Description of Three New Species of Myxosporidia (Cnidaria: Myxobolidae) Parasites of Paramormyrops kingsleyae Günther, 1896 (Mormyridae) in the Nyong Basin in Cameroon

Parasitological examination of Paramormyrops kingsleyae Günther, 1896 caught in the Djolon River in Cameroon revealed the presence of three new species of Myxosporidia of the genus Myxidium Bütschli 1882 and Henneguya Thelohan, 1892 of which complete description is given in the present study. These species are: Myxidium binguelai sp. nov., parasite of the kidney that produce fusiform spores with a turgid medial part and both ends pointed. They measured 12.1 (11.1-12.9) μm long x 5.3 (4.8-5.8) μm broad; the polar capsules are ovoid, equal in size and measure 3.6 (3.0-4.1) μm x 2.5 (2.0-2.8) μm. Myxidium djolonensis sp. nov., a parasite of the gall bladder and urinary bladder, produce fusiform and elongated spores, that measure 16.3 (15.5-18.0) μm long x 6.1 (5-7.1) μm broad; its polar capsules are equal and measure 4.9 (4-5.6) x 3.5 (3-4.2) μm. Henneguya paramormyropsi sp. nov. form cyst in gill, eye and kidney. The total spore length is 29.2 (25.5-32.5) μm. The ovoid spore body, with narrowed anterior end measure 11.9 (10.7-12.8) μm x 4.5 (3.9-5.9) μm. The two separate caudal appendages measure 17.3 (14-20.3) μm. The 2 equal-sized thin and smooth valves surrounded 2 equal-sized pyriform, elongate polar capsules (5 (4.2-5.6) ×1.5 (1.2-1.9) μm) that contained 3 to 4 coils of the filament.


INTRODUCTION
Fish farming is one of the most important human activities because of the growing need of animal protein in the world [1]. Despite its food and economic importance, fish is also a favorable biotope for the development of a large number of parasites among which Myxosporidia [2,3]. Discovered by Müller [4], Myxosporidia Bütschli [5] are ubiquitous metazoans parasites of Vertebrates and Invertebrates [2]. They are of great importance in ichthyopathology because of lethal and sublethal effects related to their development in host fish, especially in fish farming [6]. More than a century after their first description based on the morphological and metrical characteristics of the spore, the worldwide Myxosporidia fauna is now estimated at 2200 species [7]. They represent 18% of Cnidaria species currently described and are distributed in 64 genera and 17 families [7]. Myxosporidia fauna in African freshwater fishes is estimated at about 280 species [8][9][10][11][12], while that of freshwater fish from Cameroon is represented by about 80 species of which 11 belong to the genus Myxidium [11] and 12 to the genus Henneguya [8]. Molnár [13] believes that, most Myxosporidia species have strict specificity towards their hosts. In addition, Ali et al. [14] suggest the existence of geographical barriers between species of Myxosporidia. Thus, taking into consideration the fish species richness, the diversity of the environment and the geographical extent of the continent, the Myxosporidia biodiversity in Africa is certainly underestimated. Several Myxosporidia species have been described on Mormyrid fish in African [15]. However, no data are available on these parasites on Paramormyrops kingsleyae Günther [16] (Mormyridae).
In a more general study of myxosporidia parasites of some food and economic importance Teleosts in Cameroon, we found for the first time in P. kingsleyae, three new species whose full description is given in the present work. These species are: Myxidium binguelai sp. nov., Myxidium djolonensis sp. nov. and Henneguya paramormyropsi sp. nov. Fish catches were done using gill nets. In the field, once fish were caught, a buttonhole was made on the ventral surface of each individual. They were immediately immersed in a 10% formalin solution before being brought to the laboratory. Systematic position of sampled fish was taken from Stiassny et al. [17]. In the laboratory, the fish were firstly examined with naked eye (eyes, fins, operculum, scales, skin) and then with the Olympus BO61 binocular stereoscopic microscope for the presence of cysts. After dissection of the fish hosts, internal organs such as gills, heart, liver, kidneys, spleen, gall bladder, urinary bladder, gonads, intestine and urethra were taken individually and examined. The contents of the cysts were identified with the objective 100x of an IVYMEN light microscope. The content of the gall bladder, urinary bladder and swim bladder was also examined between glass and glass cover slide under the microscope. Smears from kidney, spleen, liver, gonads, heart and urethra were carefully examined at the 40x objective of the microscope. Spore smears were fixed with methanol and stained with May-Grünwald-Giemsa. Drawings of unstained spores were performed using a Wild M-20 microscope equipped with a camera Lucida. Fifty spores were measured using an objective micrometer. Variables taken into consideration were those proposed by Lom et al. [18]. Microphotographs of spores were performed using an Olympus BH-2 microscope equipped with a microphotograph device. Measurements: Measurements are given in Table 1.       Etymology: The species is related to the locality of Binguela where host fish were captured.

