First Study on Gyrodactylus (Monogenea: Gyrodactylidae) in Morocco, with Description of a New Species from Luciobarbus pallaryi and Luciobarbus ksibi (Actinopterygii: Cyprinidae)

Simple Summary Monogenean flatworms are mainly parasitic in lower aquatic vertebrates including fish, anurans and chelonians. Gyrodactylus is one of the 23 genera of Gyrodactylidae. With only 41 species described, the total number of Gyrodactylus species described from African freshwater fish still remains low. The known species represent only a fraction of the expected species richness of Gyrodactylus in Africa. In this study, we examined the gills of 738 cyprinid specimens. We isolated 26 individuals belonging to Gyrodactylus from these hosts. Twelve of these from two host species were morphologically characterized and proposed to belong to one single newly described species. In view of the importance of the cyprinid–monogenean system in studying the aquatic biodiversity and biogeography of North Africa, the present study is a substantial contribution to the parasite species inventory of these fishes. Abstract To date, 41 species of Gyrodactylus have been described from Africa. However, none of these have been reported in Morocco. After identifying and examining 738 cyprinid host specimens, 26 specimens belonging to Gyrodactylus were found to parasitize the gills of nine species of Luciobarbus, Carasobarbus, and Pterocapoeta. The current study provides new information about the presence of a new parasitic species in Morocco, the first to be characterized on a species level in the Maghreb region. It describes in detail 12 specimens of Gyrodactylus isolated from the gills of Luciobarbus pallaryi (Pellegrin, 1919) and Luciobarbus ksibi (Boulenger, 1905). Based on morphoanatomical observations, the characterization of the specimens collected indicates a species of Gyrodactylus that is new to science, described here as Gyrodactylus nyingiae n. sp. The new species is different from previously described gyrodactylids infecting African cyprinid hosts because it has a longer hamulus total length, a longer hamulus root, a downward projecting toe of the marginal hook, and a trapezium-shaped ventral bar membrane with a slightly striated median portion and small rounded anterolateral processes. This study increases the total number of Gyrodactylus spp. found in African cyprinids to four.


Introduction
Fisheries and aquaculture are important sectors that make a significant contribution by creating job opportunities for approximately 59.51 million people. These sectors consist of capture fisheries and aquaculture, which, respectively, employ 39.0 million and non-native freshwater fish species such as the silver carp Hypophthalmichthys molitrix (Valenciennes, 1844), common carp Cyprinus carpio, goldfish Carassius auratus (Linnaeus, 1758) and grass carp Ctenopharyngodon idella (Valenciennes, 1844). These fishes play a central role in aquatic ecosystems, especially with respect to their role as consumers in food chains. Their importance is increasingly recognized, making them a central focus in conservation, pollution prevention and restoration in aquatic ecosystems [23]. Despite the importance fishes offer, the introduced fishes could pose a threat to native fishes by providing a perfect opportunity for parasite transmission [24]. For this reason, it is important to have a baseline for the Moroccan native monogenean fauna of cyprinids. Therefore, and in view of the importance of the cyprinid-monogenean system in investigating the aquatic biodiversity and biogeography of North Africa, the present study aims to identify Gyrodactylus isolated from the gills of Luciobarbus pallaryi (Pellegrin, 1919) and Luciobarbus ksibi (Boulenger, 1905) in Morocco and contribute to the parasite species inventory of these fishes.

Sample Collection
During September 2014 and June 2021, a total of 28 localities covering nine different watersheds in Morocco were sampled on five different occasions for cyprinid specimens, as shown in Figure 1. The fish specimens were collected after obtaining the required permit from the Ministry of Water, Forestry and Desertification Control (sampling permit no. 62 HCEFLCD/DLCDPN/CPC/PPC). These fish samples were collected using a backpack electrofisher (Samus-725G) or gill nets when the physicochemical water parameters could not allow sampling using the electrofisher. Fish hosts were identified morphologically following [25], euthanized by severing their spinal cords and dissected immediately. The gills were fixed in accordance with [26] and some fish specimens were frozen in a portable freezer and analyzed in the laboratory. The nomenclature and the classifications of fishes are those provided in [27]. The map showing sampling localities (Figure 1) was created using QGIS v3.22.8 (QGIS Development Team 2022, QGIS Information System, Open Source Geospatial Foundation Project. http://qgis.osgeo.org, accessed on 20 January 2023).

