Monogenean fauna of alien tilapias (Cichlidae) in south China.

Tilapias are important aquaculture fishes that have been introduced widely all over the world, often carrying their monogenean parasites with them. An extensive investigation on monogeneans of invasive tilapias was conducted in 19 natural water sources in south China between July 2015 and December 2017. We found nine known species of monogeneans, i.e., Enterogyrus coronatus, E. malmbergi, Cichlidogyrus cirratus, C. halli, C. sclerosus, C. thurstonae, C. tilapiae, Scutogyrus longicornis, Gyrodactylus cichlidarum, and one unknown Gyrodactylus species. In addition to reporting ten new hosts and four new geographical records, we observed new morphological characteristics of these species. Observation on living specimens of Enterogyrus spp. demonstrated that these two species have characteristic opisthaptoral retraction capacities, while the opisthaptor glands were not observed in our specimens of E. coronatus and E. malmbergi. The morphological differences of the accessory piece of the male copulatory complex between C. cirratus and C. mbirizei (character for species differentiation) could result from the observation at different perspectives, which indicates that C. mbirizei is likely a synonym of C. cirratus. A more detailed structure of the sclerotized parts of Cichlidogyrus spp. and S. longicornis were revealed by scanning electron microscopy. As was the case for the monogeneans found on alien tilapias from other geographic regions, the present study confirmed the high potential of these monogeneans to establish populations in new habitats. © S. Zhang et al., published by EDP Sciences, 2019.

Introduction

Tilapias/Tilapia is the general name of fishes belonging to Oreochromis Günther, 1889, Sarotherodon Rüppell, 1852, Tilapia Smith, 1840 and Coptodon Gervais, 1848, all members of the Cichlidae. They are important aquaculture fishes in the world and have been introduced to at least 140 countries and have turned into worldwide invasive fishes [10]. As an important component of parasite fauna of tilapias, monogenean species reported from indigenous tilapias are very abundant, especially Cichlidogyrus species [26, 49]. In the course of tilapias introduction, monogenean species have unintentionally been brought to non-native countries, including the United States [40], Australia [57], Brazil [22, 56], China [28, 37, 58, 59], Colombia [25], Cuba [36], Iraq [1], Japan [33], Malaysia [29], Mexico [23, 46], Philippines [2], South Africa [26, 30], Thailand [27] and the UK [20], although there was a report of the whole gill parasite community loss [17].

As the country with the highest tilapias aquaculture production [16], China initially introduced Mozambique tilapia (O. mossambicus Peters, 1852) from Vietnam in 1956; other tilapias were then also introduced for culture or breed improvement [9, 60]. In the course of tilapias culture, the escapees gradually established wild populations in many natural waters of south China [21], which has become a great concern because they will not only damage the aquatic community, but also act as a refuge for aquaculture pathogens. In addition, they could acquire native parasites ([8, 23], and our unpublished data) and may spillback the parasites acquired to the endemic hosts [24]. However, extensive investigation of monogeneans of tilapias was lacking, although several sporadic reports indicated the existence of the alien gill parasites on tilapias in China [28, 37, 58, 59]. To fill this gap, an extensive investigation on the monogenean fauna of feral tilapias was carried out in south China between July 2015 and December 2017. The results presented in this paper include the monogenean fauna of tilapias and a supplementary description of new morphological features for three species.

Material and methods

Ethics

All the experimental handling was carried out in compliance with animal safety and ethics rule issued by the School of Life Sciences, Sun Yat-sen University.

Host and parasite collection

Investigation of wild tilapias and their monogenean fauna was conducted in 19 natural waters sources in south China. These sampling locations were selected based on field study and reports [21, 37, 59] to cover the distribution of wild tilapias in south China (Fig. 1, Table 1). Year-round investigations were implemented monthly in three reservoirs from April 2016 to August 2017: Nanshui reservoir (24°44′N, 113°10′E), Gaozhou reservoir (22°08′N, 111°05′E) and Songtao reservoir (19°24′N, 109°33′E) to cover the seasonality of infection (data about seasonality of infection unpublished). Fishes were identified by morphological features according to FishBase (www.fishbase.org). Nile tilapia Oreochromis niloticus and its hybrids were accepted as O. niloticus due to their indistinguishable morphologies. Oreochromis niloticus samples in Guangzhou, Guangdong Province were purchased from a local fish farm or caught from a small pond in the south campus of Sun Yat-sen University. These fishes were kept in the laboratory for observation of live parasite specimens.

Figure 1

Map of investigation sites of tilapias in south China. AM natural preservation zone, Macau; BN Lancang River, Xishuangbanna, Yunnan Province; BS Boai River, Baise, Guangxi Province; CJ Shilu reservoir, Changjiang, Hainan Province; CM Jiatan reservoir, Chengmai, Hainan Province; CZ Han River, Chaozhou, Guangdong Province; DZ Songtao reservoir, Danzhou, Hainan Province; FZ Min River, Fuzhou, Fujian Province; GM Nongba reservoir, Lincang, Yunnan Province; GZ fish farm and pond in Sun Yat-sen University, Guangzhou, Guangdong Province; HK Nandu River, Haikou, Hainan Province; HL fish market, Hualien, Taiwan; HY Xinfengjiang reservoir, Heyuan, Guangdong Province; LZ Liu River, Liuzhou, Guangxi Province; MM Gaozhou reservoir, Maoming, Guangdong Province; NN Bachi River, Nanning, Guangxi Province; QN Lancang River, Lincang, Yunnan Province; QZ Jin River, Quanzhou, Fujian Province; SG Nanshui reservoir, Shaoguan, Guangdong Province; XM Xixi River, Xiamen, Fujian Province.

