventral bar. Dorsal bar thick, relatively short. Marginal hook not observed (Figure 3). For metrical data, see

Table 3. Metrical data of Gyrodactylus cobitis from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	Cobitidae spp.	Cobitis sp.
Locality	Black Sea Basin, Ob River	Byala Reka River
	Range	Range	n
Hamuli:
TL	41–46	50	1
S	26–31	33	1
P	24–26	29	1
R	16–20	24	1
Ventral bar:
VBw	4–5	6	1
VBl	19–23	22	1
VBml	9–13	9	1
Dorsal bar:
DBl	2–6	6	1
DBw	13–17	-	-
Marginal hooks:
MHl	7–8	-	-
MHs	-	-	-
MHt	-	-	-
MHad	-	-	-

.

Remarks: This species is reported for the first time for the fauna of Bulgaria. It is a stenoxenous parasite of cobitids. Throughout its geographical range, it has been reported from C. taenia, C. melanoleuca Nichols, 1925 and Misgurnus fossilis (L.) [3]. The taxonomy of the genus Cobitis in Bulgaria is not clarified and there are several populations with uncertain taxonomic status [31].

Gyrodactylus cyprini Diarova, 1964

Record: Kakacheva-Avramova [13].

Host: Cyprinus carpio L. and Scardinius erythrophthalmus.

Locality: Reservoirs: Dolni Chiflik Reservoir (C. carpio) and Hrabrovski Reservoir (S. erythrophthalmus).

Site of infection: Gills (C. carpio) and fins (S. erythrophthalmus).

Specimens studied: Three.

From S. erythrophthalmus: Two specimens from Hrabrovski Reservoir, two slides (No. 4822, 20 April 1970—one specimen on each slide).

From C. carpio: One specimen from Dolni Chiflik Reservoir (No. 4831, one slide, 21 April 1970).

Morphology: Hamuli extra-large (Figure 4). Roots elongated, straight, ending with characteristic fist-shaped protrusions. Point straight. Dorsal bar short, thick, thinning in the middle, forming two roughly triangular halves. Ventral bar large, roughly trapezoid, edges rounded. Posterior ends of ventral bar extending into the ventral bar membrane. Ventral bar membrane not observed. Marginal hooks small. Sickle extending approximately the same length as the toe. For metrical data, see

Table 4. Metrical data of Gyrodactylus cyprini from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	C. carpio	C. carpio		S. erythrophthalmus
Locality	Palaearctic	Dolni Chiflik Reservoir		Hrabrovski Reservoir
	Range	Range	n	Range	Mean	n
Hamuli:
TL	88–140	129	1	142–147	144.5	2
S	68–84	81	1	80–90	85	2
P	34–53	54	1	56	56	2
R	56–60	55	1	69–72	71.5	2
Ventral bar:
VBl	29–38	42	1	39	-	1
VBw	8–17	12	1	13	-	1
VBml	28–44	-	1	-	-	1
Dorsal bar:
DBl	13–21	13	1	15	-	1
DBw	4–7	5	1	5	-	1
Marginal hooks:
MHl	5–7	-	-	6	6	2
MHs	-	-	-	3–4	3.5	2
MHt	-	-	-	-	-	-
MHad	-	-	-	3–4	3.5	2

.

Remarks: This species seems to be an oioxenous parasite of C. carpio, spread throughout the geographical range of its host in the Palaearctic [3]. Moravec [34] also listed Abramis brama (L.) as a host species. In Bulgaria, it was recorded from two reservoirs (Dolni Chiflik Reservoir and Hrabrovski Reservoir) and from two host species: C. carpio and S. erythrophthalmus [13]. Margaritov & Kiritsis [24] reported a single specimen of G. cyprini from Pseudorasbora parva (Temminck & Schlegel, 1846) from Chelopechene Fish Farm. According to Kakacheva-Avramova [13], the occurrence of G. cyprini on S. erythrophthalmus is accidental. She considered the small size and the slow flow of the Hrabrovski Reservoir as a reason for this accidental occurrence (i.e., more opportunities for the two host species to be in contact with one another and exchange parasites).

The morphology of the studied specimens (Figure 4) and the measurements of the haptoral hard parts (Table 4) correspond well to those of G. cyprini.

Gyrodactylus dykovae Ergens, 1991

Records: Present study.

Host: Gobio gobio (L.).

Locality: Rivers—Annadere, Palakariya and Tundzha.

Site of infection: Fins.

Specimens studied: Five, including one specimen from Annadere River near the town of Provadiya, Varna Province (No. 4779, one slide, 18 April 1970—one specimen); two specimens from the mouth of Palakariya River, Sofia City Province (No. 5991, two slides each with one specimen, 16 April 1974); and two specimens from Tundzha River near Slivenski Mineralni Bani, Sliven Province (No. 4522, two slides each with one specimen, 22 April 1969).

Morphology: Hamuli slender, medium-sized. Point long, straight. Roots with a slight inward curve. Ventral bar trapezoid, with prominent central ridge. Numerous small ridges observed along the length. Ventral bar processes not observed. Ventral bar membrane not observed. Dorsal bar thin, slightly arched. Marginal hooks with characteristic curved toe; toe pointing downwards. Marginal hook sickle curved, pointing downwards (Figure 5). For metrical data, see

Table 5. Metrical data of Gyrodactylus dykovae from Gobio gobio from various localities.

Source	Ergens [35]		Present Study
Locality	Rokytná River (Czechia)		Annadere River		Palakariya River			Tundzha River
	Range	n	Range	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	65–68	7	70	1	64–65	64.5	2	67	67	2
S	48–55	7	52	1	44–49	47.5	2	50	50	2
P	32–34	7	34	1	32	32	2	32	32	2
R	19–21	7	23	1	17	17	2	19–23	21	2
Ventral bar:
VBl	7–8	7	6	1	7	7	2	6–8	7	2
VBw	26–32	7	32	1	27–28	27.5	2	30	-	1
VBml	14–18	7	-	-	-	-	-	23	-	1
Dorsal bar:
DBl	18–22	7	-	-	15	-	1	-	-	-
DBw	2–3	7	2	1	2	-	1	-	-	-
Marginal hooks:
MHl	7	7	6	1	6	-	1	6	6	2
MHs	-	-	3	1	2	-	1	-	-	-
MHt	-	-	-	-	4	-	1	-	-	-
MHad	-	-	3	1	4	-	1	3–5	4	2

.

Remarks: This is the first report of this species for the fauna of Bulgaria. The specimens belonging to it have been reported as Gyrodactylus gobii Schulman, 1953 by Kakacheva-Avramova [12,13]. The revision of the specimens reported as G. gobii revealed that, in addition to G. gobii, some specimens belong to G. dykovae, which has been described for the same host species from Rokytná River (Czechia). The present report is the second finding of this species, which has been known from its original description only [35]. The metrical data of our specimens correspond well to those in the original description (Table 5). The observed metrical differences are in the frame of the known intraspecific variation of the species of Gyrodactylus.

Gyrodactylus fossilis Lupu & Roman, 1956

Records: Present study.

Host: Misgurnus fossilis.

Locality: Dragoman Marsh (42.9374 N, 22.9519 E).

Site of infection: Fins and skin.

Specimens studied: Three specimens from Dragoman Marsh (to be deposited, 11 May 2023, three slides, each with one specimen).

Morphology: Hamuli medium-sized. Point straight. Root elongated, thinning towards the middle, broadening in the distal ends. Ventral bar not observed. Dorsal bar long, straight. Marginal hook with long, slender shaft, ending in a thin sharp sickle (Figure 6).

Remarks: Gyrodactylus fossilis seems to be an oioxenous parasite of Misgurnus fossilis, though there is an unconfirmed record [2] from the poorly-known loach species Oxynoemacheilus lenkoranensis (Abdurakhmanov, 1962) [31] endemic in Azerbaijan. In Bulgaria, G. fossilis was reported on its original host in two sites along the Danube: near the town of Silistra [14] and at the village of Orsoya near the town of Lom [15]. Both articles reported low intensity (1–2 parasites) and the site of infection was reported as the gills. The hamuli of the specimens reported by Kakacheva-Avramova [14,15] were noticeably larger than the normal intraspecific variation of G. fossilis. Therefore, we believe that the author was dealing with a different species (for measurements, see

Table 6. Metrical data of Gyrodactylus fossilis from Misgurnus fossilis from various localities and Gyrodactylus sp. reported by Kakacheva-Avramova [14,15] as G. fossilis but representing a different (unidentified) species.

Species	Gyrodactylus fossilis								Gyrodactylus sp.
Source	Ergens [36]				Ergens et al. [3]	Present Study			Present Study
Locality	Iassy (Romania)		Dunajská Streda (Slovakia)		Black Sea Basin	Dragoman Marsh			Danube (Orsoya)		Danube (Silistra)
	Range	n	Range	n	Range	Range	Mean	n	Range	n	Range	Mean	n
Hamuli
TL	48	1	41–45	2	41–48	42–45	43.3	3	56	1	52–58	55	2
S	30	1	26–28	2	26–31	27–30	28.83	3	45	1	40–41	40.5	2
P	28	1	26–28	2	19–31	28–30	29.2	3	21	1	19–21	20	2
R	21	1	19	2	19–21	16–22	20	3	22	1	17–23	20	2
Ventral bar
VBl	18	1	16–19	2	16–21	-	-	-	-	-	-	-	-
VBw	5	1	5	2	4–6	-	-	-	-	-	-	-	-
VBml	-	-	7	2	8–12	-	-	-	-	-	-	-	-
Dorsal bar
DBl	-	1	17	2	14–17	-	-	-	-	-	-	-	-
DBw	-	1	2	2	2–3	-	-	-	-	-	-	-	-
Marginal hook
MHl	5–6	1+	5–6	2	5–8	6	6	3	5	1	6	-	1
MHs	-	-	-	-	-	2	2	2	-	-	-	-	-
MHt	-	-	-	-	-	5	5	2	-	-	-	-	-
MHad	-	-	-	-	-	3	3	2	-	-	-	-	-

). The quality of the microscopic slides made it impossible to identify the specimens from the Danube at the species level. During the course of the study, we found G. fossilis on M. fossilis from the Dragoman Marsh. It is worth noting that the fish were heavily parasitised on the fins and skin with up to 47 parasites found on a single host, which is in stark contrast to the low level of infestation on the gills reported by Kakacheva-Avramova [14,15]. The haptors of three of the specimens found in Dragoman Marsh were analysed and included in this study. The morphology (Figure 6) and the metrical data of the haptoral parts (Table 6) corresponded to the description of G. fossilis.

Gyrodactylus gobiensis Gläser, 1974

Record: Present study.

Host: Gobio gobio.

Locality: Rivers—Luda Kamchiya, Sukhoyka and Varbnishka.

Site of infection: Nasal cavity (Luda Kamchiya) and fins (Sukhoyka, Varbnishka).

Specimens studied: 8, including 1 specimen from Luda Kamchiya River (No. 4812, one slide, 20 April 1970—1 specimen); 1 specimen from Sukhoyka River at the Siniya Vir site near the town of Kotel, Sliven Province (No. 4562, one slide, 27 April 1969—1 specimen); and 6 specimens from Varbnishka River near the town of Godech, Sofia Province (No. 3521-1, one slide, 26 April 1965—6 specimens).

Morphology: Hamuli medium-sized, robust. Point long, straight. Ventral bar trapezoid, with rounded edges. Ventral bar with long median ridge and numerous small ridges. Ventral bar processes not observed. Ventral bar membrane tongue-shaped. Dorsal bar straight, thin. Marginal hook heel rounded, toe pointed, shaft long, ending in a thin sickle (Figure 7).

Remarks: This species is a stenoxenous parasite of gudgeons of the genus Gobio [2]. It was reported from Gobio gobio from the Baltic Sea and Black Sea drainages [3], Czechia [34] and the European part of Russia [37], as well as from Romanogobio kessleri (Dybowski, 1862) from Slovakia [34]. The present study is the first record of G. gobiensis from Bulgaria. Previously, the specimens of this species were misidentified as Gyrodactylus gobii [11,13]. The revision revealed that part of the specimens labelled as G. gobii exhibited the specific morphology of G. gobiensis and these are reidentified here (

Table 7. Metrical data of Gyrodactylus gobiensis from Gobio gobio from various localities.