Parasitological Examination
The fish samples were transported to the laboratory for parasitological examination. Monogeneans were isolated under a dissecting microscope (Wild Heerbrugg) from the gills (gill arches from the right side of the excised fish). With the aid of a fine needle, the parasites were picked out one by one, subsequently mounted on a glass slide and then covered with a coverslip. The slides were mounted in accordance with [28]. For worms fixed in ethanol, Hoyer's chloral hydrate was used [29] while ammonium picrate glycerine was used for frozen parasites [30]. The glass slide was left to dry for 24 h in a horizontal position before sealing the coverslip with Glyceel [31]. The type material was deposited in the collections of the research group Zoology: Biodiversity and Toxicology at Hasselt University (HU) (Diepenbeek, Belgium) (HU 838-841) and the Institut Scientifique of the Mohammed V University in Rabat (Rabat, Morocco) (ZA PPM 0101).

Identification of Representatives of Gyrodactylus
Gyrodactylus was distinguished from the other monogeneans as its members have a cylindrical body bearing two small cephalic lobes on the exterior part of the body, lack eyes and possess an opisthaptor armed with a single pair of hamuli linked by dorsal and ventral bars with 16 articulated marginal hooks (14 hooks in members of Dactylogyrus, the other monogenean genus most common on Moroccan cyprinids) [32]. The type host and additional host localities for the newly described Gyrodactylus species in the present study are depicted by blue and red circles, respectively.

Parasitological Examination
The fish samples were transported to the laboratory for parasitological examination. Monogeneans were isolated under a dissecting microscope (Wild Heerbrugg) from the gills (gill arches from the right side of the excised fish). With the aid of a fine needle, the parasites were picked out one by one, subsequently mounted on a glass slide and then covered with a coverslip. The slides were mounted in accordance with [28]. For worms fixed in ethanol, Hoyer's chloral hydrate was used [29] while ammonium picrate glycerine was used for frozen parasites [30]. The glass slide was left to dry for 24 h in a horizontal position before sealing the coverslip with Glyceel [31]. The type material was deposited in the collections of the research group Zoology: Biodiversity and Toxicology at Hasselt University (HU) (Diepenbeek, Belgium) (HU 838-841) and the Institut Scientifique of the Mohammed V University in Rabat (Rabat, Morocco) (ZA PPM 0101).

Identification of Representatives of Gyrodactylus
Gyrodactylus was distinguished from the other monogeneans as its members have a cylindrical body bearing two small cephalic lobes on the exterior part of the body, lack eyes and possess an opisthaptor armed with a single pair of hamuli linked by dorsal and ventral bars with 16 articulated marginal hooks (14 hooks in members of Dactylogyrus, the other monogenean genus most common on Moroccan cyprinids) [32].

Morphological Characterization of Members of Gyrodactylus
Light microscopy using both phase and differential interference contrast approaches was used to study the shape and dimensions of sclerotized structures, which were viewed under a ×100 oil immersion objective on a Leica DM2500 optical microscope using Las X software v3.6.0.20104 fitted with a Leica DMC4500 camera. The whole mount, attachment organ, and male copulatory organ (MCO) (when present) on each specimen were photographed. The haptoral morphometrics (26 point-to-point measurements) followed the measurements proposed by [34]; these were taken using ImageJ v1.53k software (available at http://imagej.nih.gov/ij accessed on 15 September 2021) and only for specimens preserved using Hoyer's solution. These measurements were given in micrometers (µm) as the mean, followed by the range in parentheses and the number of structures (n) measured for each metric. The micrographs taken were used to draw taxonomically important structures using Inkscape v1.2.