Table 1

Monogeneans of invasive tilapias with mean abundance in south China.

Sites	Abbr.	Waters	Host species	No.	Cci	Cha	Csc	Cth	Cti	Slo	Eco	Ema
Baise	BS	River	C. zillii	30	0.3	0.1	×	×	0.8	×	0.3	×
			S. galilaeus	30	×	<0.1	0.6	×	11.4	×	×	×
			O. niloticus	30	2	6.6	6.1	1.6	12.9	3.4	×	0.1
Changjiang	CJ	Reservoir	C. zillii	20	0.9	0.1	×	×	3.4	1.3	×	×
			O. niloticus	22	1.5	2.3	0.1	1.4	42.7	0.9	×	×
Chengmai	CM	Reservoir	C. zillii	128	0.9	0.2	×	<0.1	5.1	<0.1	×	×
			O. niloticus	39	0.1	3.5	2.5	3	9.8	6.1	×	<0.1
Chaozhou	CZ	River	C. zillii	108	0.1	×	<0.1	×	0.4	<0.1	×	×
			S. galilaeus	13	×	×	0.4	×	6.5	×	×	0.2
			O. niloticus	26	0.3	×	1.2	×	7.1	1.3	×	0.1
Danzhou	DZ	Reservoir	C. zillii	23	×	×	×	×	0.4	×	×	×
			O. mossambicus	15	0.3	0.1	8.9	0.4	5.5	1.3	×	1.6
			O. niloticus	492	5.2	7.7	3.7	7.5	11.9	6.9	×	1.6
Fuzhou	FZ	River	C. zillii	25	×	×	×	×	1	×	–	–
			S. galilaeus	13	×	×	0.2	×	1.5	×	–	–
			O. niloticus	17	×	×	0.1	×	0.6	×	–	–
Gengma	GM	Reservoir	O. niloticus	31	0.6	×	1.3	×	5.9	2.9	×	×
Guangzhou	GZ	Captivity	O. niloticus	>50	×	×	√	√	√	√	×	√
Haikou	HK	Estuary	C. zillii	10	×	×	×	×	4.8	×	×	×
			O. mossambicus	32	×	×	×	×	×	×	×	×
			O. niloticus	32	×	×	×	×	1	×	×	×
Heyuan	HY	Reservoir	C. zillii	29	<0.1	×	×	×	3.7	×	0.2	×
			S. galilaeus	6	×	×	×	×	6.3	×	×	×
			O. niloticus	7	2	3.4	0.7	0.3	26.9	2.1	×	×
Hualien	HL	Captivity	O. niloticus	1	1	×	×	1	1	1	–	–
Liuzhou	LZ	River	C. zillii	40	×	×	×	×	0.3	×	1.8	×
			O. niloticus	13	×	0.3	1.4	×	4.1	1.2	0.4	0.5
Macau	AM	Estuary	C. zillii	6	×	×	×	×	×	×	×	×
			O. niloticus	6	×	×	×	×	×	×	×	×
Maoming	MM	Reservoir	C. zillii	553	0.6	0.3	<0.1	0.1	4	0.1	0.8	<0.1
			O. niloticus	599	2.5	5	1	4.6	15.6	5	×	0.3
Nanning	NN	River	C. zillii	30	<0.1	0.1	×	×	5.1	×	0.1	×
			S. galilaeus	6	×	×	0.3	×	14.2	×	×	×
			O. niloticus	32	0.4	6	4.4	4.1	19.6	10.4	×	0.1
Quannei	QN	River	C. zillii	34	0.7	×	×	×	3.8	0.1	3	×
			O. niloticus	30	2.2	×	1.2	0.8	9.5	3.8	×	1
Quanzhou	QZ	River	C. zillii	42	×	×	<0.1	×	0.3	×	<0.1	×
			S. galilaeus	26	×	×	0.7	×	7.1	×	×	×
			O. niloticus	31	0.1	×	2.7	0.3	4.5	1.1	×	0.2
Shaoguan	SG	Reservoir	C. zillii	683	<0.1	×	×	<0.1	1	×	1.8	×
Xiamen	XM	River	C. zillii	5	×	×	×	×	2	×	–	–
			S. galilaeus	19	×	×	0.1	×	0.6	×	–	–
			O. niloticus	28	×	×	1.5	0.6	5	1.2	–	–
Xishuangbanna	BN	River	C. zillii	23	×	×	×	×	0.2	<0.1	1	×
			O. niloticus	42	0.1	5.6	3.5	1.7	2	1.5	×	0.1

Abbr., abbreviation of sites; No., sampling number; Cti, Cichlidogyrus tilapiae; Cci, C. cirratus; Cth, C. thurstonae; Csc, C. sclerosus; Cha, C. halli; Slo, Scutogyrus longicornis; Eco, E. coronatus; Ema, E. malmbergi. √, sampled, but data not recorded; ×, not sampled; –, not examined. There were twelve G. cichlidarum sampled, including two G. cichlidarum collected from Gaozhou reservoir, Maoming (MM) and Songtao reservoir, Danzhou (DZ), respectively, and eight collected from laboratory reared O. niloticus in Guangzhou (GZ). Only one Gyrodactylus sp1. collected from Songtao reservoir, Danzhou.

Fish samples caught in the wild were individually killed and examined for parasites in the gills, stomach and urinary bladder. The parasite examination for the fish samples kept in the laboratory included the body surface. For identification, live monogeneans were detached with a dissecting needle, pipetted out, and mounted in a drop of ammonium picrate glycerin (GAP) on a slide under a coverslip, which was sealed using nail polish. After identification, target slides were rinsed in distilled water for 12–24 h until the nail polish could be easily removed, and the detached specimens were stored in vessels for further studies.