Source	Ergens et al. [3]	Present Study
Locality	Baltic Sea and Black Sea Basins	Luda Kamchiya River		Sukhoyka River		Varbnishka River
	Range	Range	n	Range	n	Range	Mean	n
Hamuli:
TL	59–79	65	1	66	1	62–68	65.6	6
S	42–56	48	1	47	1	46–52	48.5	6
P	30–37	32	1	31	1	31–34	32.3	6
R	15–26	21	1	26	1	15–21	17.1	6
Ventral bar:
VBw	26–31	27	1	-	-	27–29	28	5
VBl	6–7	7	1	-	-	6–7	6.6	5
VBml	15–19	-	-	-	-	17	-	1
Dorsal bar:
DBl	25–35	20	1	-	-	15–20	18.5	4
DBw	2–3	2	1	2	1	2	2	4
Marginal hooks:
MHl	6–7	7	1	-	-	6	6	5
MHs	-	4	1	-	-	2–3	2.4	5
MHt	-	-	-	-	-	-	-	-
MHad	-	4	1	-	-	3–4	3.4	5

).

Gyrodactylus gobii Schulman, 1953

Records: Kakacheva-Avramova [11] (1), Kakacheva-Avramova [13] (2).

Host: Gobio gobio.

Locality: Rivers—Annadere (2), Eleshnitsa (2), Kotleshnitsa (2), Varbnishka (1) and Zlatina (2).

Site of infection: Fins.

Specimens studied: 15, including 1 specimen from Annadere River (No. 4781, 18 March 1970); 2 specimens from Eleshnitsa River (No. 4811, two slides each with 1 specimen, 20 April 1970), 9 specimens from Kotleshnitsa River (No. 4550, one slide, 27 April 1969—1 specimen; No. 4551, seven slides, each with 1 specimen, 25 April 1969); 1 specimen from Varbnishka River (No. 3521-2, one slide, 26 April 1965—1 specimen); and 2 specimens from Zlatina River (No. 4773, two slides each with 1 specimen, 17 April 1970).

Morphology: Hamuli medium-sized, stout. Root slightly curving inwards, end rounded. Point straight. Ventral bar trapezoid, with prominent processes. Ventral bar with a median ridge and numerous vertical ridges. Ventral bar membrane mostly trapezoid. Dorsal bar thick, slightly arched. Marginal hook with a rounded heel, pointed toe and arched sickle (Figure 8).

Remarks: This species has been described by Schulman (1953) as a parasite of G. gobio from several localities (

Table 8. Metrical data of Gyrodactylus gobii from Gobio gobio from various localities.

Source	Schulman [38]	Ergens et al. [3]	Kakacheva-Avramova [11]		Present Study
Locality	Neman River, Rezka Lake, Drevyata Lake	Palaearctic	Western Stara Planina		Annadere River		Eleshnitsa River			Kotleshnitsa River			Varbnishka River		Zlatina River
	Range	Range	Range	n	Range	n	Range	Mean	n	Range	Mean	n	Range	n	Range	Mean	n
Hamuli:
TL	46–61	47–60	48–72	23	60	1	56–57	56.5	2	55–60	57.2	9	55	1	55	-	1
S	36–40	34–44	34–52	23	44	1	40–41	40.5	2	42–45	43.8	9	44	1	37–41	39	2
P	16–24	22–28	21–34	23	31	1	27–28	27.5	2	26–30	28.4	9	26	1	28–29	28.5	2
R	9–12	13–19	15–18	23	19	1	15–17	16	2	13–21	15.9	9	13	1	16–18	17	2
Ventral bar:
VBl	21–24	21–27	16–28	23	27	1	23–25	24	2	24–31	27	9	26	1	25	-	1
VBw	5–6	5–7	5–6	23	6	1	7	7	2	5–7	6.3	9	5	1	6	-	1
VBml	-	11–15	-	-	-	-	14	-	1	13	-	1	-	-	-	-	-
Dorsal bar:
DBl	18–20	15–20	14–20	23	15	1	15	-	1	19–23	21.3	7	19	1	-	-	-
DBw	2	2–3	2–3	23	2	1	2	-	1	2–3	2.3	8	2	1	2–3	2.5	2
Marginal hooks:
MHl	-	5–7	4–6	23	-	-	6	6	2	6–7	6.6	9	6	1	6	6	2
MHs	-	-	-	-	-	-	2	-	1	2	2	7	-	-	2	-	1
MHt	-	-	-	-	-	-	4–5	4.5	2	4–5	4.6	7	-	-	4–5	4.5	2
MHad	-	-	-	-	-	-	3	3	2	3–4	3.4	7	-	-	3	3	2

). It was subsequently reported as a parasite of other fish species of the genus Gobio, as well as from Hemibarbus maculatus Bleeker, 1871 and Oncorhynchus mykiss (Walbaum, 1792) [2]. In Bulgaria, it was recorded from its type host G. gobio from the rivers Annadere, Eleshnitsa, Kotleshnitsa, Luda Kamchiya, Palakariya, Sukhoyka, Tundzha, Varbnishka and Zlatina [11,13]. Revising the materials in the collection of IBER-BAS revealed that some of the specimens were misidentified, especially in the case when there were coinfections with several gyrodactylid species. The wide range of measurements of the haptoral hard parts given by Kakacheva-Avramova [11] confirms the presence of a multispecies infection as the ranges of the metrical characteristics are wider than those in the original description by Schulman [38]. See Table 8 for comparative data.

Among the specimens identified as G. gobii, there are the following.

Five specimens of G. dykovae: No. 4522, two slides, two specimens, Tundzha River [13]; No. 4779, one slide, one specimen, Annadere River [13]; No. 5991, two slides, two specimens, Palakariya River [16].

Ten specimens of G. gobiensis: No. 3517, one slide, one specimen; No. 3521-1, one slide with six specimens, Varbnishka River [11]; No. 4562, one slide, one specimen, Sukhoyka River [13]; No. 4812, one slide, one specimen, Luda Kamchiya River [13].

One specimen of G. markakulensis: No. 4812, Luda Kamchiya River [13].

Gyrodactylus gracilihamatus Malmberg, 1964

Syn. Gyrodactylus wageneri scardinii Malmberg, 1957

Records: Kakacheva-Avramova [11].

Host: Squalius cephalus (L.).

Locality: Rivers—Luda Kamchiya and Shiposhnitsa.

Site of infection: Fins and gills.

Specimens studied: Two, including one specimen from Luda Kamchiya (No. 4568, one slide, 27 April 1965) and one specimen from the mouth of Shiposhnitsa River (No. 6515, one slide, 14 October 1975).

Morphology: Hamuli robust, straight, medium-sized (Figure 9). Point straight. Root straight. Ventral bar almost rectangular, with oval lateral sides. Proximal ends of ventral bar with prominent processes. Ventral bar with central ridge along its length. Ventral bar membrane not observed. Dorsal bar mostly straight, thin. Marginal hooks slender. For metrical data, see

Table 9. Metrical data of Gyrodactylus gracilihamatus from various hosts and localities.

Species	Gyrodactylus wageneri scardinii	Gyrodactylus gracilihamatus
Host	A. alburnus	Various Cyprinids	S. cephalus
Source	Malmberg [32]	Ergens et al. [3]	Present Study
Locality	Nämdö, Sweden	Basins of Black, Baltic and Caspian Seas	Luda Kamchiya River		Shiposhnitsa River
	Mean	Range	Range	n	Range	n
Hamuli:
TL	63	57–80	66	1	59	1
S	-	43–49	47	1	44	1
P	29	24–31	28	1	28	1
R	19	18–26	18	1	18	1
Ventral bar:
VBl	10	6–8	7	1	6	1
VBw	24	23–29	27	1	22	1
VBml	15	13–18	-	1	-	-
Dorsal bar:
DBl	28	17–25	17	-	-	-
DBw	2	1–2	2	1	1	1
Marginal hooks:
MHl	6	5–7	-	-	6	1
MHs	-	-	-	-	-	-
MHt	-	-	-	-	-	-
MHad	-	-	-	-	-	1

.

Remarks: This species was first described as a parasite of Alburnus alburnus [39] and recorded from several other cyprinid species such as Rutilus rutilus (L.) [1], Alburnoides bipunctatus (Bloch, 1782) and Squalius cephalus [34], Blicca bjoerkna (L.) and others [3]. In Bulgaria, it was reported with its synonymous name G. wageneri scardinii and as G. gracilihamatus from A. alburnus, R. rutilus and S. cephalus (Table S1).

Gyrodactylus katharineri Malmberg, 1964

Records: Present study.

Host: Barbus petenyi (L.), Cyprinus carpio, Vimba melanops (Heckel, 1837).

Locality: Blagoevgrad Fish Farm; rivers Tundzha and Varbnishka.

Site of infection: Fins.

Specimens studied: Five.

From B. petenyi—One specimen from Varbnishka River (No. 3507, one slide, 25 April 1965).

From V. melanops—One specimen from Tundzha River (No. 4519, one slide, 22 April 1969).

From C. carpio—Three specimens, including one from Blagoevgrad Fish Farm (No. 7003, one slide, 14 March 1984) and two from Varbnishka River (No. 3502, two slides each with one specimen, 25 April 1965).

Morphology: Hamuli large to extra-large, robust. Point straight. Root mostly straight, slightly curving inwards. Ventral bar with median ridge and characteristic elongated loop-shaped processes. Dorsal bar wide, slightly arched in the middle. Marginal hooks large, thick, with the characteristic shape for the species. Toe pointed, shaft thick, sickle compact, slightly extending over toe (Figure 10). For metrical data, see

Table 10. Metrical data of Gyrodactylus katharineri from various hosts and localities.

Source	Malmberg [1]			Ergens et al. [3]	Present Study
Host	C. carpio			Cyprinids	B. petenyi		V. melanops		C. carpio		C. carpio
Locality	Bokelholm Fish-Culture Farm, West Germany			“Europe and Asia”	Varbnishka River		Tundzha River		Blagoevgrad Fish Farm		Varbnishka River
	Range	Mean	n	Range	Range	n	Range	n	Range	n	Range	Mean	n
Hamuli:
TL	87–97	91.7	10	71–112	89	1	101	1	96	1	100–103	101.3	2
S	62–71	65.8	10	51–84	65	1	72	1	71	1	73–75	74	2
P	39–43	40.9	10	27–52	43	1	44	1	43	1	45–51	48.3	2
R	26–31	29	10	16–35	-	-	29	1	25	1	30–31	30.3	2
Ventral bar:
VBw	39–41	40.6	10	33–50	45	1	46	1	47	1	42–44	43	2
VBl	8–13	10.4	10	8–14	10	1	11	1	10	1	12–15	13.5	2
VBml	24–28	25.8	10	18–40	-	-	-	-	-	-	-	-	-
Dorsal bar:
DBl	34–50	42.7	10	20–48	21	1	31	1	33	1	29–32	30.5	2
DBw	2–4	3.2	10	2–5	4	1	5	1	4	1	3–5	4	2
Marginal hooks:
MHl	8–9	8.7	10	8–10	8	1	9	1	10	1	9	9	2
MHs	-	-	-	-	4	1	4	1	4	1	4	4	2
MHt	-	-	-	-	-	-	-	-	7	1	6	6	2
MHad	-	-	-	-	4	1	5	1	5	1	4–5	4.5	2

.

Remarks: This species was described from C. carpio as the “main host” and other cyprinids were listed as “temporary hosts” [1]. Ergens [7] pointed out that G. katharineri was a parasite on a variety of fish species, among which the common carp, crucian carp, Prussian carp, common gudgeon, common bleak and common rudd. However, he listed C. carpio, Carassius carassius (L.) and C. gibelio (Bloch, 1782) as the main hosts. In Bulgaria, this species was published as Gyrodactylus sp. from B. petenyi in the rivers Buchinska, Nishava, Varbnishka [11], Palakariya [16] and Blagoevgradska Bistritsa [18], as well as from Barbus barbus (L.) from the rivers Blagoevgradska Bistritsa, Struma and Zheleznitsa [18]. It was also recorded by Margaritov & Kiritsis [24] from Pseudorasbora parva at Chelopechene Fish Farm near Sofia. According to Kakacheva-Avramova [8], Margaritov [22] found G. katharineri on C. carpio in the fish farms at Belene, Blagoevgrad, Chelopechene, Plovdiv and Yambol and misidentified it as G. elegans. The morphological and metrical data of the specimens in the collection of IBER-BAS correspond to those of G. katharineri.