Myxidium djolonensis sp. nov.
Vegetative form: Not observed, only mature and free spores were found in the bile and urinary bladder.
Spores: Fusiform, elongated (2.7 times longer than wide) and sometimes slightly arched, the spores have truncated ends (Figures 6-8). Each valve has 7 to 9 longitudinal striations (Figures 7 and 8). The polar capsules are ovoid and equal in size ( Figures 9 and 10). Within each of them, there are 5 to 7 coils of the filament ( Figure 10). The sporoplasm occupies the extracapsular space.
Measurements: Measurements are given in Table 2.  Averages of the parameters measured are followed by minimal and maximal values in brackets.

Henneguya paramormyropsi sp. nov.
Vegetative form: Elongated, spherical or subspherical whitish cysts were observed in the secondary gill lamellae and the aqueous humor of the eye. These cysts are polysporous and measure 590-396 x 310-252 μm. On a parasitized host fish, one can count an average    of 10 cysts. Vegetative stages were not observed in the kidney but isolated or grouped spores were found.
Spore: Spore body ovoid with narrowed anterior end ( Figure 11). Shell valves smooth and extend posteriorly with two separate caudal appendages ( Figure 11). Polar capsules are pyriform, elongated (3.3 times longer than wide) and of equal size (Figures 12 and 13). In each of them, there are three to four coils of the filament ( Figure  14). The sporoplasm occupies the rest of the spore cavity ( Figures  11 and 14).
Measurements: Measurements are given in Table 3.
Type host: Paramormyrops kingsleyae Günther [16] (Mormyridae).  Averages of the parameters measured are followed by minimal and maximal values in brackets.         The parasite of Paramormyrops kingsleyae differs from species of Myxosporidia previously described by many characters. We believe that, this parasite is new and propose to name it Myxidium binguelai sp. nov., a name that refers to the locality of Binguela where fish hosts were captured.

Myxidium djolonensis sp. nov.
With the general shape of its spore, the presently described Myxosporidia approaches species previously described in Africa or other continents. M. distichodi Kostoïngué, Faye and Toguebaye [24] (host: Distichodus engyephalus (Cyprinidae) in Chad) forms spores whose length (16.3 x 6.5 µm on average) is comparable to that of the spores of the species presently describe. However, the spores of M. distichodi are spindle-shaped, with pointed ends and a turgid medial part.
Myxidium brienomyri forms large trophozoites in the gall bladder of Brienomyrus brachyistus (Mormyridae) in Cameroon [27]. The spores of this species, being ellipsoid with rounded ends measured 13.7 x 6.47 µm on average. The maximum number of striations found on the valves is greater (12 striations) compared to 9 striations found on spores of the presently described species. In addition, their polar capsules are spherical.
These differences between the parasite of Paramormyrops kingsleyae and previously described species lead to the creation of a new species. We propose to name it Myxidium djolonensis sp. nov., a name that refers to the Djolon River in which host fish were caught.

CONCLUSION
In ichthyopathology, it is important to report new parasites and pathological conditions when they are discovered because such information may be useful in the future as a baseline for assessing the health of ecosystems in the face of global warming. Thus, the present work makes available new data on Myxosporidia parasites of Paramormyrops kinsleyae. Myxidium binguelai sp. nov., a parasite of the kidney of P. kinsleyae contains a large iodinophilous vacuole in its sporoplasm. Although the iodinophilous vacuole is not a species identification criterion for Myxobolidae, it is important to note that this is the first time that the iodinophilous vacuole is reported in a species of the genus Myxidium. The description of Myxidium binguelai sp. nov. and Myxidium djolonensis sp. nov. bring to 20 the number of species of Myxidium known in African freshwater fish and to 13 the number of species of the genus described in freshwater teleosts of Cameroon. With the description of Henneguya paramormyropsi sp. nov., 28 Henneguya species are known to infest freshwater fish in Africa while 13 are parasites of freshwater fish in Cameroon. In case of heavy infection, the rupture of cysts of Henneguya paramormyropsi sp. nov. implanted in the secondary gill lamellae and the aqueous humor of the eye can lead to a malfunction of these organs and may create lesions that would be gateways for secondary pathogens (Bacteria and Fungi). In view of the above-mentioned pathogenic effects, particular attention should be paid to the epidemiology of these parasite species in order to prevent their adverse effects on the population of Paramormyrops kinsleyae in the Nyong basin so as to handle this fish species in cultivation and promote fish yields.