Light microscopy and scanning electron microscopy

For SEM studies, worms were processed for scanning electron microscopy (SEM) according to Mo and Appleby [38] or Fannes et al. [15], sputter coated with gold and finally examined under Quanta 400 (FEI, Netherlands) in the Instrumental Analysis & Research Center, Sun Yat-sen University. For light microscopy studies, identified worms were digested following the protocol depicted by Fannes et al. [15], and later remounted in GAP on slides. Or alternatively, specimens were rinsed several times with water before being stained in Modified Gomori’s Trichrome, dehydrated in an ethanol gradient, cleared in clove oil, and finally mounted in neutral Canada balsam. For Gyrodactylus species, GAP preserved specimens were digested in situ: proteinase K solution was dripped on one side of the coverslip, while a piece of filter paper was placed on the opposite side until the GAP solution was entirely replaced by proteinase K solution; later the same method was used to replace the digestive fluid by GAP solution.

Morphological analyses

The numbering of the sclerotized parts of the species in genera Scutogyrus, Cichlidogyrus and Enterogyrus was adopted from ICOPA IV [14] and the terminology followed Pariselle and Euzet [49]. For Gyrodactylus species, the measurements of sclerotized parts and terminology followed Shinn et al. [54]. The terminology was employed as follows: anchor instead of gripus or hamulus; hooks rather than marginal hook, uncinulus or hooklet, and ventral bar instead of ventral transverse bar. Additionally, the ventral bar length represents the length of one branch rather than the whole length (with that of Gyrodactylus as an exception). The metrics are shown in Figure 2.

Figure 2

(A–B) Measurements studied. (A) Measurements used to study Cichlidogyrus spp. and Scutogyrus spp. DB dorsal bar: h, length of dorsal bar auricle; w, dorsal bar maximum width; x, dorsal bar total length; y, distance between auricles; z, dorsal bar base end length. A anchor: a, anchor total length; b, anchor blade length; c, anchor shaft length; d, anchor guard length; e, anchor point length. Vg vagina: x, vagina total length, w, vagina maximum width. MCC male copulatory complex: Ap, accessory piece straight length; St, stalk length; He, heel straight length; Pe, penis: x, penis total curved length; w, penis maximum width. Pl auxiliary plate: x, auxiliary plate total length; w, auxiliary plate maximum width. H hook straight length. VB ventral bar: w, ventral bar maximum width; x, length of one ventral bar branch. (B) Measurements used to study Enterogyrus spp. VB ventral bar: x, length of one ventral bar branch; w, ventral bar maximum width. A anchor: a, anchor total length; b, anchor blade length; c, anchor shaft length; d, anchor guard length; e, anchor point length. Pe penis: x, penis total curved length; w, penis base maximum width.

Photographs and measurements of the sclerotized parts were taken under an Olympus DX41 microscope equipped a DP73 Olympus camera (Olympus, Japan), and processed by the software cellSens Standard 1.7.1. Illustrations were drawn freehand with the aid of an Olympus U-DA drawing attachment and then digitized and processed using Adobe Illustrator CS6 (Adobe, USA). All measurements were taken in micrometers and presented in the following order: mean ± standard deviation (minimum – maximum, number of measurements). Voucher specimens were stored in the Research Center for Parasitic Organisms, School of Life Sciences, Sun Yat-sen University (SYSU) and a set of whole-mount specimens was also deposited in the Muséum National d’Histoire Naturelle, France (MNHN).

Results

A total of 3,426 feral tilapias (including 1,789 Coptodon zillii, 113 Sarotherodon galilaeus, 1,477 O. niloticus and 47 O. mossambicus) and more than 50 cultured O. niloticus from south China were examined for monogenean parasites (see Table 1). The ten species of monogeneans collected belong to two families:

Ancyrocephalidae Bychowsky & Nagibina, 1968 with three genera Enterogyrus Paperna, 1963, Cichlidogyrus Paperna, 1960 and Scutogyrus Pariselle & Euzet, 1995.

From the stomach of the fish:

Enterogyrus coronatus Pariselle, Lambert & Euzet, 1991 and E. malmbergi Bilong Bilong, 1988.

From gills of the fish:

Cichlidogyrus cirratus Paperna, 1964; C. halli Price & Kirk, 1967; C. sclerosus Paperna & Thurston, 1969; C. thurstonae Ergens, 1981 and C. tilapiae Paperna, 1960.

Scutogyrus longicornis Paperna & Thurston, 1969.

Gyrodactylidae Cobbold, 1864 with the genus Gyrodactylus von Nordmann, 1832 and the species G. cichlidarum Paperna, 1968 and Gyrodactylus sp1.

The urinary bladders did not host monogenean species.

Family Ancyrocephalidae Bychowsky & Nagibina, 1968

Genus Enterogyrus Paperna, 1963

Enterogyrus coronatus Pariselle, Lambert & Euzet, 1991

Type host: Coptodon guineensis (Perciformes: Cichlidae).

Hosts: Oreochromis niloticus and Coptodon zillii.

Site of infection: Stomach.

Type locality: Ebrié lagoon, Cote d’Ivoire.

Localities: Nanshui reservoir, Shaoguan, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Xinfengjiang reservoir, Heyuan, Guangdong Province; River Liu, Liuzhou, Guangxi province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Jin River, Quanzhou, Fujian Province; Lancang River, Lincang, Yunnan Province; Lancang River, Xishuangbanna, Yunnan Province.