Gyrodactylus laevis Malmberg, 1957

Records: Present study.

Host: Phoxinus phoxinus.

Locality: Nishava River.

Site of infection: Gills.

Specimens studied: Three from Nishava River (No. 3557, two slides, 28 April 1965—three specimens, one slide with one specimen and one slide with two specimens).

Morphology: Hamuli small to medium-sized. Roots thick, slightly bending outwards. Shaft and point slender. Ventral bar almost rectangular. Ventral bar membrane not observed. Dorsal bar thin, short. Marginal hook with distinct heel and toe; sickle slender (Figure 11).

Remarks: This species seems euryxenous, reported from various hosts [2]. According to Ergens et al. [3], it is a specific parasite of P. phoxinus and it is unclear whether the reports of this parasite from other fish species are G. laevis or another species with similar morphology. In Bulgaria, it has been reported on a variety of hosts (Table S1). Reviewing the available slides in the collection of IBER-BAS revealed that none of the specimens identified as G. laevis belonged to this species. Gyrodactylus laevis was present on two slides, one identified as G. aphyae (one of the slides with No. 3555) and one identified as G. macronychus (No. 3557). The measurements of the haptoral hard parts and the overall morphology of the haptor of these two specimens corresponded well with G. laevis (

Table 11. Metrical data of Gyrodactylus laevis from Phoxinus phoxinus from various localities.

Source	Malmberg [32]		Ergens et al. [3]	Present Study
Locality	Fiolenbäcken, Aneboda (Sweden)		Palaearctic	Nishava River
	Range	n	Range	Range	Mean	n
Hamuli:
TL	36	1	33–43	37–44	39.8	3
S	-	1	27–34	27–35	31.5	3
P	18	1	15–18	12–15	13.8	3
R	-	1	10–16	11–12	11.8	3
Ventral bar:
VBw	-	1	4–6	4	-	1
VBl	-	1	9–15	-	-	-
VBml	-	1	9–16	-	-	-
Dorsal bar:
DBl	-	1	7–9	-	-	-
DBw	-	1	1	-	-	-
Marginal hooks:
MHl	6	1	5–6	5–6	5.3	3
MHs	-	-	-	2–3	2.7	3
MHt	-	-	-	-	-	-
MHad	-	-	-	2–4	2.5	3

).

Gyrodactylus latus Bychowsky, 1933

Records: Present study.

Host: Cobitis sp.

Locality: Byala Reka River (near the village of Byalgradets, 41.4240 N, 25.9160 E).

Site of infection: Skin.

Specimens studied: Three (to be deposited, 5 July 2017).

Morphology: Hamuli medium-sized, robust. Root short. Ventral bar small, trapezium-shaped. Ventral bar membrane with multiple ridges. Dorsal bar thick. Marginal hooks with small blade and large basal width. (Figure 12). For metrical data, see

Table 12. Metrical data of Gyrodactylus latus from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	C. taenia	Cobitis sp.
Locality	Volga, Danube, Oder, Elbe River Estuaries	Byala Reka River
	Range	Range	Mean	n
Hamuli:
TL	50–57	47–55	50	3
S	40–44	38–43	39.7	3
P	22–28	25–30	26.3	3
R	15–19	16–18	16.8	3
Ventral bar:
VBl	6–7	6–7	6.5	2
VBw	13–19	13–14	13.5	2
VBml	12–14	10–15	12.5	2
Dorsal bar:
DBl	9–14	10–12	11	2
DBw	1–2	1–2	1.7	3
Marginal hooks:
MHl	7	6–7	6.3	3
MHs	-	1	1	3
MHt	-	5	5	3
MHad	-	2–3	2.3	3

.

Remarks: In the course of the present study, this species was recorded for the first time for the fauna of Bulgaria. The two specimens that were published as G. latus (No. 3488, two slides) by Kakacheva-Avramova [11] corresponded to the morphology of G. papernai Ergens & Bychowsky, 1967, a specific parasite of Barbatula barbatula (L.). Their identity is discussed in the section for G. papernai.

Gyrodactylus leucisci Žitňan, 1964

Records: Kakacheva-Avramova [11] (1), present study (2).

Host: Squalius cephalus.

Locality: Rivers—Kotleshnitsa (2), Nishava (1), Shiposhnitsa (2) and Varbnishka (1); Zhrebchevo Reservoir (2).

Site of infection: Fins.

Specimens studied: Nine, including three from Kotleshnitsa River (No. 4542, two slides, each with one specimen, 25 April 1969); one from Nishava River near the town of Godech (No. 3451, one slide, 20 April 1965—one specimen); one from Shiposhnitsa River (No. 6520, one slide, 14 October 1975—one specimen); three from Varbnishka River near the site of Murgashko Poishte (No. 3525, two slides, each with one specimen 24 April 1965; No. 3540, one slide, 25.04.1965—one specimen); and one from Zhrebchevo Reservoir (No. 13685, one slide, 16 May 1995—one specimen).

Morphology: Hamuli robust, medium-sized to large. Point long, straight. Roots mostly straight, slightly curving inwards. Ventral bar slightly trapezoid, with many small median ridges. Ventral bar processes prominent. Ventral bar membrane tongue-shaped. Dorsal bar thick, arched. Marginal hook heel angular; toe pointed, sickle extending over toe (Figure 13). For metrical data, see Table 13.

Remarks: This species is reported mostly as a parasite of S. cephalus but there are several reports from two other host species: Rutilus rutilus and Leuciscus leuciscus (L.) [2]. It was recorded from S. cephalus from Czechia and Slovakia [34], the former USSR [40] and Bulgaria [11,13]. The specimens from Kotleshnitsa River were published as Gyrodactylus scardinii [13] and the specimen from Shiposhnitsa River was published as G. gracilihamatus [19]. The specimen from Zhrebchevo Reservoir was not published.

Table 13. Metrical data of Gyrodactylus leucisci from Squalius cephalus from various localities.

Source	Žitňan [41]	Ergens et al. [3]	Kakacheva-Avramova [11]		Present Study
Locality	Hron River (Slovakia)	Palaearctic	Western Stara Planina		Kotleshnitsa River			Nishava River		Shiposhnitsa River		Varbnishka River			Zhrebchevo Reservoir
	Range	Range	Range	n	Range	Mean	n	Range	n	Range	n	Range	Mean	n	Range	n
Hamuli:
TL	75	63–73	62–70	6	67–70	68.6	3	68	1	64	1	63–69	66	3	66	1
S	58	44–51	46–50	6	48–50	49.6	3	45	1	46	1	42–46	44.6	3	43	1
P	36	31–33	30–32	6	32–35	36	3	33	1	31	1	30–33	32	3	29	1
R	25	21–24	-	6	20–25	22	3	21	1	20	1	15–23	19.2	3	22	1
Ventral bar:
VBw	10	7–8	6	6	9	-	1	6	1	7	1	6–8	7	2	8	1
VBl	30	28–32	26–28	6	30	-	1	29	1	27	1	30	30	2	-	-
VBml	10	17–19	-	-	-	-	-	-	-	16	1	-	-	-	-	-
Dorsal bar:
DBl	15	19–21	24	6	4	-	1	20	1	15	1	17–20	18.5	2	-	-
DBw	4	2–3	3	6	20	-	1	2	1	3	1	2–3	2.5	2	5	1
Marginal hooks:
MHl	6.0–6.5	7	6–7	6	5–7	6	2	7	1	6	1	6	6	3	7	1
MHs	-	-	-	-	2	-	1	2	1	2	1	3–4	3.3	3	2	1
MHt	-	-	-	-	4	-	1	4	1	4	1	-	-	-	4	1
MHad	-	-	-	-	4	-	1	3	1	3	1	3	3	3	4	1

Table 13. Metrical data of Gyrodactylus leucisci from Squalius cephalus from various localities.

Source	Žitňan [41]	Ergens et al. [3]	Kakacheva-Avramova [11]		Present Study
Locality	Hron River (Slovakia)	Palaearctic	Western Stara Planina		Kotleshnitsa River			Nishava River		Shiposhnitsa River		Varbnishka River			Zhrebchevo Reservoir
	Range	Range	Range	n	Range	Mean	n	Range	n	Range	n	Range	Mean	n	Range	n
Hamuli:
TL	75	63–73	62–70	6	67–70	68.6	3	68	1	64	1	63–69	66	3	66	1
S	58	44–51	46–50	6	48–50	49.6	3	45	1	46	1	42–46	44.6	3	43	1
P	36	31–33	30–32	6	32–35	36	3	33	1	31	1	30–33	32	3	29	1
R	25	21–24	-	6	20–25	22	3	21	1	20	1	15–23	19.2	3	22	1
Ventral bar:
VBw	10	7–8	6	6	9	-	1	6	1	7	1	6–8	7	2	8	1
VBl	30	28–32	26–28	6	30	-	1	29	1	27	1	30	30	2	-	-
VBml	10	17–19	-	-	-	-	-	-	-	16	1	-	-	-	-	-
Dorsal bar:
DBl	15	19–21	24	6	4	-	1	20	1	15	1	17–20	18.5	2	-	-
DBw	4	2–3	3	6	20	-	1	2	1	3	1	2–3	2.5	2	5	1
Marginal hooks:
MHl	6.0–6.5	7	6–7	6	5–7	6	2	7	1	6	1	6	6	3	7	1
MHs	-	-	-	-	2	-	1	2	1	2	1	3–4	3.3	3	2	1
MHt	-	-	-	-	4	-	1	4	1	4	1	-	-	-	4	1
MHad	-	-	-	-	4	-	1	3	1	3	1	3	3	3	4	1

Gyrodactylus luciopercae Gussev, 1962

Records: Kakacheva-Avramova [14] (1), Kakacheva-Avramova [15] (2), present study (3).

Host: Sander lucioperca (L.) (1, 2) and Gymnocephalus cernua (3).

Locality: Rivers—Bulgarian section of the Danube near the villages of Dolni Tsibar (Montana Province) (2), Dunavets (Silistra Province) (3) and near the town of Svishtov (1, 2).

Site of infection: Fins (1) and gills (1, 3).

Specimens studied: 32.

From S. lucioperca—29 specimens, including 23 from near the town of Svishtov (No. 5617, 23 slides, 14 August 1972—23 specimens) and 6 from near Dolni Tsibar (No. 5947, 3 slides, 22 November 1973—4 specimens: 1, 1 and 2 specimens; No. 5948, 1 slide, 22 November 1973—1 specimen; No. 5949, 1 slide, 22 November 1973—1 specimen).

From G. cernua: Three specimens from near Dunavets Village (No. 5397, three slides, 16 October 1971).

Morphology: Hamuli large, robust. Shaft long, point straight, root thinner than shaft, slightly bending inwards. Ventral bar trapezoid, proximal ends triangular. Ventral bar with median horizontal ridge and numerous small vertical ridges. Ventral bar membrane tongue-shaped. Dorsal bar long, thin and slightly bent. Marginal hooks with pointed toe, rounded heel and long sickle (Figure 14). For comparative measurements, see

Table 14. Metrical data of Gyrodactylus luciopercae from various hosts and localities.