Specimens Examined and Individuals of Gyrodactylus Isolated
A total of 738 fish specimens belonging to three genera (Luciobarbus Heckel, 1843, Carasobarbus Karaman, 1871, and Pterocapoeta Günther, 1902) were collected. Thirteen cyprinid fish species were identified and their gills were examined for infection with species of Gyrodactylus (Table 1). A total of nine out of the 13 species were found to be infected with representatives of Gyrodactylus (n = 26).

Infection Parameters
The infection parameters of examined hosts are shown in Table 2.

Characterization of a New Species of Gyrodactylus
All the isolated flatworms belonging to Gyrodactylus showed the diagnostic features of this genus: gyrodactylid monogeneans with an opisthaptor with one pair of haptoral anchors surrounded by 16 marginal hooks. The measurements are given in Table 3 (Table 3) and the similarity in the shape of the marginal hook sickle ( Figure 4) suggest that the worms infecting the two host species are conspecific. Table 1. Cyprinid specimens collected, their localities and number of individuals belonging to Gyrodactylus infecting the hosts (note that the number of parasites collected might be an underestimation because only the right gill arches were screened for parasites).  6.2 (5.5-6.5) 9 6.6 6.3 (5.5-6.6) 10 Marginal hook sickle proximal width (MHSPW) 4.6 (3.9-5) 9 5.

Remarks
The comparison with other gyrodactylid species is based on the phenotypic similar ities to known parasite species and their occurrence from related hosts. From the three species of Gyrodactylus recorded from cyprinids in Africa, the newly described species o Gyrodactylus can be differentiated by the longer hamuli; G. nyingiae n. sp. 76.5 (65.9-88.2 compared to a hamulus total length in G. ivindoensis of 55 (52-58), 32.1 (23-33) in G. kyogae and 43.3 (35.1-51.5) in G. paludinosus. Like G. paludinosus, G. kyogae has an upward pro jecting toe, in contrast to that of G. nyingiae n. sp. whose toe points downwards. Addition ally, the MCO of G. nyingiae n. sp. has one principal spine and five to six smaller spines arranged in a single row (Figures 2 ad 3), in contrast to G. kyogae, which has an unarmed MCO [35]. Gyrodactylus kyogae, in contrast to the other three species, lacks a ventral bar membrane. Gyrodactylus ivindoensis has shorter marginal hooks and a total marginal hook length of 22 (21)(22)(23)(24) compared to that of G. nyingiae n. sp., which is 34.8 (31.7-42.1). When comparing the relative length of the root to the hamulus total length respectively, G nyingiae n. sp. (26.7 vs. 76.4), G. ivindoensis (19.4 vs. 55) and G. paludinosus (15.4 vs. 43.3 have similar ratios of the root length to the total hamulus length (ca. 1:2.8). Gyrodactylus kyogae (9.2 vs. 33.1) on the other hand has a different ratio of the relative root length to the total hamulus length (1:3.5).
Due to the important biogeographical connections between the Middle East and the Maghreb region during the Cenozoic period in the dispersal of freshwater fish fauna, it is
Due to the important biogeographical connections between the Middle East an Maghreb region during the Cenozoic period in the dispersal of freshwater fish fauna