Number of voucher specimens observed: 31 (SYSUECO1–30; MNHN HEL906).

Description (Figs. 3, 4a–c, 10j): Four eyespots well developed without lenses. Tegument thick and transversally striated. Opisthaptor shape variable (tongue- or cup-shaped). Adults 421 ± 76.2 (292–574, 30) long and 149 ± 26.7 (101–205, 30) wide. Pharynx globular 32 ± 6.9 (22–56, 30) in diameter. Dorsal anchor with shaft longer than blade: a = 10 ± 2 (8–20, 29), b = 14 ± 2.6 (10–27, 29), c = 18 ± 3 (13–30, 29), d = 7 ± 1.2 (5–13, 29), e = 5 ± 1.3 (3–9, 29). Ventral anchor smaller than dorsal one: a = 14 ± 2 (9–23, 29), b = 12 ± 1.6 (9–20, 29), c = 5 ± 1 (4–10, 29), d = 5 ± 1.1 (3–9, 29), e = 4 ± 0.9 (2–7, 28). Thin and weak V-shaped ventral bar: x = 9 ± 2.3 (7–16, 23), w = 1 ± 0.2 (0–1, 23). Hooks (marginal hooks) robust except first and second pairs (thinner): I = 12 ± 0.9 (11–13.7, 25), II = 12 ± 0.8 (10–13.1, 25), III = 13 ± 0.9 (10–13.8, 23), IV = 13 ± 0.9 (10–14.3, 27), V = 14 ± 0.9 (11–15.7, 28), VI = 14 ± 0.7 (12–14.7, 27), VII = 13 ± 0.7 (12–14.8, 27). Penis spiral pattern: 4–2–3, 52 ± 13.5 (46–123, 30) long and 6 ± 1.7 (3–13, 30) wide at the base. Eggs oval, length 72 (n = 1) and width 66 (n = 1).

Figure 3

Drawings of sclerotized parts of E. coronatus Pariselle, Lambert & Euzet, 1991. DA, dorsal anchor; VA, ventral anchor; VB, ventral bar; Pe, penis; I–VII, hooks.

Figure 4

Different body shape of E. coronatus Pariselle, Lambert & Euzet, 1991 under coverslip and the motion of opisthaptoral sclerotized parts depicted in drawings. (a) Cup-shaped body (retracted); (b) interim body shape; (c) tongue-shaped body (relaxed).

Figure 10

Opisthaptoral sclerotized parts of Gyrodactylus sp1. (a) Central hook complex; (b) ventral bar; (c) hook; (d) hook sickle; (e) dorsal bar.

Remarks: According to the measurements and descriptions of the sclerotized parts, the present specimens resemble E. coronatus Pariselle et al. [50]. The hooks were almost identical in length but pairs I and II are discernibly slenderer than others [30]. The parasite can stretch out its retractable opisthaptor to anchor the stomach wall and withdraw it to relocate to a new site during movement (based on the observations in situ and GAP preserved specimens). As a result, E. coronatus can present two body shapes depending on the different status of opisthaptor: cup- or tongue-shaped (Fig. 4). No opisthaptor glands were observed in the stained specimens, which was different from the description of E. cichlidarum by Paperna [42].

In the eight locations (Baise, Heyuan, Liuzhou, Maoming, Nanning, Quannei, Quanzhou and Xishuangbanna) where E. coronatus coexisted with the hosts O. niloticus and C. zillii, C. zillii was always found with E. coronatus infection, while O. niloticus was only found to be infected in Liuzhou (see Table 1). Especially in Maoming, where both O. niloticus and C. zillii were monthly sampled for year-round, E. coronatus was exclusively collected from C. zillii (prevalence: 32.2%; mean intensity: 2.1). In the whole investigation, E. coronatus was not collected from S. galilaeus and O. mossambicus. This species had previously been reported from Tilapia guineensis (Coptodon guineensis) [50], Tilapia dageti (Coptodon dageti) [34], and Pseudocrenilabrus philander philander [30]. In a word, E. coronatus shows host preference to C. zillii in China and it possesses the potential to infect other cichlids. The occurrence of this species in the stomach of O. niloticus and C. zillii from China provides new localities and new host records.

Enterogyrus malmbergi Bilong Bilong, 1988

Type host: Oreochromis niloticus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Stomach.

Type locality: Sanaga River, Cameroon.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Han River, Chaozhou, Guangdong Province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Liu River, Liuzhou, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Jiatan reservoir, Chengmai, Hainan Province; Jin River, Quanzhou, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Lancang River, Lincang, Yunnan Province.

Voucher specimens observed and deposited: 32 (SYSUEMA1–31; MNHN HEL907).