Source	Ergens et al. [3]	Present Study
		Gyrodactylus luciopercae						Gyrodactylus longiradix sensu Kakacheva-Avramova [14]
Host	G. cernua, P. fluviatilis, S. lucioperca	S. lucioperca						G. cernua
Locality	Basins of Caspian, Black and Baltic Seas	Danube (Svishtov)			Danube (Dolni Tsibar)			Danube (Dunavets)
	Range	Range	Mean	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	64–80	60–70	66.5	23	65–79	73.3	6	60–67	64.2	3
S	45–51	45–50	47.7	23	45–57	53.8	6	47–51	48.6	3
P	27–31	24–31	28.4	23	28–36	32.4	6	25–29	26.5	3
R	22–28	17–26	22.3	20	20–31	25	6	20–24	22.5	3
Ventral bar:
VBl	5–8	6–8	6.3	21	7	7	4	5–7	5.7	3
VBw	26–33	24–32	27.5	17	24–25	24.5	2	23–24	23.5	2
VBml	14–19	13–16	14.8	9	15	-	1	-	-	-
Dorsal bar:
DBl	21–29	16–26	22.2	12	-	-	-	2	2	2
DBw	1–3	1–2	1.8	18	1–3	2.1	5	22–25	23.5	2
Marginal hooks:
MHl	6–7	6–7	6.7	20	6–7	6.8	6	6–7	6.5	2
MHs	-	2–3	2.2	17	2–3	2.4	5	2	2	2
MHt	-	4–6	4.7	15	3–5	4.6	5	5	5	2
MHad	-	3–5	3.9	17	3–4	3.8	5	3–4	3.5	2

.

Remarks: This species is a stenoxenous parasite of fishes of the family Percidae in the Palaearctic [2]. It was recorded throughout Europe mostly from S. lucioperca but also from G. cernua and Perca fluviatilis L. [3]. There is a record from the alien species Percottus glenii Dybowsky, 1877 in Germany [42]. In Bulgaria, it was recorded from S. lucioperca at two sites along the Danube.

During the revision, we also found that three specimens from G. cernua published as Gyrodactylus longiradix, Coll. No. 5397 [14,15], belonged to G. luciopercae.

Gyrodactylus macrocornis Ergens, 1963

Syn. Gyrodactylus chondrostomatis Žitňan, 1964

Record: Kakacheva-Avramova & Menkova [18].

Host: Chondrostoma nasus (L.).

Locality: River—Zheleznitsa.

Site of infection: Fins.

Specimens studied: 12 (No. 7057, eight slides, undated: six slides with 1 specimen each; two slides with 3 specimens each).

Morphology: Hamuli large, robust. Point straight. Root mostly straight. Ventral bar trapezoid, with pointed proximal ends. Distal end of ventral bar uneven, forming a jagged border with the ventral bar membrane. Ventral bar membrane not observed. Dorsal bar thin, arched. Marginal hook heel thick, rounded. Toe sharp, pointing downwards (Figure 15).

Remarks: This species is an oioxenous parasite of C. nasus [2]. In Bulgaria, it was reported by Kakacheva-Avramova & Menkova [18] from a single location—Zheleznitsa River (West-Aegean Sea Basin)—with the synonymous name Gyrodactylus chondrostomatis. Re-examining the slides and comparing the metric data show that the available collection specimens belong to G. macrocornis (

Table 15. Metrical data of Gyrodactylus macrocornis from Chondrostoma nasus from various localities.

Source	Ergens et al. [3]	Kakacheva-Avramova & Menkova [18]		Present Study
Locality	Danube River, Oder River	Zheleznitsa River		Zheleznitsa River
	Range	Range	n	Range	Mean	n
Hamuli:
TL	74–85	72	1	71–78	75.3	12
S	54–60	54	1	52–59	54.7	12
P	30–48	33	1	35–39	36.3	12
R	23–29	24	1	18–26	23.2	12
Ventral bar:
VBl	7–11	7	1	8–11	9.3	12
VBw	29–36	28	1	31–39	31.3	12
VBml	18–25	-	-	13–20	16.5	2
Dorsal bar:
DBl	20–28	21	1	15–26	20.9	8
DBw	2–4	2	1	3–6	4.2	12
Marginal hooks:
MHl	5–7	6	1	5–6	5.9	9
MHs	-	-	-	2	2	8
MHt	-	-	-	3–6	4.6	7
MHad	-	-	-	3–4	3.2	6

).

Gyrodactylus macronychus Malmberg, 1957

Records: Kakacheva-Avramova [11].

Host: Phoxinus phoxinus.

Locality: River—Nishava.

Site of infection: Fins and gills.

Specimens studied: One (No. 3778, one slide, 27 October 1965).

Morphology: Hamuli large, long and slender. Point long and straight. Root long and straight. Ventral bar and dorsal bar not observed. Marginal hooks with angular toe. Marginal hooks with long crescent blade (Figure 16). For metrical data, see

Table 16. Metrical data of Gyrodactylus macronychus from various hosts and localities.

Source	Malmberg [32]						Ergens et al. [3]	Kakacheva- Avramova [11]		Present Study
Host	P. phoxinus						P. phoxinus, R. percnura, R. rutilus	P. phoxinus
Locality	Nämdö (Sweden)			Svartbäken, Jämtland (Sweden)			Palaearctic	Nishava River		Nishava River
	Range	Mean	n	Range	Mean	n	Range	Range	n	Range	n
Hamuli:
TL	68–75	71	6	71–78	75	6	68–96	74–84	7	80	1
S	-	-	-	-	-	-	40–62	54–66	7	56	1
P	29–36	33	6	35–36	35	6	26–40	32–40	7	35	1
R	23–26	24	6	20–24	23	6	22–33	-	-	27	1
Ventral bar:
VBl	6–12	10	6	11–15	12	6	6–12	12	7	-	-
VBw	27–33	30	6	34–40	36	6	26–45	42	7	-	-
VBml	12–20	16	6	16–20	18	6	13–25	-	-	-	-
Dorsal bar:
DBl	27–28	28	6	29–43	30	6	22–33	-	-	-	-
DBw	1	1	6	1–2	2	6	2–5	-	-	-	-
Marginal hooks:
MHl	8–10	9	6	9–10	10	6	8–12	9–12	7	10	1
MHs	-	-	-	-	-	-	-	-	-	8	1
MHt	-	-	-	-	-	-	-	-	-	-	-
MHad	-	-	-	-	-	-	-	-	-	5	1

.

Remarks: This species was described based on specimens from P. phoxinus from Sweden [32]. It seems to be an oioxenous parasite of P. phoxinus, widespread in the geographic range of its host [3]. There are also data about G. macronychus parasitising Rhynchocypris percnura (Pallas, 1814) and Rutilus rutilus [3], as well as unverified data about its occurrence on Salmo trutta L. (cited by Harris et al. [2]). In Bulgaria, G. macronychus was recorded from Nishava River [11] and Sukhoyka River [13]. The specimens from Sukhoyka River were not available in the collection.

Gyrodactylus malmbergi Ergens, 1961

Records: Present study.

Host: Barbus petenyi and B. cyclolepis Heckel, 1837.

Locality: Rivers, from B. cyclolepis—Glazne (at Banya Village, 41.8876 N, 23.5202 E); and from B. petenyi—Malki Iskar (near the town of Roman, 43.1357 N, 23.9261 E).

Site of infection: Fins and skin.

Specimens studied: Six (to be deposited, two from Glazne River, 10 September 2020, four from Malki Iskar River, 23 October 2020).

Morphology: Hamuli medium-sized, robust. Point straight. Roots characteristically bent inwards, with irregular shape with numerous ridges. Ventral bar with loop-like protrusions, median ridge and numerous small vertical ridges. Ventral bar with jagged lower half, forming into a tongue-shaped membrane. Dorsal bar straight. Marginal hook blade slightly bent forward; toe curved and pointed (Figure 17).

Remarks: The specimens from Barbus barbus from the rivers Struma and Gradevska (SW Bulgaria) [18] as well as those from B. petenyi from Palakariya River (Southwestern Bulgaria) and the rivers Sarneshka and Shirokolashka, Rhodope Mts. [16,17], were not available in the collection of IBER-BAS. In the course of the present study, G. malmbergi was found on B. petenyi from Malki Iskar River and on B. cyclolepis from Glazne River. The comparison with the metrical data [3] showed similarity (

Table 17. Metrical data of Gyrodactylus malmbergi from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	B. barbus, B. petenyi	B. petenyi
Locality	Slovakia, Hungary, Bulgaria	Malak Iskar River			Glazne River
	Range	Range	Mean	n	Range	Mean	n
Hamuli:
TL	-	-	-	-	-	-
S	38–43	39–40	39.8	4	35–37	35.8	2
P	27–29	22–25	23.4	4	20–24	21.8	2
R	-	-	-	-	-	-	-
Ventral bar:
VBw	45–46	30–33	31.3	4	28–31	29.5	2
VBl	7–9	5–7	5.88	4	5–7	6	2
VBml	14–15	-	-	-	14	14	2
Dorsal bar:
DBl	26–33	25–29	26.5	4	22–30	26	2
DBw	2–3	2–3	2.75	4	2–4	3	2
Marginal hooks:
MHl	6–8	6	6	3	6–7	6.5	2
MHs	-	3	3	2	3–4	3.5	2
MHt	-	4	4	2	5	-	1
MHad	-	4	4	2	3–4	3.5	2

), with the exception of the width of the ventral bar, which might be due to the different method of measurement.

Gyrodactylus markakulensis Gvosdev, 1950

Records: Kakacheva-Avramova [13] (1), present study (2).

Host: Gobio gobio (1, 2).

Locality: Rivers—Eleshnitsa (1), Kotleshnitsa (1) and Luda Kamchiya (2).

Site of infection: Gills.

Specimens studied: Three, including one from Luda Kamchiya River (No. 4812, one slide, 20 April 1970); one from Eleshnitsa River (No. 4810, one slide, 20 April 1970); and one from Kotleshnitsa River near the town of Kotel (No. 4547, one slide, 25 April 1969).

Morphology: Hamuli large, robust, widening between the root and the shaft connecting point. Point straight. Ventral bar mostly rectangular, with no distinct processes. Ventral bar membrane not observed. Marginal hook with characteristic thick flat heel, toe pointing downwards, shaft thick, ending with a small delicate sickle (Figure 18).

Remarks: This species seems to be a specific parasite on gudgeons of the genera Gobio and Romanogobio [2]. According to Ergens et al. [3], it is widespread throughout the Palaearctic. In Bulgaria, it was reported from G. gobio from rivers in Stara Planina [11,13]. During the present study, one of the specimens (No. 4812) from Luda Kamchiya River identified as G. gobii by Kakacheva-Avramova [13] was re-identified as G. markakulensis (

Table 18. Metrical data of Gyrodactylus markakulensis from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Locality	Palaearctic	Luda Kamchiya River		Kotleshnitsa River		Eleshnitsa River
	Range	Range	n	Range	n	Range	n
Hamuli:
TL	46–65	61	1	62	1	67	1
S	37–49	45	1	44	1	46	1
P	22–30	30	1	30	1	31	1
R	12–22	25	1	22	1	21	1
Ventral bar:
VBl	7–11	-	-	9	1	-	-
VBw	13–17	-	-	18	1	-	-
VBml	10–20	-	-	-	-	-	-
Dorsal bar:
DBl	2–4	-	-	-	-	-	-
DBw	6–11	3	1	-	-	-	-
Marginal hooks:
MHl	6–9	6	1	6	1	-	-
MHs	-	-	-	4	1	-	-
MHt	-	-	-	-	-	-	-
MHad	-	4	1	2	1	-	-

).

Gyrodactylus medius Kathariner, 1894

Records: Present study.

Host: Cyprinus carpio.

Locality: Zhrebchevo Reservoir.

Site of infection: Fins.

Specimens studied: One (No. 13591, one slide, 10 June 1994).

Morphology: Hamuli medium-sized. Point slightly curved inwards. Ventral bar trapezoid, with long median ridge. Ventral bar membrane not distinct. Dorsal bar mostly straight. Marginal hooks small; toe sharp, heel rounded (Figure 19). For metrical data, see Table 19.

Remarks: Gyrodactylus medius was described as a parasite of Cyprinus carpio and Cobitis taenia [43]. The validity of this taxon has been a subject of controversy, not only because the original description is vague and lacks metrical data of the haptoral hard parts, but because it obviously combined characteristics of two species [44]. This has caused researchers to report G. medius from various cyprinids [2,44]. Ergens [45] provides an amended redescription (including measurements) of the haptoral hard parts based on the analysis of numerous specimens from the former Czechoslovakia. Currently, G. medius is treated as a specific parasite of C. carpio and records of this parasite on other fish species should be regarded as accidental infections [3]. For the purposes of the present study, we used the morphological data of G. medius provided by Ergens [45].