Remarks
The comparison with other gyrodactylid species is based on the phenotypic similarities to known parasite species and their occurrence from related hosts. From the three species of Gyrodactylus recorded from cyprinids in Africa, the newly described species of Gyrodactylus can be differentiated by the longer hamuli; G. nyingiae n. sp. 76.5 (65.9-88.2) compared to a hamulus total length in G. ivindoensis of 55 (52-58), 32.1 (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) in G. kyogae and 43.3 (35.1-51.5) in G. paludinosus. Like G. paludinosus, G. kyogae has an upward projecting toe, in contrast to that of G. nyingiae n. sp. whose toe points downwards. Additionally, the MCO of G. nyingiae n. sp. has one principal spine and five to six smaller spines arranged in a single row (Figures 2 and 3), in contrast to G. kyogae, which has an unarmed MCO [35]. Gyrodactylus kyogae, in contrast to the other three species, lacks a ventral bar membrane. Gyrodactylus ivindoensis has shorter marginal hooks and a total marginal hook length of 22 (21)(22)(23)(24) compared to that of G. nyingiae n. sp., which is 34.8 (31.7-42.1). When comparing the relative length of the root to the hamulus total length respectively, G. nyingiae n. sp. Due to the important biogeographical connections between the Middle East and the Maghreb region during the Cenozoic period in the dispersal of freshwater fish fauna, it is interesting to compare the Gyrodactylus fauna of the Iranian region with the North African ones [4,36]. The freshwater species of Gyrodactylus mentioned by [37] and [38] were either known to be from Europe or Central Asia, or undescribed. It is therefore productive to compare G. nyingiae n. sp. with widespread Palearctic species of Gyrodactylusinfecting cyprinids, several of which are reminiscent of G. nyingiae n. sp. in marginal hook morphology. This includes Gyrodactylus mutabilitas Bychowsky, 1957, and Gyrodactylus scardiniensis Glaeser, 1974, which can both be distinguished from G. nyingiae n. sp. by virtue of their shorter hamulus root (max. of 20 in G. mutabilitas and max. of 23 in G. scardiniensis versus min. of 24 in G. nyingiae n. sp.), and Gyrodactylus schulmani Ling, 1962, which has a hamulus of a total length of a max. of 44, shorter than the minimum of 66 of G. nyingiae n. sp. A Gyrodactylus species described from a fish species endemic to Iran is Gyrodactylus jalalii Vanhove, Boeger, Muterezi Bukinga, Volckaert, Huyse and Pariselle, 2012, a parasite of the cichlid host Iranocichla hormuzensis Coad, 1982. It can easily be distinguished from G. nyingiae n. sp. by its more pronounced ventral bar auricles and the sub rectangular ventral bar membrane, which contrast the properties of G. nyingiae n. sp. including its small rounded anterolateral processes and trapezium-shaped ventral bar membrane. Following [39], Gyrodactylus molnari Ergens, 1978, infecting Cyprinus carpio Linnaeus, 1758, in Iraq has a shorter hamulus length (55-65) compared to G. nyingiae n. sp. (65.9-88.2). Additionally, G. molnari has a longer dorsal bar (15)(16)(17)(18), compared to G. nyingiae n. sp. (9.9-13.4), and an entirely different shape of the marginal hook sickle. Following [40,41], Gyrodactylus sprostonae Ling, 1962, was found on Cyprinus carpio in Iran. It has a longer dorsal bar (17.4-20) compared to G. nyingiae n. sp. (9.9-13.4) and shorter total hamulus length (48.47-54.23) compared to G. nyingiae n. sp. (65.9-88.2). Due to the fact that the goldfish (Carassius auratus) has been widely introduced in many countries including Morocco, it is also interesting to compare G. nyingiae n. sp. with Gyrodactylus kobayashii Hukuda, 1940, previously isolated from goldfish in central China [42]. G. nyingiae n. sp. has a MCO which resembles that of G. kobayashii with both possessing one principal spine and 5-6 smaller spines. Both species also have slightly curved marginal hook sickles. However, the marginal hook sickle in G. kobayashii has a tip that terminates beyond the limits of its toe while that of G. nyingiae n. sp. has its tip in line with the distal end of the toe. Additionally, G. nyingiae n. sp. has a longer hamulus (76.4 vs. 59.3), a longer sickle (47.4 vs. 40.5), a longer hamulus root (26.7 vs. 21.6), and a longer marginal hook shaft (28.7 vs. 23.3) than G. kobayashii has. Therefore, G. nyingiae n. sp. can be distinguished from the aforementioned Gyrodactylus species by virtue of its longer total hamulus length, longer hamulus root, small rounded anterolateral process and trapezium-shaped ventral bar membrane.