Description (Figs. 5, 6a–c, 10i): Four eyespots well developed without lenses. Tegument thick and striated transversally. Opisthaptor slightly retractable, body cup-shaped. Adults 721 ± 94.2 (481–854, 31) long and 284 ± 43.3 (218–361, 31) wide. Pharynx globular 56 ± 10.3 (38–77, 31) in diameter. Dorsal anchor with shaft shorter than blade: a = 26 ± 1.3 (23–29, 31), b = 36 ± 1.1 (33–38, 31), c = 26 ± 1.4 (22–29, 31), d = 16 ± 1.1 (13–18, 31), e = 7 ± 0.7 (5–8, 31). Ventral anchor smaller than dorsal one: a = 20 ± 1 (18–22, 31), b = 15 ± 0.6 (14–16, 31), c = 10 ± 0.9 (7–12, 31), d = 10 ± 0.8 (7–11, 31), e = 5 ± 0.7 (4–7, 31). Straight-shaped robust ventral bar: x = 25 ± 1.5 (21–28, 31), w = 3 ± 0.4 (2–4, 31). Hooks short, robust and basically identical: I = 15 ± 1.1 (13–18, 29), II = 14 ± 1 (12–17, 29), III = 14 ± 0.7 (13–15, 29), IV = 15 ± 1.2 (12–17, 29), V = 15 ± 1 (13–17, 29), VI = 15 ± 1.1 (12–17, 29), VII = 15 ± 0.8 (14–17, 30). Penis spiral pattern: 3–2–1/3–1–2, 49 ± 3.9 (43–58, 31) long and 6 ± 0.8 (5–8, 31) wide at the base. Eggs oval, length 91 ± 2.8 (86–93, 5) and width 77 ± 2.7 (72–79, 5).

Figure 5

Drawings of sclerotized parts of E. malmbergi Bilong Bilong, 1988. DA, dorsal anchor; VA, ventral anchor; VB, ventral bar; Pe, penis; I–VII, hooks.

Figure 6

Different body shape of E. malmbergi Bilong Bilong, 1988 under coverslip and the motion of opisthaptoral sclerotized parts depicted in drawings. (a) Cup-shaped body (retracted); (b) interim body shape; (c) cup-shaped body (relaxed).

Remarks: The morphologies and measurements of the sclerotized parts of our specimens basically agree with the previous description of E. malmbergi, but are slightly larger [7], probably influenced by environmental conditions [11]. The in situ observation of E. malmbergi found that the wound in the stomach of hosts was larger than those caused by E. coronatus [31] and the persistence of infection will enlarge the wound. No opisthaptor glands were observed in this species, which was consistent with descriptions of other Enterogyrus species [3, 5–7, 30, 50] except E. cichlidarum which was ever described with opisthaptor glands [42]. In addition, E. malmbergi has a much less retractable opisthaptor (only cup-shaped) than E. coronatus (Figs. 4, 6).

In the ten sampling sites (Baise, Chengmai, Chaozhou, Danzhou, Liuzhou, Maoming, Nanning, Quannei, Quanzhou and Xishuangbanna), where E. malmbergi coexisted with the hosts O. niloticus and C. zillii, this parasite was always collected from O. niloticus, but not from C. zillii except in Maoming where both O. niloticus and C. zillii were found to be infected (see Table 1). Even in Maoming, year-round investigations revealed that E. malmbergi had much higher infection levels in O. niloticus (prevalence: 16.4%; mean intensity: 1.4) than in C. zillii (prevalence: 1.4%; mean intensity: 1.1). In addition, E. malmbergi was also sampled from S. galilaeus and O. mossambicus in the present study, and had previously been reported from Cichlasoma callolepis [23]. These results indicate that E. malmbergi presents host preference to O. niloticus in China, but has the potential to infect other cichlids. It is the first record of E. malmbergi in China, and with C. zillii, S. galilaeus and O. mossambicus as new host records.

Genus Cichlidogyrus Paperna, 1960

Cichlidogyrus cirratus Paperna, 1964

Type host: Sarotherodon galilaeus.

Hosts: Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Tiberias Lake, Israel.

Localities: Nanshui reservoir, Shaoguan, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Han River, Chaozhou, Guangdong Province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Shilu reservoir, Changjiang, Hainan Province; Jiatan reservoir, Chengmai, Hainan Province; Jin River, Quanzhou, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Nongba reservoir, Lincang, Yunnan Province; Lancang River, Lincang, Yunnan Province.

Voucher specimens observed and deposited: 31 (SYSUCCI1–30; MNHN HEL902).

Description (Figs. 7, 8a, b, 10a, b): Adults 809 ± 199 (362–1088, 30) long and 129 ± 21.2 (89–180, 30) wide at level of ovary. Pharynx globular 31 ± 5 (21–39, 27) in diameter. Dorsal anchor with short shaft and regularly curved blade: a = 46 ± 1.9 (42–50, 30), b = 39 ± 1.4 (36–41, 30), c = 3 ± 1.2 (1–6, 30), d = 12 ± 2.4 (7–17, 30), e = 14 ± 1.4 (11–17, 30). Arched dorsal bar: h = 16 ± 1.3 (14–19, 29), w = 9 ± 1.4 (5–13, 30), x = 41 ± 2.4 (36–45, 30), y = 14 ± 1.5 (11–18, 28). Ventral anchor with undeveloped shaft: a = 49 ± 2.1 (44–52, 30), b = 45 ± 2.3 (40–48, 30), c = 1 ± 0.9 (0–3, 30), d = 9 ± 1.5 (6–12, 30), e = 17 ± 0.8 (15–18, 30). V-shaped ventral bar: x = 37 ± 2 (32–41, 30), w = 6 ± 0.6 (5–7, 30). Hooks short: I = 15 ± 0.6 (14–17, 29), II = 13 ± 0.9 (11–15, 24), III = 16 ± 1.2 (14–20, 26), IV = 22 ± 1.3 (19–24, 29), V = 24 ± 1.3 (21–27, 28), VI = 22 ± 1.8 (15–23, 28), VII = 18 ± 1 (15–20, 27). Very long and thin coiled penis, starting in a bulb with marked heel. Accessory piece, connected with the penis bulb by a rod, coated by a large, oval and thin membrane extended to the bifurcate ends: Pe = 210 ± 20.9 (165–240, 26), He = 12 ± 3.4 (8–25, 30), Ap = 41 ± 3.3 (35–49, 30). Penis end double sharped, not blunt. Very long and thin spirally coiled vagina, no valuable length could be taken.

