Revisiting the type material of two African Diplozoinae (Diplozoidae: Monogenea), with remarks on morphology, systematics and diplozoid specificity.

The morphological characterisation of Diplozoidae spp. is highly reliant on the details of the sclerotised components of the hooks and clamps in the haptor. Only six species of Paradiplozoon (Diplozoinae) have been described from Africa, four of which have adequate morphological and even comparative ITS2 rDNA data available. However, the descriptions of Paradiplozoon ghanense (Thomas, 1957) and Paradiplozoon aegyptense (Fischthal & Kuntz, 1963) lack essential taxonomic information, specifically the details for their haptoral sclerites. As such, all available material from museum collections for these two species were studied using light microscopy to supplement the original morphometric descriptions. The holotype and paratypes of P. aegyptense were studied, but only voucher material for P. ghanense could be sourced. However, this voucher material for P. ghanense was deposited by the species authority and bore a striking resemblance to the illustrations and collection details from the original description. They were thus identified as the type series for the taxon, with a lectotype and paralectotype designated. Both P. ghanense and P. aegyptense could be readily distinguished from other taxa based on the supplementary data generated here, supporting their distinctness. The haptoral sclerites of P. aegyptense were most similar to those of Paradiplozoon krugerense Dos Santos & Avenant-Oldewage, 2016, also described from Labeo spp., while the sclerites of P. ghanense were most similar to Paradiplozoon bingolense Civánová, Koyun & Koubková, 2013 and Paradiplozoon iraqense Al-Nasiri & Balbuena, 2016. Additionally, a voucher of P. aegyptense collected from the alestid type host of P. ghanense was reidentified as the latter species here. This greatly simplified the known host specificity for Paradiplozoon spp. in Africa, with P. aegyptense now exclusively reported from Cypriniformes (Cyprinidae and Danionidae), and P. ghanense restricted to Characiformes (Alestidae). The occurrence of all diplozoids from non-cyprinoid hosts was also investigated and several records of diplozoids occurring on non-cyprinoid hosts were collated and scrutinised. Excluding the two instances of diplozoids described and exclusively occurring on Characiformes fishes (P. ghanense and Paradiplozoon tetragonopterini (Sterba, 1957)), most other non-cyprinoid collections appear sporadic and unsubstantiated, but warrant further investigation supported by diligent taxonomic data. Even though the morphometric descriptions of both P. ghanense and P. aegyptense were fully reported on here, additional material will be needed to study their genetic profiles and phylogeny.

Additional data on Spinitectus petterae (Nematoda: Rhabditida) from Clarias gariepinus (Siluriformes: Clariidae) in the Vaal River system: conserved morphology or high intraspecific genetic variability?

Two species of Spinitectus Fourment, 1884 have been recorded from southern Africa, namely Spinitectus polli Campana-Rouget, 1961 and Spinitectus petterae Boomker, 1993, both from the Limpopo River system. Spinitectus petterae was described from North African catfish, Clarias gariepinus (Burchell), whereas S. polli infects squeakers, Synodontis spp. During parasitological surveys in the Vaal River system (Orange River catchment), Spinitectus specimens were collected from C. gariepinus. These systems are adjacent but not connected. Therefore, this study aimed to identify the specimens collected using morphological and molecular techniques. The morphological study included light and scanning electron microscopy of whole specimens and excised spicules. Specimens were genetically characterised using 18S rDNA, 28S rDNA and cox1 mtDNA. Additionally, immature specimens of S. petterae were collected near the type locality. Morphological characteristics were most similar to S. petterae from C. gariepinus, whereas genetic data were dissimilar to all available data for the genus. Additional morphological characteristics noted for S. petterae in the present study were the details of the left and right spicule structure and the porous structures on the pseudolabia. Specimens from the Vaal River system differed from those originally described as S. petterae by additional spines posterior to the third ring, lacking caudal alae and variable total body and male oesophagus length. Based on 18S rDNA, haplotypes from the type locality varied only slightly from the study material, supporting the morphological identification. However, 28S rDNA and, more conspicuously, cox1 mtDNA displayed substantial variation between specimens from these localities, which needs further investigation. Haplotypes generated in the present study were highly dissimilar to those characterised for S. petterae from Tanzania and Egypt. Nevertheless, the nematodes collected from C. gariepinus in the Vaal River system are considered S. petterae. This study expands the geographical distribution and adds additional morphological and genetic information for S. petterae, contributing to the limited knowledge of African species of Spinitectus.

Aspects of the pathology induced by Spinitectus petterae Boomker, 1993 in the stomach of Clarias gariepinus (Burchell, 1822) using light and scanning electron microscopy.