Table 19. Metrical data of Gyrodactylus medius from Cyprinus carpio from various localities.

Source	Ergens [45]	Ergens et al. [3]	Present Study
Locality	Jindřichův Hradec (Czechia)	Palaearctic	Zhrebchevo Reservoir
	Range	Range	Range	n
Hamuli:
TL	49–50	45–51	49	1
S	38–39	34–39	38	1
P	21–22	18–20	23	1
R	17–18	12–19	16	1
Ventral bar:
VBl	4–6	5–7	5	1
VBw	18–20	18–21	21	1
VBml	13–14	12–13	-	-
Dorsal bar:
DBl	14–15	12–18	-	-
DBw	1	1–2	-	-
Marginal hooks:
MHl	6	4	5	1
MHs	-	-	-	-
MHt	-	-	-	-
MHad	-	-	-	-

Table 19. Metrical data of Gyrodactylus medius from Cyprinus carpio from various localities.

Source	Ergens [45]	Ergens et al. [3]	Present Study
Locality	Jindřichův Hradec (Czechia)	Palaearctic	Zhrebchevo Reservoir
	Range	Range	Range	n
Hamuli:
TL	49–50	45–51	49	1
S	38–39	34–39	38	1
P	21–22	18–20	23	1
R	17–18	12–19	16	1
Ventral bar:
VBl	4–6	5–7	5	1
VBw	18–20	18–21	21	1
VBml	13–14	12–13	-	-
Dorsal bar:
DBl	14–15	12–18	-	-
DBw	1	1–2	-	-
Marginal hooks:
MHl	6	4	5	1
MHs	-	-	-	-
MHt	-	-	-	-
MHad	-	-	-	-

Gyrodactylus melas Ondračková, Seifertová & Leis, 2020

Record: Vancheva et al. [28].

Host: Ameiurus melas (Rafinesque, 1820).

Locality: Lake Srebarna Reserve (44.1035 N, 27.0667 E).

Site of infection: Skin, fins and eye surface.

Specimens studied: 15 (1 of which deposited in the collection of IBER-BAS No. M0163.1.2).

Remarks: This species was described as an oioxenous parasite of A. melas in both its native range in the Nearctic and its invasive range in the Palaearctic [46]. Prior to the description, specimens from Bulgaria have been reported as Gyrodactylus nebulosus Kritsky & Mizelle, 1968 [28]. However, additional molecular and morphological data demonstrated that it is a closely related but distinct species, differing from G. nebulosus [46]. The metrical and morphological data of the specimens from Lake Srebarna are published elsewhere [28].

Gyrodactylus papernai Ergens & Bychowsky, 1967

Records: Present study.

Host: “Cobitis taenia (L.)” (Cobitis sp., probably C. elongata Heckel & Kner, 1858 or C. elongatoides Băcescu & R. F. Mayer, 1969, see Froese & Pauly [31]).

Locality: Buchinska River.

Site of infection: Fins.

Specimens studied: Two from Buchinska River at the site Tsafti Tran near the town of Godech (No. 3488, two slides, one specimen on each slide, 23 April 1965).

Morphology: Hamuli medium-sized, slender. Point long and straight. Roots bent, pointing inwards. Ventral bar slightly trapezoid, with median ridge. Ventral bar processes not distinct. Dorsal bar long, thin, straight. Marginal hook with long crooked thin blade; heel rounded, toe pointed (Figure 20). For metrical data, see

Table 20. Metrical data of Gyrodactylus papernai from various hosts and localities.

Source	Ergens & Bychowsky [47]		Přikrylová et al. [48]			Ergens et al. [3]	Present Study
Host	B. barbatula					B. barbatula, B. toni	“C. taenia”
Locality	Pond “Hubenov”, Písek (Czechia)		Czechia			Palaearctic	Buchinska River
	Holotype	Range	Range	Mean	n	Range	Range	Mean	n
Hamuli:
TL	60	52–60	-	-	-	-	-	-	-
S	42	36–42	35–45.5	39.7	158	33–44	40–41	40.5	2
P	32	29–32	25–32	28.8	158	24–32	29–30	29.5	2
R	-	-	-	-	158	-	-	-	-
Ventral bar:
VBw	30	25–30	22.5–29.5	26.2	158	22–31	26	-	1
VBl	9	6–9	5.5–10	7.8	158	5–11	7	-	1
VBml	19	16–19	11–20.5	16.7	158	10–19	-	-	-
Dorsal bar:
DBl	29	23–29	21–30	26.6	158	19–29	30	-	1
DBw	3	2–3	1.5–3	2.2	158	1–3	2	-	1
Marginal hooks:
MHl	8	6–8	5–7.5	6.4	158	5–8	6	6	2
MHs	-	-	-	-	158	-	4–5	4.5	2
MHt	-	-	-	-	158	-	5	5	2
MHad	-	-	-	-	158	-	6–7	6.5	2

.

Remarks: This species is an oioxenous parasite of Barbatula barbatula in Europe [47]. While it had been synonymised with Gyrodactylus jiroveci Ergens & Bychowsky, 1967 [7], its status as a valid species was reinstated on the basis of morphological and molecular evidence [48]. According to Ergens et al. [3], it was also found on Barbatula toni (Dybowski, 1869). In Bulgaria, it was published from Buchinska River at the site Tsafti Tran near the town of Godech as G. latus Bychowsky, 1933 [11]. The revision of the collection specimens revealed the presence of G. papernai on both slides. The occurrence of this parasite on “C. taenia” is most likely accidental in view of the low intensity (2 specimens) and prevalence (on 1 out of 27 examined fish specimens [11]). The type host of G. papernai—B. barbatula—was examined from the same sampling site as fish specimens identified as C. taenia [11] but no G. papernai was found. For the identification of G. papernai, we compared the metrical data of our specimens with the original description [47] as well as with the data provided by Přikrylová et al. [48] (Table 20). The morphology of our specimens corresponds well to that of G. papernai and the measurements of the haptoral hard parts are well within the range of variation of this species. The hamuli of our specimens are more robust and the haptoral parts are larger compared to those of G. jiroveci. The marginal hook sickle of both specimens is slenderer but the toe is more bent (Figure 20) compared to that of G. jiroveci.

Gyrodactylus perccotti Ergens & Yukhimenko, 1973

Records: Vancheva & Georgiev [29].

Host: Perccottus glenii.

Locality: Lake Srebarna Reserve (44.1035 N, 27.0667 E).

Site of infection: Fins.

Specimens studied: Two (one of which deposited in the collection of IBER-BAS, No. BG-IBER-INV-000043644).

Remarks: This oioxenous parasite on P. glenii was described from Amur River [49]. Since its host P. glenii has an extensive invasive range in Europe [50], G. perccotti has been reported from Poland [51], Russia [52], Hungary [53] and Ukraine [54]. In Bulgaria, G. perccotti was found on P. glenii from Lake Srebarna, Silistra Province. For description and molecular data, see Vancheva & Georgiev [29].

Gyrodactylus prostae Ergens, 1963

Records: Kakacheva-Avramova [11] (1), Nedeva-Menkova [19] (2), Kakacheva-Avramova & Nedeva-Menkova [16] (3), present study (4).

Host: Chondrostoma nasus (4), Rhodeus amarus (Bloch, 1782) (4), Rutilus rutilus (2, 3) and Squalius cephalus (1, 2, 4).

Localities: Rivers—Leva (4), Lom (1), Ogosta (1, 4), Palakariya (3, 4), Shiposhnitsa (2) and Zheleznitsa (4). Reservoirs Pchelina (4) and Zhrebchevo (4).

Site of infection: Fins (for all host species) and gills (for S. cephalus).

Specimens studied: 35.

From S. cephalus—19 specimens, including 2 from Pchelina Reservoir (No. 9243, one slide, 14.05.1985—1 specimen; No. 10300, one slide, 16.05.1985—1 specimen); 5 from Zhrebchevo Reservoir (No. 13685, four slides, 16.05.1995—three slides each with 1 specimen and one with 2 specimens); 2 from Lom River (No. 3374, one slide, 14.10.1964—1 specimen; No. 3376, one slide, 14.10.1964—1 specimen); 8 from Ogosta River (No. 3014, three slides, 17.04.1964—3 specimens; No. 3015, one slide, 17.04.1964—1 specimen; No. 3030, four slides, 17.04.1964—4 specimens); 1 from Shiposhnitsa River (No. 6524, one slide, 14.10.1975); and 1 from Zheleznitsa River (No. 7059, one slide, undated).

From R. amarus—Four specimens, including one from Leva River (No. 3101, one slide, 23.04.1964) and three from Ogosta River near the mouth of Barziya River (No. 3020, three slides, each with one specimen 17.04.1964).

From R. rutilus—10 specimens, including 4 from Palakariya River (No. 5995, one slide, 16.04.1974—1 specimen; No. 6207, one slide, 07.05.1975—1 specimen; No. 6208, two slides each with 1 specimen, 07.05.1975) and 6 from Shiposhnitsa River (No. 6218, one slide, 07.05.1975—1 specimen; No. 6235 one slide, 07.05.1975—1 specimen; No. 6239, one slide, 11.05.1975—1 specimen; No. 6240, two slides, each with 1 specimen, 11.05.1975; No. 6241, one slide, 11.05.1975—1 specimen).

From C. nasus—Two from Palakariya River (No. 6334, two slides, each with one specimen, 26.06.1975).

Morphology: Hamuli small to medium-sized, robust, thinning towards the point (Figure 21). Roots slightly bending outwards. Point straight, almost parallel to root. Ventral bar mostly rectangular. Anterior side of ventral bar with lower mid-section and rounded protruding sides. Ventral bar membrane mostly rectangular. Dorsal bar thick and short. Marginal hook with large rounded heel, toe pointed, shaft and sickle slender. For metrical data, see

Table 21. Metrical data of Gyrodactylus prostae from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	Various Cyprinids	S. cephalus																R. amarus					R. rutilus						C. nasus
Locality	Palae- arctic	Pchelina Reservoir			Zhrebchevo Reservoir			Lom River			Ogosta River			Shiposhnitsa River		Zhelez-nitsa River		Leva River		Ogosta River			Palakariya River			Shiposhnitsa River			Palakariya River
	Range	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	n	Range	n	Range	n	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	44–60	38–43	41	2	45–54	48.6	5	43–46	44.5	2	41–47	44.4	8	40	1	37	1	40	1	41–43	41.7	3	47–48	47.3	4	42–47	44.4	6	41–44	42.5	2
S	34–44	28–32	30	2	33–40	35.8	5	33–35	34	2	29–35	32.8	8	29	1	27	1	31	1	30–31	30.2	3	32–37	34.9	4	32–36	33.8	6	32–35	33.3	2
P	20–30	15	-	1	17–26	20.6	5	19–20	19.5	2	15–20	18.8	8	17	1	15	1	18	1	20–21	20.2	3	22–23	22.1	4	19–23	20.8	6	15–16	15.5	2
R	16–22	13–15	14	2	14–20	16.1	5	15	15	2	13–17	15.4	8	13	1	13	1	16	1	15–16	15.5	3	17–18	17.6	4	15–19	16.7	6	14–15	14.3	2
Ventral bar:
VBl	4–7	-	-	-	4–7	5.3	3	6	-	1	5–7	5.5	4	-	-	5	1	3	1	5–6	5.3	3	-	-	-	4–6	5	2	4–5	4.5	2
VBw	15–19	-	-	-	16–20	17.6	3	12–15	13.5	2	15–17	15.8	5	-	-	13	1	19	1	13–15	14	2	16	-	1	15–17	15.8	4	15	15	2
VBml	12–16	-	-	-	-	-	-	-	-	-	10–12	11	2	-	-	-	-	9	1	-	-	-	12	-	1	10–13	11.7	3	12	-	1
Dorsal bar:
DBl	9–11	-	-	-	7	-	1	7–9	8	2	6–9	7.6	8	-	-	-	-	-	-	3–5	4	2	7–8	7.5	2	6–8	7	4	8–9	8.5	2
DBw	2–3	3	-	1	3	3	2	2–4	3	2	3–4	3.1	8	3	1	3	1	2	1	2–3	2.5	2	3	3	4	2–3	2.4	6	3	3	2
Marginal hooks:
MHl	7–8	6	-	1	6	6	3	6	6	2	6–7	6.2	6	6	1	-	-	5	1	7	7	3	5–7	6	3	6–7	6.8	5	6	-	1
MHs	-	-	-	-	2	2	2	1–2	1.5	2	1–2	1.3	5	-	-	-	-	1	1	1	1	3	1–2	1.8	4	1–2	1.4	5	2	-	1
MHt	-	-	-	-	-	-	-	4	-	1	4–5	4.3	4	-	-	-	-	4	1	4–5	4.5	2	4–5	4.7	3	4–5	4.3	5	4	-	1
MHad	-	-	-	-	4	4	2	4	4	2	4	c	5	-	-	-	-	3	1	3–4	3.5	2	3–4	3.7	3	4	4	5	3	-	1

.