Figure 7

Opisthaptoral and genital sclerotized parts of Cichlidogyrus cirratus Paperna, 1964. Ap, accessory piece; DB, dorsal bar; DA, dorsal anchor; He, heel; Pe, penis; VA, ventral anchor; VB, ventral bar; Vg, vagina; I–VII, hooks.

Figure 8

Morphology of the male copulatory complex of the same specimen of Cichlidogyrus cirratus observed in different angles. (a) undigested C. cirratus, (b) digested C. cirratus.

Remarks: Cichlidogyrus cirratus was first described by Paperna [43] from the gills of Tilapia galilaea (Sarotherodon galilaeus) in Lake Tiberias, Israel, and redescribed by Ergens [13] from the gills of Tilapia nilotica (Oreochromis niloticus) in River Nile, Egypt. In the present study, C. cirratus was collected from O. mossambicus (new host), O. niloticus and Coptodon zillii, but was not found in its type host (S. galilaeus).

The long winding penis and the short first pair of hooks differentiates C. cirratus from all other congeneric species from cichlid hosts, except C. mbirizei Muterezi Bukinga et al., 2012 [39, 49]. The measurements of C. cirratus were consistent with those of C. mbirizei, and the latter was distinguished from C. cirratus in the original description by the shape of the accessory piece of the male copulatory complex (C. mbirizei without long expansion at mid-length and with two ends of rounded outgrowth versus C. cirratus with long expansion and hooked ends) and the vagina (double pitch in C. mbirizei versus sinuous in C. cirratus). However, the thin, long and transparent expansion in the middle of the accessory piece of C. cirratus was variable due to the different perspectives (Fig. 8). Moreover, two types of accessory piece extremity (hooked versus rounded outgrowth) could transform in digested specimens, when the accessory piece turns over (Fig. 8). These morphological features suggest that C. mbirizei and C. cirratus are likely synonymous. Pending genetic study, these two species are kept valid in the present study.

Cichlidogyrus cirratus (or C. mbirizei) was also recorded from non-native tilapias in Malaysia and Thailand [27, 29]. In addition, Cichlidogyrus sp. (named as C. bananensis by Xiao [59]) found in Lancang River in China was likely a misidentified C. cirratus as it shows close morphological and morphometric similarities to the latter, which was also collected from the same locality in the present study.

Cichlidogyrus halli Price & Kirk, 1967

Type host: Oreochromis shiranus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Upper Shire River, Malawi.

Localities: Gaozhou reservoir, Maoming, Guangdong province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Liu River, Liuzhou, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Jiatan reservoir, Chengmai, Hainan Province; Shilu reservoir, Changjiang, Hainan Province; Lancang River, Xishuangbanna, Yunnan Province; Hualien, Taiwan.

Voucher specimens observed and deposited: 31 (SYSUCHA1–30; MNHN HEL903).

Remarks: The morphologies and measurements of the voucher specimens in the present study agree with the previous ones from C. halli [12]. This species was also described from non-native tilapias in Brazil [22], South Africa [32], Thailand [27], Malaysia [29], Japan [33] and China [37, 59]. It is the first record of C. halli in Coptodon zillii and O. mossambicus.

Cichlidogyrus sclerosus Paperna & Thurston, 1969

Type host: Oreochromis mossambicus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Kajansi, Uganda.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Han River, Chaozhou, Guangdong Province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Liu River, Liuzhou, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Shilu reservoir, Changjiang, Hainan Province; Jiatan reservoir, Chengmai, Hainan Province; Xixi River, Xiamen, Fujian Province; Jin River, Quanzhou, Fujian Province; Min River, Fuzhou, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Nongba reservoir, Lincang, Yunnan Province; Lancang River, Lincang, Yunnan Province.

Voucher specimens observed and deposited: 31 (SYSUCSC1-30; MNHN HELxxxx).

Remarks: The morphologies and measurements of specimens in the present study agree with the previous ones of C. sclerosus [12, 45]. This species has been reported from non-native tilapias in Iraq [1], Mexico [23, 46], Colombia [25], Thailand [27], Malaysia [29], South Africa [32], Brazil [22], Japan [33] and China [28, 37, 58, 59]. Sarotherodon galilaeus represents a new host record of this parasite.

Cichlidogyrus thurstonae Ergens, 1981

Type host: Oreochromis niloticus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Nile River, Egypt.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong province; Nanshui reservoir, Shaoguan, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Boai River, Baise, Guangxi Province; Bachi River, Nanning, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Shilu reservoir, Changjiang, Hainan Province; Jiatan reservoir, Chengmai, Hainan Province; Xixi River, Xiamen, Fujian Province; Jin River, Quanzhou, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Lancang River, Lincang, Yunnan Province.

Voucher specimens observed and deposited: 32 (SYSUCTH1–31; MNHN HEL904).

Remarks: The morphologies and measurements agree with the previous ones of C. thurstonae [13, 47]. This species has been reported from non-native tilapias in Thailand [27], Malaysia [29], Brazil [22] and China [28, 37]. This is the first record of C. thurstonae from Coptodon zillii.