Spinitectus spp. (Rhabdochonidae) are enteric nematodes characterized by annular spines. At the anterior end, these spines assist attachment and aid penetration into the host tissue. During parasitological surveys of the Vaal River system from three localities, below the Vaal River Barrage in the Vaal Dam surrounding UJ island and below the Grootdraai Dam, Spinitectus specimens were collected from the stomach lining of the sharptooth catfish, Clarias gariepinus (Burchell, 1822). Histopathological effects induced by Spinitectus petterae Boomker, 1993 on C. gariepinus has not been studied. Thus, the aim of this study was to describe the histopathology induced by S. petterae. For light microscopic examination, tissue samples with attached S. petterae were sectioned and stained with haematoxylin and eosin. Additionally, attached nematodes were also studied using scanning electron microscopy. Leukocytes were counted with the Disector principle. Standard infection parameters (prevalence, mean intensity and abundance) were calculated and compared to host parameters. Prevalence varied greatly (11.77% to 100%) between localities. Histopathology induced by S. petterae to C. gariepinus stomach (cardiac region) consisted of significant leukocyte infiltration, acute ulcerations and chronic granuloma formation. This was similar to the pathology of other Spinitectus occurring in host anterior intestine and stomach, but granuloma formation had not been previously reported and this suggests chronic infection in wild caught fish.

First isolation and scanning electron microscopy of haptoral sclerites of Macrogyrodactylus (Monogenea).

Macrogyrodactylus congolensis (Prudhoe, 1957) is one of six species of Macrogyrodactylus, all of which are endemic to Africa. This monogenean is a host-specific ectoparasite of the African sharptooth catfish, Clarias gariepinus (Burchell, 1822). It attaches to the host with a posterior haptor armed with sclerites. The specific morphology of sclerites is taxonomically significant and usually studied using light microscopy. The aim of the present study was to confirm the identification of macrogyrodactylid parasites using classic morphology (light microscopy of glycerine ammonium picrate mounted specimens) and molecular techniques (18S rDNA, ITS rDNA and cytochrome oxidase subunit 1 (COI) mtDNA). Additionally, the sclerites were accurately described with a technique not previously used for the genus, whereby haptoral sclerites were isolated by removing the encapsulating soft tissue with a digestion buffer and studied with scanning electron microscopy (SEM). Morphology and morphometry of studied specimens corresponded to available data for M. congolensis, confirming the identity of the parasite. All previous descriptions were summarized in a table and discrepancies discussed. Molecular analysis also confirmed the specimens to be M. congolensis, but ITS rDNA and COI mtDNA was more reliable than 18S rDNA in this regard. The isolation of haptoral sclerites and their study using SEM was successful, resolving the morphology of all sclerites. This study provided the first reconstruction of the haptor of a Macrogyrodactylus species following SEM analysis, as well as the first mtDNA for M. congolensis. Further study of isolated haptoral sclerites of other macrogyrodactylids is required to determine the full benefits of studying their isolated sclerites.

Light and scanning electron microscopy of the effects of Macrogyrodactylus congolensis (Prudhoe, 1957) on the skin of the African sharptooth catfish Clarias gariepinus (Burchell, 1822).

Clarias gariepinus (Burchell, 1822) is one of the two most actively cultured freshwater fish in Africa and therefore, economically important. Specimens of this species were purchased from a fish farm near Hartbeespoort Dam (North West, South Africa) and introduced into the tanks of the research aquarium in the Department of Zoology at the University of Johannesburg. However, the skin of these fish was infected with Macrogyrodactylus congolensis (Prudhoe, 1957), which proliferated profusely in the favourable conditions of the aquarium, posing a potential threat to its host. The current study was aimed at examining the pathology caused by M. congolensis on the skin of C. gariepinus. Species identification of the parasite was confirmed using light microscopy (LM), scanning electron microscopy (SEM) and DNA barcoding of the internal transcribed spacer region. Examination of the pathology was studied using LM of haematoxylin and eosin-stained sections (epoxy embedded) and SEM of parasites attached to the hosts' skin. Infected skin exhibited excessive mucus production, corroborated by an increased number of mucus cells alongside proliferated and abnormally enlarged club cells, resulting in varying thickness of the epidermal layer. At the site of attachment, the basement membrane detached from the dermis. Hamulus points and marginal hooks of the parasite pierce through the hosts' skin resulting in tearing. Epidermal cells and melanin granules were observed in the intestinal lumen of the parasite. Melanin granules were absorbed by the parasite's intestinal epithelium confirming that the parasite feeds on host skin tissue.

An alien parasite affects local fauna-Confirmation of Sinergasilus major (Copepoda: Ergasilidae) switching hosts and infecting native Silurus glanis (Actinopterygii: Siluridae) in Hungary.

In 2016, an intense copepod infection was recorded from a reservoir in proximity to the Danube River in Hungary from visibly emaciated wels catfish, Silurus glanis. The parasite-induced pathology was described but parasite identity was not conclusive. Additional sample collections in 2017 and 2018 allowed for identification using both light and scanning electron microscopy, alongside genetic characterisation. The copepods were confirmed to be ergasilids, Sinergasilus major, distinctly different from any previous infection on silurids in Europe. This is the first record of this parasite from Hungary and the first host record from wels catfish.