Multivariate Statistics
The morphological variation of the 12 specimens of Gyrodactylus was visualized based on a PCA performed on 19 standardized haptoral morphometric characters. The first two principal component axes contributed to 25.9% and 19.6% of the variation, respectively ( Figure 5).
The biplot shows no clear separation which includes all the 12 specimens belonging to Gyrodactylus. From the PCA biplot, we can confirm that we are dealing with a single species described herein as Gyrodactylus nyingiae n. sp. To better illustrate how the newly identified Gyrodactylus nyingiae n. sp. compares to other previously described species of Gyrodactylus, we performed a PCA analysis based on the mean values of 11 haptoral morphometric parameters, i.e., HPL, HSL, HRL, HTL, MHTL, MHSHL, MHSL, MHSPW, MHSDW, MHAD and VBTL (Table 4).

Multivariate Statistics
The morphological variation of the 12 specimens of Gyrodactylus was visualized based on a PCA performed on 19 standardized haptoral morphometric characters. The first two principal component axes contributed to 25.9% and 19.6% of the variation, respectively ( Figure 5).  Table 3). The first two principal components explained 29.7% and 23.0% of the total variation, respectively ( Figure 6).
With the average values from the supplementary individuals, the results of the PCA indicate a distinct difference between the Gyrodactylus species that were previously described and the newly described species mentioned in this study. The first two principal components explained 29.7% and 23.0% of the total variation, respectively ( Figure 6).

Discussion
Gyrodactylus nyingiae n. sp. is the first described species of Gyrodactylus in Morocco and to the best of our knowledge is the first member of this genus to be identified on a species level in the Maghreb region. Monogenean parasites have been reported in Tunisian aquatic vertebrates, including both marine and freshwater hosts. However, not even a single Gyrodactylus species has been recorded from the examined hosts [43]. The species reported in the current study is also the first gyrodactylid to be described from Luciobarbus in Africa, as previous studies on gyrodactylids infecting cyprinids in Africa have focused on the small barbs belonging to Enteromius, with only three Gyrodactylus species having being described so far [14]. Since Gyrodactylus is a genus of monogeneans with high diversity and minimal morphological variation, it has become a common practice to use a combination of morphological and molecular information for describing and delimiting new species [44,45]. However, due to the limited number of specimens in our study, we opted to use all of the individuals for phenotypic characterization. This decision was based on the fact that our study aimed to unravel the diversity of branchial monogeneans in the hosts, which had not been previously documented.
The current study's low number of gyrodactylids isolated from cyprinid hosts could be due to the fact that only the gills were examined for parasites. Similarly, Louizi et al. [46] found a species depauperate fauna and low abundances of gill-infecting monogeneans on native cichlid fishes in Morocco. In addition to the low prevalence and possible seasonality of members of Gyrodactylus, less research, a lack of reports on infections, a lack of understanding of relationships between these monogeneans and cyprinid hosts [14], and environmental conditions in Morocco's freshwater ecosystems might limit the species richness and abundance of certain monogenean taxa. On the other hand, Dactylogyrus reaches higher species richness and higher infection intensities in Moroccan cyprinid-monogenean systems [5,47]. It is also worth noting that the low sample size of the present study is only an indicative value of the population size of the new species of Gyrodactylus in the host.
More research is needed on the African continent to understand the relationship, evolutionary history, and development of gyrodactylids and their hosts, as it is endowed with a diverse endemic fish fauna that undoubtedly possess undiscovered parasite diversity [48,49].

Conclusion
Based on the morphoanatomical observation of opisthaptoral parts of 12 individuals of Gyrodactylus in the current study, we describe a new species infecting two cyprinid hosts for the first time in Morocco and the Maghreb region. The new species is different from previously described gyrodactylids infecting cyprinid hosts because it has a longer total hamulus length, a longer hamulus root, a downward projecting toe, trapeziumshaped ventral bar membrane with slightly striated median portion and small rounded anterolateral processes.