Cichlidogyrus tilapiae Paperna, 1960

Type host: Sarotherodon galilaeus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Jordan and coastal system, Israel.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong Province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Han River, Chaozhou, Guangdong Province; Gaozhou reservoir, Maoming, Guangdong Province; Nanshui reservoir, Shaoguan, Guangdong Province; Boai River, Baise, Guangxi Province; Liu River, Liuzhou, Guangxi Province; Bachi River, Nanning, Guangxi Province; Shilu reservoir, Changjiang, Hainan Province; Jiatan reservoir, Chengmai, Hainan Province; Songtao reservoir, Danzhou, Hainan Province; Nandu River, Haikou, Hainan Province; Min River, Fuzhou, Fujian Province; Jin River, Quanzhou, Fujian Province; Xixi River, Xiamen, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Nongba reservoir, Lincang, Yunnan Province; Lancang River, Lincang, Yunnan Province; Hualien, Taiwan.

Voucher specimens observed and deposited: 33 (SYSUCTI1–32; MNHN HEL905).

Remarks: The morphologies and measurements of the present specimens agree with the previous ones of C. tilapiae [12, 13, 41], which has been reported from non-native tilapias in Iraq [1], Mexico [23], Colombia [25], Thailand [27], Malaysia [29], Japan [33], South Africa [32], Australia [57] and Brazil [22, 56]. Cichlidogyrus haplochromii Paperna & Thurston, 1969 found in Lancang River and Guangzhou in China by Li et al. [28], Meng [37] and Xiao [59], was obviously a misidentification of C. tilapiae due to their high degree of similarities in both measurements and morphologies.

Genus Scutogyrus Pariselle & Euzet, 1995

Scutogyrus longicornis Paperna & Thurston, 1969

Type host: Sarotherodon galilaeus.

Hosts: Sarotherodon galilaeus, Oreochromis mossambicus, Oreochromis niloticus and Coptodon zillii.

Site of infection: Gills.

Type locality: Lakes Georges and Albert, Uganda.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Han River, Chaozhou, Guangdong Province; Xinfengjiang reservoir, Heyuan, Guangdong Province; Bachi River, Nanning, Guangxi Province; Boai River, Baise, Guangxi Province; Liu River, Liuzhou, Guangxi Province; Songtao reservoir, Danzhou, Hainan province; Shilu reservoir, Changjiang, Hainan Province; Jiatan reservoir, Chengmai, Hainan Province; Xixi River, Xiamen, Fujian Province; Jin River, Quanzhou, Fujian Province; Lancang River, Xishuangbanna, Yunnan Province; Nongba reservoir, Lincang, Yunnan Province; Lancang River, Lincang, Yunnan Province; Hualien, Taiwan.

Voucher specimens observed and deposited: 31 (SYSUSLO1–30; MNHN HEL908).

Remarks: The description of specimens in the present study generally agrees with the previous ones of S. longicornis in morphologies and measurements [12, 45], except that a single large and numerous very small holes were seen on the basal portion of the male copulatory organ (see Fig. 9f) which were not previously described. This species had been reported from non-native tilapias in the Philippines [2], Mexico [23], Thailand [27], Malaysia [29], South Africa [32], Brazil [22] and China [28, 37, 58, 59].

Figure 9

Scanning electron micrographs of genital sclerotized parts of species of Cichlidogyrus, Scutogyrus and Enterogyrus. (a) Male copulatory complex of C. cirratus; (b) penis end of male copulatory complex of C. cirratus; (c) male copulatory complex of C. sclerosus; (d) male copulatory complex of C. thurstonae; (e) male copulatory complex of S. longicornis; (f) penis basement of S. longicornis; (g) vagina of S. longicornis; (h) male copulatory complex of C. halli; (i) penis of E. malmbergi; (j) penis of E. coronatus.

Family Gyrodactylidae Cobbold, 1864

Genus Gyrodactylus von Nordmann, 1832

Gyrodactylus cichlidarum Paperna, 1968

Type host: Sarotherodon galilaeus.

Hosts: Oreochromis niloticus.

Site of infection: Skin, fins and rarely gills.

Type locality: Accra plain, Ghana.

Localities: A pond in Sun Yat-sen University and a fish farm in Guangzhou, Guangdong province; Gaozhou reservoir, Maoming, Guangdong province; Songtao reservoir, Danzhou, Hainan province.

Voucher specimens observed and deposited: 13 (SYSUGCH1–13).

Remarks: The morphologies and measurements of voucher specimens in the present study agree with the previous descriptions of G. cichlidarum which was firstly described by Paperna [44] in Ghana and redescribed by García-Vásquez et al. [20]. This species had also been reported from non-native tilapias in the Philippines [2] (G. niloticus was synonymized with G. cichlidarum [20]) and Mexico [46].

Gyrodactylus sp1.

Hosts: Oreochromis niloticus.

Site of infection: Gills.

Localities: Songtao reservoir, Danzhou, Hainan province.

Voucher specimens observed and deposited: 1 (SYSUSP1-1).

Description (Fig. 10): Only one GAP mounted specimen was measured under coverslip pressure. Body 373 long, 77 wide at level of uterus. Haptor, pharynx bulb and penis not measurable. Total length of anchor (hamulus) 50, shaft 32 long, point 22 long, root 22 long, aperture distance 16, proximal shaft width 7, distal shaft width 3, inner curve length 3. Anchor aperture angle 38°, anchor point curve angle 10° and inner anchor aperture angle 43°. Dorsal bar with two protuberances, 1.4 wide, 14 long. Ventral bar with two big rounded auricular processes, 19 wide, 35 long, ventral bar processes 9 long, mid-length of ventral bar processes 12 long, median portion 5 wide, ventral bar membrane 18 long. Hooks total length 23, shaft length 18, filament loop length 11, hook aperture length 3, sickle length 4, sickle proximal width 3, sickle distal width 3, instep height 0.4 and toe length 1.2.