Remarks: This species is euryxenous. It was reported from basins of the rivers Danube, Elbe and Oder from Abramis brama, Blicca bjoerkna, Leucaspius delineatus Heckel, 1843, Leuciscus leuciscus, Squalius cephalus and Rutilus rutilus [55]. According to Ergens et al. [3], the occurrence of G. prostae on L. delineatus was accidental or doubtful. In Bulgaria, G. prostae was reported from B. bjoerkna, R. rutilus and S. cephalus from various localities (Table S1). Revising the materials in the collection of IBER-BAS, we found out that the specimens of G. prostae from R. amarus from Leva River and Ogosta River and those from S. cephalus from Zheleznitsa River were published as G. laevis [11,18]. There were two slides in the collection containing specimens of G. prostae from Chondrostoma nasus from Palakariya River that had not been mentioned in any publication.

Gyrodactylus rhodei Žitňan, 1964

Records: Kakacheva-Avramova [11].

Host: Rhodeus amarus.

Locality: River—Leva.

Site of infection: Fins and gills.

Specimens studied: One (No. 2981, one slide, 21 April 1964).

Morphology: Hamuli medium-sized, stout. Root short, shaft slightly curved, point straight. Ventral bar mostly trapezoid, with rounded lateral sides. Ventral bar membrane mostly tongue-shaped. Dorsal bar thin, uniform. Marginal hook with angular pointed toe, heel rounded (Figure 22).

Remarks: This species is a stenoxenous parasite on Rhodeus sericeus (Pallas, 1776) and R. amarus [3]. It was reported from Czechia and Slovakia [34] and Russia [3]. In Bulgaria, it was recorded in low numbers from R. amarus, i.e., two and three specimens, in the two infected bitterlings sampled from Leva River [11]. In the helminthological collection of IBER-BAS, there are two slides labelled as G. rhodei with Serial No. 2981. Examination of these slides revealed one specimen of G. rhodei (

Table 22. Metrical data of Gyrodactylus rhodei from various hosts and localities.

Source	Žitňan [56]		Ergens et al. [3]	Present Study
Host	R. amarus		R. amarus, R. sericeus	R. amarus
Locality	Hron River (Slovakia)		Danube River, Elbe River, Maritime Territory (Russia)	Leva River
	Range	n	Range	Range	n
Hamuli:
TL	54–59	8	53–59	53	1
S	42–46	8	42–46	41	1
P	26–29	8	23–30	27	1
R	17–18	8	16–18	16	1
Ventral bar:
VBl	5–6	8	5–9	5	1
VBw	23–26	8	23–26	21	1
VBml	17–19	8	15–19	-	-
Dorsal bar:
DBl	19–22	8	19–22	-	-
DBw	2–3	8	2–3	2	1
Marginal hooks:
MHl	5.8–6.2	8	5–6	6	1
MHs	-	8	-	2	1
MHt	-	8	-	5	1
MHad	-	8	-	3	1

). Another slide with the same number contained one specimen of Gyrodactylus vimbi Schulman, 1953.

Gyrodactylus shulmani Ling, 1962

Records: Present study.

Host: Cyprinus carpio.

Locality: Zhrebchevo Reservoir.

Site of infection: Gills.

Specimens studied: Three from Zhrebchevo Reservoir (No. 11374, one slide, 9 June 1994—two specimens; No. 11375, one slide, 9 June 1994—one specimen).

Morphology: Hamuli small. Root and point short and straight. Ventral bar mostly trapezoid, with median ridge and numerous smaller perpendicular ridges. Ventral bar membrane not observed. Dorsal bar straight and thin. Marginal hook small, with rounded heel (Figure 23).

Remarks: Gyrodactylus shulmani was described as a parasite of Carassius auratus (L.) [57]. It is a stenoxenous parasite reported from Carassius carassius, C. gibelio, Cyprinus rubrofuscus Lacepède, 1803 and C. carpio [3]. In Bulgaria, it was recorded from C. carassius from the Danube at Koshava Village (Vidin Province) [11], from Hypophthalmichthys molitrix (Valenciennes, 1844) at Polikraishte Fish Farm [23] and from C. gibelio from Zhrebchevo Reservoir [20]. In the present study, we found G. shulmani among the unpublished materials from C. carpio from Zhrebchevo Reservoir. The measurements of the haptors of the specimens of G. shulmani corresponded well with previously published data (

Table 23. Metrical data of Gyrodactylus shulmani from various hosts and localities.

Source	Ling [57]	Ergens et al. [3]	Present Study
Host	C. auratus	C. carassius, C. gibelio, C. carpio and C. rubrofuscus	C. carpio
Locality	Liao Ho (China)	Palaearctic	Zhrebchevo Reservoir
	Range	Range	Range	Mean	n
Hamuli:
TL	34.2–38	34–44	34–40	37.5	3
S	24.7–28.5	24–33	28–29	28.7	3
P	15.2–17.1	13–19	17–19	17.8	3
R	9.5–11.4	9–13	11–12	11.2	3
Ventral bar:
VBl	-	3–6	4–5	4.3	2
VBw	13.3–16.1	10–18	16	-	1
VBml	-	7–11	-	-	-
Dorsal bar:
DBl	9.5–13.3	9–15	-	-	-
DBw	-	1–2	-	-	-
Marginal hook:
MHl	-	4	5	5	3
MHs	-	-	2–3	2.5	2
MHt	-	-	2–4	3	2
MHad	-	-	2	2	2

).

Gyrodactylus sprostonae Ling, 1962

Records: Present study.

Host: Carassius carassius, Carassius sp., Cyprinus carpio, Scardinius erythrophthalmus and Squalius cephalus.

Locality: Blagoevgrad and Chelopechene Fish Farms; Pchelina and Zhrebchevo Reservoirs.

Site of infection: Fins (all), gills (all) and skin (C. carassius, C. carpio).

Specimens studied: 127.

From S. cephalus: One specimen from Pchelina Reservoir (No. 10375, one slide, 18 May 1985—one specimen).

From C. carassius: 16 specimens from Pchelina Reservoir (No. 10302, one slide, 16 May 1985—1 specimen, No. 10303, one slide, 16 May 1985—1 specimen, No. 10304, one slide, undated—1 specimen, No. 10323, one slide, 16 May 1985—1 specimen, No. 10361, one slide, 18 May 1985—1 specimen, No. 10381, two slides, 18 May 1985—2 specimens, No. 10382, two slides, 18 May 1985—2 specimens, No. 10400, two slides, 19 May 1985—2 specimens, No. 10401, two slides, 19 May 1985—2 specimens, No. 10402, one slide, 19 May 1985—1 specimen, No. 10403, two slides, 19 May 1985—2 specimens).

From Carassius sp.: One from Chelopechene Fish Farm (No. 7091, one slide, 11 April 1984—one specimen).

From C. carpio: 94 specimens, including 30 from Blagoevgrad Fish Farm (No. 7131, 2 slides, undated—2 specimens, No. 7137, 4 slides, undated—4 specimens, No. 7139, 3 slides, undated—3 specimens, No. 7140, 3 slides, undated—3 specimens, No. 7141, 4 slides, undated—6 specimens, 2 slides with 1 specimen and 2 slides with 2 specimens, No. 7142, 1 slide, undated—1 specimen, No. 7201, 2 slides, undated—2 specimens, No. 7202, 1 slide, undated—1 specimen, No. 7204, 1 slide, undated—1 specimen, No. 7206, 3 slides, undated—3 specimens, No. 7208, 4 slides, undated, 4 specimens); 28 from Pchelina Reservoir (No. 6941, 7 slides, 13 July 1983—8 specimens, 1 slide with 2 specimens and 6 slides with 1 specimen, No. 10257, 5 slides, 15 May 1985—5 specimens, No. 10290, 1 slide, 16 May 1985—3 specimens, No. 10350, 1 slide, 18 May 1985—1 specimen, No. 11069, 10 slides, 12 May 1986—10 specimens, No. 11550, 1 slide, undated, 1 specimen); and 36 from Zhrebchevo Reservoir (No. 11373, 1 slide, 9 June 1994—1 specimen, No. 13647, 1 slide, 27 May 1994—1 specimen, No. 13707, 8 slides, 18 May 1995—8 specimens, No. 13727, 1 slide, 19 May 1995—1 specimen, No. 13756, 12 slides, 21 May 1995—12 specimens, No. 13764, 5 slides, 21 May 1995—4 slides with 1 specimen, 1 slide with 2 specimens, No. 13766, 7 slides, 22 May 1995—7 specimens).

From S. erythrophthalmus: 15 specimens from Pchelina Reservoir (No. 11058, 10 slides, 20 May 1985—1 slide with 3 specimens, 3 slides with 2 specimens, 6 slides with 1 specimen each).

Morphology: Hamuli medium-sized, slender. Roots proximal ends angular. Shaft long and thin. Point mostly straight with a slight inward curve. Ventral bar trapezoid, with thin mid-section and protruding ends. Ventral bar with a median ridge and numerous small ridges. Ventral bar membrane mostly triangular. Dorsal bar thin, arching proximally in the mid-section. Marginal hook slender, with thin shaft and blade, toe pointed, heel rounded (Figure 24).

Remarks: Gyrodactylus sprostonae was described as a gill parasite of Carassius auratus and Cyprinus carpio from Liao River in China [57]. It is an euryxenous parasite that has been reported from a number of cyprinid hosts [2]. It is mainly found on C. carpio, Carassius gibelio and C. carassius and is considered widespread throughout the geographical range of its hosts [3]. In Bulgaria, G. sprostonae was reported on a hybrid of Hypophthalmichthys molitrix and H. nobilis (J. Richardson, 1845) at Mechka Fish Farm [23]. It was also recorded from C. gibelio in Zhrebchevo Reservoir [20] but no specimens from this host were available in the collection. In the present study, we revised the specimens of G. sprostonae, which were misidentified as G. medius from C. carpio from Blagoevgrad Fish Farm. The specimens from C. carpio from Pchelina Reservoir were reported by Nedeva [58] as G. medius. The rest of the specimens were not published. All of the specimens correspond well to the morphology and the known metrical variability of the haptoral hard parts of G. sprostonae (

Table 24. Metrical data of Gyrodactylus sprostonae from various hosts and localities.