Remarks: Although only one specimen has been collected, its characteristics of dorsal bar (with two protuberances) and ventral bar (with two large rounded auricular processes) made it resemble Gyrodactylus yacatli García-Vásquez et al., 2011 [19], which was first described from the gills and fins of O. niloticus cultured in Mexico and also from the fins of O. niloticus and Pseudocrenilabrus philander in Zimbabwe [61]. The marginal hook sickles of the present specimen are different from former descriptions (i.e., with a larger angle) [19, 61]. However, the drawings of dorsal bar in these descriptions were not consistent; the former had a straight dorsal bar but the latter possessed a dorsal bar with two protuberances. Our specimen was more like the description of Zahradníčková et al. [61], but could not be definitively identified.

Discussion

Morphological characteristics of relevant monogenean species

To date, there are eight valid African species of Enterogyrus, namely E. cichlidarum Paperna, 1963; E. malmbergi; E. melenensis Bilong Bilong, Birgi & Lambert, 1989; E. barombiensis Bilong Bilong, Birgi & Euzet, 1991; E. foratus Pariselle, Lambert & Euzet, 1991; E. coronatus; E. amieti Bilong Bilong, Euzet & Birgi, 1996, and E. crassus Bilong Bilong, Birgi & Euzet, 1996 [3, 5–7, 49]. These species were initially described with two different opisthaptor features (cup- or tongue-shaped), which were used for the division of Enterogyrus into two groups [6, 30, 50]. However, the results in the present study, based on the observation of live worms of E. coronatus and E. malmbergi in situ, did not support this hypothesis. Enterogyrus coronatus presented a variable opisthaptoral shape during anchoring on the stomach wall (opisthaptor tongue-shaped) and shifting from one location to another (opisthaptor cup-shaped). The opisthaptor of E. coronatus was more variable than that of E. malmbergi which could only present a cup-shaped opisthaptor. In addition, the opisthaptoral sclerotized parts (posterior hooks (pairs I and II), ventral anchors and ventral bar) of E. coronatus were discernibly slenderer than those of E. malmbergi. This might facilitate the extension of the opisthaptor and its penetration into the stomach wall. Pathologically, E. malmbergi caused larger wounds than E. coronatus [31] in the stomach of the host, which might be ascribed to the larger body size and less extensible opisthaptor. This inference needs further confirmation by comparing the pathologies of other Enterogyrus species with slenderer opisthaptor sclerotized parts (E. cichlidarum, E. melenensis, E. barombiensis, E. foratus and E. amieti), and that of E. crassus which possesses larger opisthaptoral sclerotized parts.

The identification of Cichlidogyrus species was primarily based on the two-dimensional morphologies of the sclerotized parts in the whole-mount specimens, e.g., GAP preserved specimens. Based on the three-dimensional morphologies of the accessory piece terminal of the male copulatory complex of C. cirratus, considerable change was detected as a result of different view angle in the present study. This hints that three-dimensional morphologies of isolated sclerotized parts by modern technical methods such as laser scanning confocal fluorescence microscopy can provide more comprehensive information for taxonomic studies [18, 53].

Monogenean fauna of exotic tilapias

Gill monogeneans from introduced tilapias have been studied widely around the world [1, 2, 22, 23, 25–29, 32, 46, 52, 56, 58], whereas few reports are available about stomach [2, 23, 30, 40], skin or fins parasites [20, 46]. However, there are no reports about urinary bladder monogeneans in invasive tilapias, though Tilapia sp. can be infected by Urogyrus cichlidarum Bilong Bilong, Birgi & Euzet, 1994 in Cameroon [4].

The introduction and spread of non-indigenous tilapias could be associated with the introduction and spread of their parasites, but the parasite species richness often decreased in comparison with that in their native range [55]. For example, it was reported that the monogenean species of O. niloticus numbered 18 in its native range [26, 48], while in the introduced areas they numbered between 1 and 7 although the species presented were similar [1, 2, 21, 22, 26–28, 32, 44, 50, 54, 56]. The similar monogenean species composition might be related to the similarity of tilapia strains cultured in different areas, e.g., genetically improved farmed tilapia (GIFT), which was widely introduced and cultured around the world. It was ever reported once that the monogenean species were completely lost as a result of tilapia introduction [17]. In the present study, the monogenean fauna of tilapias also shows different species loss in the different locations (see Table 1), e.g., only C. tilapiae was found in two sites (Haikou and Gengma) and even no parasites in Macau.

This study reported ten new host records of several monogeneans on tilapias (E. coronatus from Coptodon zillii and O. niloticus; E. malmbergi from Coptodon zillii, S. galilaeus and O. mossambicus; C. halli from Coptodon zillii and O. mossambicus; C. thurstonae from Coptodon zillii; C. cirratus from O. mossambicus; C. sclerosus from S. galilaeus), which demonstrated their lower host specificity. However, the host specificities of these species were basically consistent with previous reports [35, 51]. In addition, the distinct host preference of Enterogyrus species (E. malmbergi prefers to infect O. niloticus; E. coronatus prefers to infect Coptodon zillii), together with the preferences of Cichlidogyrus and Scutogyrus species to O. niloticus in the present study, could be ascribed to the considerable intergeneric and parental care behavior differences of hosts [35].

In China, Cichlidogyrus levequei Pariselle & Euzet, 1996 was previously reported from O. niloticus [28], but it was not collected in the present study. However, the existence of this species in China is really doubtful because authors could not provide specimens and the description was too simple to judge the species [28]. In addition, C. levequei was recorded to be specific to the host Coptodon coffea which is endemic in West Africa [48].