Source	Ling [57]	Ergens et al. [3]	Present Study
Host	C. auratus, C. carpio	Cyprinids	S. cephalus		Carassius sp.		C. carassius			C. carpio									S. erythrophthalmus
Locality	Liao Ho (China)	Palaearctic	Pchelina Reservoir		Chelopechene Fish Farm		Pchelina Reservoir			Blagoevgrad Fish Farm			Pchelina Reservoir			Zhrebchevo Reservoir			Pchelina Reservoir
	Range	Range	Range	n	Range	n	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	40.8–51.3	41–62	50	1	60	1	51–60	57.1	16	43–59	51.3	29	51–60	56.8	28	53–62	55.9	36	45–61	55.1	15
S	36.1–39.9	35–45	42	1	45	1	40–45	42.8	15	32–44	38.4	30	40–43	41.8	28	38–45	41.25	34	41–45	42.8	15
P	17.1–22.8	17–25	24	1	26	1	21–27	25.4	14	18–25	21.7	26	21–27	23.2	28	19–26	23.4	36	20–26	23.7	15
R	13.3–20.9	13–24	15	1	16	1	15–23	20.5	14	12–23	17.3	28	15–24	20.5	28	16–24	20.7	32	13–22	16.4	15
Ventral bar:
VBl	-	4–7	5	1	5	1	3–5	4.2	16	3–4	3.2	27	3–6	4.2	22	4–6	4.6	22	3–5	4.2	15
VBw	14.2–20.9	13–26	22	1	23	1	18–24	21.8	14	18–21	19.4	27	19–29	22.1	15	18–25	21.7	17	21–25	22.3	13
VBml	-	15–18	-	-	-	-	13–15	14	2	21–26	23.5	2	-	-	-	-	-	-	13	-	1
Dorsal bar:
DBl	9.5–19	9–20	-	-	-	-	19–20	19.5	2	14–20	15.6	7	12–18	14	5	11–23	18	4	11–17	14.6	5
DBw	-	1–2	1	1	-	-	1–2	1.3	11	1–2	1.7	14	1–2	1.8	16	1–3	1.5	13	1–2	1.8	12
Marginal hook:
MHl	-	4–6	5	1	6	1	5–6	5.7	12	5–6	5.3	15	5–6	5.6	15	5–6	5.7	19	5–6	5.2	6
MHs	-	-	-	-	3	1	2–4	2.6	9	1–3	1.9	6	1–3	2.1	9	2	2	12	2	2	6
MHt	-	-	-	-	4	1	3–4	3.8	8	3–4	3.4	5	3–4	3.8	9	3–4	3.8	12	3–4	3.8	6
MHad	-	-	-	-	4	1	3–4	3.4	8	3	3	5	3–4	3.8	9	3–4	3.2	10	3–4	3.4	6

).

Gyrodactylus stankovici Ergens, 1970

Records: Present study.

Host: Cyprinus carpio.

Locality: Varbnishka River.

Site of infection: Gills.

Specimens studied: Three from Varbnishka River (No. 3502, three slides, 15 April 1965—one specimen on each slide).

Morphology: Hamuli medium-sized, robust. Point and root straight. Ventral bar trapezoid. Median ridge inconspicuous. Ventral bar membrane not observed. Dorsal bar thick, thinning towards the ends. Marginal hook with arched blade, pointed toe and rounded heel (Figure 25).

Remarks: This species seems to be an oioxenous parasite of C. carpio [2], which is considered widespread in the geographical range of its host [3]. It is uncertain whether it infects Carassius gibelio [3]. In Bulgaria, this parasite has been reported by Margaritov [26] in Ictalurus punctatus (Rafinesque, 1818). The specimens included in the present study were reported as G. medius by Kakacheva-Avramova [11]. The measurements of the specimens from Varbnishka River corresponded well with the known variability of the haptoral hard parts for G. stankovici (

Table 25. Metrical data of Gyrodactylus stankovici from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	Cyprinus carpio, Carassius gibelio	C. carpio
Locality	Palaearctic	Varbnishka River
	Range	Range	Mean	n
Hamuli:
TL	50–63	62–68	66.2	3
S	35–46	47–51	49.5	3
P	23–33	34–38	35.3	3
R	16–23	21–24	22.8	3
Ventral bar:
VBl	6–8	7–9	8	3
VBw	20–25	26–28	27	3
VBml	12–18	-	-	-
Dorsal bar:
DBl	13–19	10	-	1
DBw	2	2–3	2.3	3
Marginal hook:
MHl	5–6	5–6	5.7	3
MHs	-	2–3	2.3	3
MHt	-	4–5	4.3	3
MHad	-	3	3	3

).

Gyrodactylus truttae Gläser, 1974

Records: Kakacheva-Avramova & Nedeva-Menkova [17] (1), Kakacheva-Avramova & Menkova [18] (2).

Host: Salmo trutta fario.

Locality: Rivers—Bistritsa (2), Demyanitsa (2), Rilska (2) and Shirokolashka (1).

Morphology: Hamuli medium-sized, robust. Root relatively short. Ventral bar trapezoid. Ventral bar membrane tongue shaped. Dorsal bar thin, straight. Marginal hook sickle extending over the base of the toe (Figure 26). For metrical data, see

Table 26. Metrical data of Gyrodactylus truttae from various hosts and localities.

Source	Ergens et al. [3]	Present Study
Host	Salmonids	S. trutta
Locality	Palaearctic	Bistritsa River		Demyanitsa River			Rilska River			Shirokolashka River
	Range	Range	n	Range	Mean	n	Range	Mean	n	Range	Mean	n
Hamuli:
TL	54–65	64	1	60–65	62.7	6	61–62	61.3	2	62–69	64.3	8
S	40–49	48	1	45–47	46.1	6	47	47	2	45–50	47.1	8
P	29–34	33	1	30–33	31.6	6	30–31	30.5	2	32–36	33.1	8
R	16–21	16	1	16–19	18.3	6	15–18	16.5	2	17–20	18.3	8
Ventral bar:
VBl	7–9	8	1	6–9	8.1	6	7	7	2	6–9	7.9	7
VBw	24–32	30	1	27–30	28.8	6	28	28	2	28–31	29.5	8
VBml	10–17	-	-	10–11	10.5	2	-	-	-	12–14	13	2
Dorsal bar:
DBl	17–30	-	-	20–24	21.3	3	18–21	19.5	2	16–21	18.4	5
DBw	1–2	-	-	2–3	2.3	4	2	-	1	2	2	7
Marginal hook:
MHl	6–8	-	-	6–7	6.6	6	7–8	7.5	2	5–7	6.1	8
MHs	-	-	-	3–5	4.2	3	4–5	4.5	2	3–4	3.2	3
MHt	-	-	-	-	-	-	-	-	-	-	-	-
MHad	-	-	-	3–4	3.8	6	4	4	2	3–4	3.7	8

.

Site of infection: Fins.

Specimens studied: 17, including 1 from Bistritsa River (No. 6468, one slide, 28 September 1976); 2 from Rilska River (No. 6650, one slide, 20 May 1976—1 specimen, No. 6659, one slide, 21 May 1976—1 specimen); 8 from Shirokolashka River (No. 6252, five slides, 6 July 1975—5 specimens, No. 6258, two slides, 6 July 1975—2 specimens; No. 6384, one slide, 23 July 1975—1 specimen); and 6 from Demyanitsa River (No. 8, one slide, undated—1 specimen, No. 9, one slide, undated—1 specimen, No. 21, one slide, undated—1 specimen, No. 24, one slide, 16 June 1977—1 specimen, No. 39, one slide, undated—1 specimen, No. 64, one slide, 16 June 1977—1 specimen).

Remarks: This species seems to be a stenoxenous parasite of salmonid fishes [2] which is widespread through the range of its hosts [3]. In Bulgaria, it has been recorded from populations of S. trutta in the wild. The measurements of the haptoral hard parts of the specimens in the collection of IBER-BAS corresponded well with the known variability for the species reported by Ergens et al. [3].

Gyrodactylus vimbi Schulman, 1953

Syn. Gyrodactylus scardinii Malmberg, 1957

Records: Kakacheva-Avramova [13] (1) and present study (2).

Host: Rhodeus amarus (2), Vimba melanops (2) and Scardinius erythrophthalmus (1).

Locality: Hrabrovski Reservoir; rivers Leva and Maritsa.

Site of infection: Fins.

Specimens studied: Four.

From R. amarus: One specimen from Leva River (No. 2981, one slide, 21 April 1964).

From V. melanops: Two specimens from Maritsa River near the town of Harmanli (No. 2221, two slides, 18.07.1962—one specimen on each slide).

From S. erythrophtalmus: One specimen from Hrabrovski Reservoir (No. 4783, one slide, 18 April 1970).

Morphology: Hamuli medium-sized. Point slightly curved. Ventral bar mostly trapezoid, with visible processes. Ventral bar with median ridge and numerous small perpendicular ridges along the length of the median ridge. Ventral bar membrane tongue-shaped. Dorsal bar straight, thick. Marginal hook shaft almost perpendicular to the base of the hook, toe pointing downwards (Figure 27).

Remarks: This species has been described from the gills of V. vimba (L.) from Western Dvina River [38]. It is an euryxenous parasite, known from variety of cyprinid hosts [2]. In Bulgaria, it was reported from V. vimba from Luda Kamchiya River and from S. erythrophthalmus from Hrabrovski Reservoir in Eastern Stara Planina [13]. The specimens from Luda Kamchiya River were not found in the collection. The specimen from S. erythrophtalmus from Hrabrovski Reservoir was published with the synonymous name G. scardinii [13]. The specimens from Maritsa River were reported as G. medius (Kakacheva-Avramova 1962). The specimen from Leva River was identified as G. rhodei [11]. All specimens correspond well with the measurements and morphology of the species (

Table 27. Metrical data of Gyrodactylus vimbi from various hosts and localities.

Source	Schulman [38]	Ergens et al. [3]	Present Study
Host	V. vimba	Cyprinids	R. amarus		S. erythrophtalmus		V. melanops
Locality	Western Dvina River	Baltic, Black, Caspian Sea Drainages, Ob River	Leva		Hrabrovski Reservoir		Maritsa River
	Range	Range	Range	n	Range	n	Range	Mean	n
Hamuli:
TL	64–66	56–67	63	1	67	1	55–56	55.5	2
S	48–57	41–51	46	1	50	1	43–44	43.5	2
P	31–33	24–31	31	1	35	1	26–27	26.5	2
R	18	18–21	21	1	25	1	18	-	1
Ventral bar:
VBl	7	6–8	7	1	7	1	6–7	6.5	2
VBw	29–31	24–28	27	1	28	1	24	-	1
VBml	-	12–16	15	1	17	1	-	-	-
Dorsal bar:
DBl	18–20	18–23	18	1	22	1	15	-	1
DBw	3	1–3	3	1	2	1	2	2	2
Marginal hook:
MHl	-	5–6	6	1	5	1	6	6	2
MHs	-	-	2	1	2	1	-	-	-
MHt	-	-	4	1	4	1	-	-	-
MHad	-	-	3	1	3	1	-	-	-

).

3.2. Unidentifiable Specimens

In addition to the above-described 29 species, some specimens could not be identified at the species level due to the small number or poor condition of the specimens. These are as follows.

Gyrodactylus sp. 1

Host: Barbus petenyi.

Locality: Varbnishka River.

Site of infection: Fins.

Specimens studied: One (No 3507, 25 April 1965).

Morphology: Hamuli medium-sized. Roots slightly curved inwards. Point long and straight. Ventral bar trapezoid, laterally rounded, with median ridge. Ventral bar posterior edge uneven. Dorsal bar uniform. Marginal hook not observed. Measurements: TL = 67, S = 49, P = 34, R = 19; VBl = 6, VBw = 30; DBl = 17, DBw = 2 (Figure 28).

Remarks: This specimen was identified as G. markewitschi Kulakovskaya, 1952 by Kakacheva-Avramova [11]. Its metrical data correspond to the measurements presented by Kakacheva-Avramova [8] for this species but differ from the metrical data provided by Ergens et al. [3]. Therefore, we prefer to consider this specimen as needing further studies in order to be identified.

Gyrodactylus sp. 2

Host: Gobio gobio.

Locality: Kotleshnitsa River near the town of Kotel.

Site of infection: Not specified.

Specimens studied: One (No. 4551, 25 April 1969).

Morphology: Hamuli medium-sized, stout. Point straight. Root folded. Ventral bar trapezoid with prominent median ridge. Ventral bar membrane not observed. Dorsal bar arched. Marginal hook with rounded toe and heel. Measurements: S = 44, P = 29; VBl = 7, VBw = 28; DBl = 18, DBw = 2; MHl = 7, MHs = 2, MHt = 3, MHad = 4 (Figure 29).

Remarks: The position of the root did not allow for the total length of the hamuli and the root length to be measured. Therefore, this specimen could not be identified at the species level.

Gyrodactylus sp. 3

Host: Rhodeus amarus.

Locality: Leva River.

Site of infection: Gills.

Specimens studied: One (No. 5101, 23 April 1964)

Morphology: Hamuli large, slender. Root same width as hamulus, straight. Point long and straight. Ventral bar trapezoid, with median ridge. Ventral bar membrane not observed. Dorsal bar thick. Marginal hook toe pointed, heel slightly angular, shaft long. Measurements: TL = 71, S = 51, P = 33, R = 22; VBl = 7, VBw = 29, DBl = 19, DBw = 2, MHl = 6, MHs = 2, MHt = 4, MHad = 3 (Figure 30).

Remarks: This species was published as G. laevis from Leva River [11]. This specimen does not correspond to the morphology and metrical data of G. laevis (Figure 11, Table 11). Possibly, it is an undescribed species and further specimens are needed for a reliable description.

3.3. Revised Host–Parasite List of Gyrodactylus spp. As Revealed by the Present Study

• Family Acheilognathidae
• Rhodeus amarus

  -

  G. prostae

  -

  G. rhodei

  -

  G. vimbi

  -

  Gyrodactylus sp. 3

• Family Cobitidae
• Cobitis taenia

  -

  G. papernai

• Cobitis sp.

  -

  G. cobitis

  -

  G. latus

• Misgurnus fossilis

  -

  G. fossilis

• Family Cyprinidae
• Barbus cyclolepis

  -

  G. malmbergi

• Barbus petenyi

  -

  G. katharineri

  -

  G. malmbergi

  -

  Gyrodactylus sp. 1

• Carassius carassius

  -

  G. sprostonae

• Cyprinus carpio

  -

  G. cyprini

  -

  G. katharineri

  -

  G. medius

  -

  G. shulmani

  -

  G. sprostonae

  -

  G. stankovici

• Family Gobionidae
• Gobio gobio

  -

  G. dykovae

  -

  G. gobiensis

  -

  G. gobii

  -

  G. markakulensis

  -

  Gyrodactylus sp. 2

• Family Ictaluridae
• Ameiurus melas

  -

  G. melas

• Family Leuciscidae
• Chondrostoma nasus

  -

  G. macrocornis

  -

  G. prostae

• Phoxinus phoxinus

  -

  G. aphyae

  -

  G. laevis

  -

  G. macronychus

• Rutilus rutilus

  -

  G. prostae

• Scardinius erythrophthalmus

  -

  G. cyprini

  -

  G. sprostonae

  -

  G. vimbi

• Squalius cephalus

  -

  G. gracilihamatus

  -

  G. leucisci

  -

  G. prostae

  -

  G. sprostonae

• Vimba melanops

  -

  G. katharineri

  -

  G. vimbi

• Family Odontobutidae
• Perccottus glenii

  -

  G. perccotti

• Family Percidae
• Gymnocephalus cernua

  -

  G. luciopercae

• Sander lucioperca

  -

  G. luciopercae

• Family Salmonidae
• Salmo trutta fario

  -

  G. truttae

4. Discussion

In the present study, we revised the specimens of Gyrodactylus deposited in the Helminthological Collection of IBER-BAS. These were mostly specimens collected by D. Kakacheva-Avramova and I. Nedeva-Menkova. We also added newly-collected specimens of G. cobitis, G. fossilis, G. latus and G. malmbergi as well as two species that were recently recorded for the country—G. melas [28,46] and G. perccotti [29]. The revision of the available specimens based on the morphological and metrical data of the haptoral hard parts gives more insight into the diversity of the genus in the freshwater fishes in Bulgaria.

In light of the abovementioned results, the recorded species of Gyrodactylus from freshwater fish in Bulgaria can be divided into four categories:

Species with verified (confirmed) status—22: G. aphyae, G. cyprini, G. fossilis, G. gobii, G. gracilihamatus, G. katharineri, G. laevis, G. latus, G. leucisci, G. luciopercae, G. macrocornis, G. macronychus, G. malmbergi, G. markakulensis, G. medius, G. prostae, G. rhodei, G. shulmani, G. sprostonae, G. stankovici, G. truttae and G. vimbi.

Recently reported species—two: G. melas and G. perccotti. These are parasites of alien species that have been introduced together with their hosts to Bulgaria during the last 20 years [28,29].

New species for the fauna of Bulgaria—four: G. dykovae, G. gobiensis and G. papernai (all three reported based on the revision of the collection) and G. cobitis (reported based on the recently collected specimens). Most likely, these species are not “newcomers”. The former three species were not properly identified in the course of the previous studies while the hosts of G. cobitis (species of the family Cobitidae) were poorly studied for parasites in this country in the past.

Species with unverified status—nine: G. carassii, G. elegans, G. hronosus, G. matovi, G. pannonicus, G. sedelnikowi (no available specimens in the collection for any of them); G. cernuae (poor quality of the slides); G. longiradix (specimens re-identified as G. luciopercae, which is already reported for the fauna of Bulgaria); and G. markevitschi (the only available specimen does not correspond to the morphology of the species).

There is a discrepancy between the reported amounts of specimens collected by Kakacheva-Avramova and Nedeva-Menkova [11,12,13,14,15,16,17,18,19] and the number of specimens available in the Helminthological Collection of IBER-BAS. A possible explanation for this is that the majority of the collected individuals of Gyrodactylus were examined in temporary mounts on site and only some of them were prepared in permanent microscopic mounts [8].

The specimens of ten species of Gyrodactylus reported by Margaritov and his collaborators (Table S1) were not available for re-examination. However, the majority of them (seven species) were recorded by other authors from Bulgaria. The remaining three species were not recorded by other studies in the country. These are as follows:

G. rarus Wegener, 1910—originally reported for Bulgaria from Gasterosteus aculeatus L. in captivity (in a freshwater tank in Varna Aquarium [9]). This species is considered a specific parasite of Pungitius spp. and its records on G. aculeatus are characterised as “accidental or doubtful” [3].

G. gobioninum Gusev, 1955—reported from Pseudorasbora parva at Chelopechene Fish Farm (near Sofia) [24]. This species identification contradicts with the published metrical data: the ventral bar as 23–28 μm wide and hamuli 52–60 μm long (versus 17 μm and 51 μm, respectively, in G. gobioninum, according to Ergens et al. [3]). Two species of Gyrodactylus were described as specific parasites of P. parva: G. parvae You, Easy & Cone, 2008 [59] and G. pseudorasborae Ondračková, Seifertová & Tkachenko, 2023 [60]; however, neither of them clearly corresponded to the metrical data provided by Margaritov & Kiritsis [24].

G. kherulensis Ergens, 1974—a parasite of Cyprinus carpio through its geographical range [3]. In Bulgaria, it was initially reported in a conference abstract by Margaritov [23] among the parasites of carp in fish farms, without specifying exact localities. Margaritov [24], in a dissertation summary, mentioned this species from the fish farms of Polikraishte and Batin. Nedeva [58] also reported this species from carps in Pchelina Reservoir in a conference abstract. No exact data on the prevalence, intensity and number of specimens studied were given in either abstract.

No specimens of these three species (G. rarus, G. gobioninum and G. kherulensis) were available in the Helminthological Collection of IBER-BAS. Based on the aforementioned account, we assume that their occurrence in the fauna of Bulgaria requires further confirmation.

Summarising the result of the present study, the actual number of taxa of Gyrodactylus confirmed as parasites of freshwater fishes in Bulgaria includes 28 species. The most species-rich region in Bulgaria seems to be the Danube Drainage, with 21 species. The most possible reasons for this are that it also occupies half of the country’s territory and coincides with the region where the most studies have been conducted (Figure S1, Table S1). In the East-Aegean Drainage, which is the second river basin in size in Bulgaria, 12 species of Gyrodactylus have been recorded. In the Black Sea Drainage and the West-Aegean Sea Drainage, only nine and four species, respectively, have been recorded, which reflects not only their smaller sizes but also the smaller research effort dedicated to them. Further studies are needed in the latter two drainages in order to more comprehensively reveal their fish parasite fauna.

The most widespread gyrodactylid species in Bulgaria is G. prostae, being found on four host species from eight localities. It is followed by G. gobii (one host species, five localities) and G. sprostonae (four host species, four localities). Gyrodactylus prostae and G. sprostonae are euryxenous parasites. These results are expected in view of the fact that their hosts are mostly cyprinid species, often with economic importance, which are frequently in the focus of the research activities. The common carp (C. carpio) was the main subject of many studies carried out by Bulgarian researchers because it is the most economically significant species in Bulgaria [61]. Therefore, it is not surprising that C. carpio is the most species-rich in terms of gyrodactylids, with six recorded species. The species of less commercial value, which have often been neglected in parasitological studies in the past in this country, need much more research attention in view of their importance for the ecosystem and biodiversity studies.

Compared to Bulgaria, the fauna of Gyrodactylus from freshwater fishes in neighbouring countries is relatively less studied. The Northern Balkans and the Lower Danube (including Danube Delta) are known as the most species-rich region in Europe in terms of freshwater fish [62]. Historically, the most detailed research in the Western Balkans was carried out for the Danube on the border between Serbia and Croatia [63], reporting 25 species of Gyrodactylus from a variety of host species, but the findings were presented in the form of a host–parasite list only. Without any supporting evidence for the occurrence of the parasites (such as photographs, illustrations or metric data), it is hard to verify the authenticity of those records. The checklist of the parasites of fishes of Serbia [64] reported the species mentioned in the former publication [63] and further records for two of the species reported in an unpublished dissertation. In recent years, there has been a detailed examination of alien fish species from the Ukrainian part of the Danube Delta reporting the alien G. avalonia Hanek & Threlfall, 1969 from Lepomis gibbosus L. and G. perccotti from Perccottus glenii [54].

From the Republic of North Macedonia, there are several reports on single species occurrences of Gyrodactylus spp. [65,66]. However, a recent thesis involving gyrodactylids from Lake Prespa and Lake Ohrid [67] reported abundant molecular data, identifying 6 species, providing tentative identification for further 5 species and an additional 13 species that cannot be identified at the species level (no matching GenBank sequences). The latter study demonstrated that the diversity of Gyrodactylus spp. in the Balkan Peninsula is much greater than currently recognised.

In Romania, several studies have been conducted, with some of their results being similar to those of our study. Gyrodactylus katharineri, G. malmbergi and G. barbi Ergens, 1970 were reported from Barbus peloponnesius Valenciennes, 1842 [68], the former two known also from Bulgaria. In another study, mainly consisting of an analysis of Gyrodactylus spp. on farmed salmonids [69], G. salaris and G. truttae were reported, with G. salaris dominating in the majority of the samples. Interestingly, G. truttae was found with the highest numbers in a wild population of S. trutta. Similarly, this parasite was found in wild populations of river trout in Bulgaria [17].

One of the most detailed studies on Gyrodactylus in the Balkans was carried out in Greece [70], with reports of seven new species from sand gobies. Unfortunately, the fish species in this study have not been studied in Bulgarian freshwater basins. Therefore, no parallel can be made with the results of that study and ours.

This study emphasises the value of museum collections for the examination of parasite diversity. The studied group of freshwater fish parasites has been the focus of the researchers between 1959 and 1999 in Bulgaria. Substantial parts of the specimens examined by previous authors have been preserved as museum specimens, which allowed the re-assessment of their morphology, identification, distribution data and host–parasite associations tens of years later. Unfortunately, the studied material, which has been fixed in formalin and preserved in glycerine–gelatine microscopic slides over decades, is not usable for molecular genetic studies. However, it allows us to update the data in view of the latter development of the taxonomy of the group [2,3,59,60]. The examination of museum collections is a powerful approach in the assessment of information on parasite specificity, geographical distribution and pathogen diagnostics, as well as for discovering new taxa [71].

5. Conclusions

We believe that the present revision is an important step in the examination of the diversity of the gyrodactylids of freshwater fishes in Bulgaria. It verifies the data on taxonomic identification, host range and geographical distribution in the territory of the country for 28 species and provides a baseline for further studies on their ecology, host ranges, phylogenetic relationships and host–parasite interactions.