Parasite diversity of introduced fish Lates niloticus, Oreochromis niloticus and endemic Haplochromis spp. of Lake Victoria, Kenya.

This study reports on the status of metazoan fish parasites in Lake Victoria following the establishment of introduced Lates niloticus (Latidae) and Oreochromis niloticus (Cichlidae) and changes in environmental quality. For this study, 412 fish specimens were examined for parasites: 103 L. niloticus, 165 O. niloticus, 82 Haplochromis piceatus and 62 H. humilior (endemic cichlids). In total, 25 parasite taxa were identified: Lates niloticus (6), O. niloticus (19) and Haplochromis spp. (13). The myxosporean Henneguya ghaffari, prevalence (P) = 79% and the monogenean Diplectanum lacustris (P = 34%), were the dominant parasites on L. niloticus. Myxobolus sp. (P = 44%) was dominant on O. niloticus while for the haplochromines, metacercariae of 'Neascus' sp. (Diplostomidae) was dominant (P = 37%). Contrary to reports of high diversity of monogeneans on endemic species, the haplochromines harboured only Cichlidogyrus gillardinae (P = 6.9%). Oreochromis niloticus harboured seven monogenean species: Cichlidogyrus sclerosus, C. halli, C. tilapiae, C. quaestio, Scutogyrus longicornis, Gyrodactylus cichlidarum and G. malalai-they appear to have been co-introduced with the fish. Cichlidogyrus gillardinae, C. quaestio, G. malalai and the acanthocephalan Acanthogyrus (Acathosentis) tilapiae are recorded for the first time in Lake Victoria, representing new geographical records. Presence of A. (A) tilapiae in Haplochromis spp. indicates possible spillover from O. niloticus. Low prevalence and diversity of monogeneans, crustaceans and glochidia on the fish corresponded with increased pollution in the lake. Overall, changing environmental conditions and introductions of fish species have contributed to parasite community changes in Lake Victoria.

Review on the molecular study of the Diplozoidae: analyses of currently available genetic data, what it tells us, and where to go from here.

The use of molecular tools in the study of parasite taxonomy and systematics have become a substantial and crucial component of parasitology. Having genetic characterisation at the disposal of researchers has produced mostly useful, and arguably more objective conclusions. However, there are several groups for which limited genetic information is available and, coupled with the lack of standardised protocols, renders molecular study of these groups challenging. The Diplozoidae are fascinating and unique monogeneans parasitizing mainly freshwater cyprinid fishes in Europe, Asia and Africa. This group was studied from a molecular aspect since the turn of the century and as such, limitations and variability concerning the use of these techniques have not been clearly defined. In this review, all literature and molecular information, primarily from online databases such as GenBank, were compiled and scrupulously analysed for the Diplozoidae. This was done to review the information, detect possible pitfalls, and provide a "checkpoint" for future molecular studies of the family. Hindrances detected are the availability of sequence data for only a limited number of species, frequently limited to a single sequence per species, and the heavy reliance on one non-coding ribosomal marker (ITS2 rDNA) which is difficult to align objectively and displays massive divergences between taxa. Challenging species identification and limited understanding of diplozoid species diversity and plasticity are also likely restricting factors, all of which hamper the accurate taxonomic and phylogenetic study of this group. Thus, a more integrated taxonomic approach through the inclusion of additional markers, application of more rigorous morphological assessment, more structured barcoding techniques, alongside thorough capturing of species descriptions including genetypes, genophore vouchers and reference collections in open sources are encouraged. The pitfalls highlighted are not singular to the Diplozoidae, and the study of other groups may benefit from the points raised here as well.

Additional Morphological Features and Molecular Data of Paracamallanus cyathopharynx (Nematoda: Camallanidae) Infecting Clarias gariepinus (Actinopterygii: Clariidae) in Kenya.

This study provides additional taxonomic features based on scanning electron microscopy (SEM) and molecular data for Paracamallanus cyathopharynx ( Baylis, 1923 ) (Nematoda: Camallanidae). Parasites were collected from the posterior end of the intestine of cultured freshwater Sharptooth catfish Clarias gariepinus (Burchell, 1822) from Kibos fish farm, Kisumu County, Kenya, from December 2017 to March 2018. Additional taxonomic features recorded for P. cyathopharynx include the occurrence of 4-5 equal length digitate processes on the caudal end of the female, 4 processes (2 smaller and 2 larger) on the male caudal end, and a description of the shape of the distal tip of the right spicule. The study provides SEM images of the exposed sclerotized buccal capsule. This gives more information on the tridents, the sclerotized plate that extends laterally from the buccal capsule, and the narrow isthmus separating the anterior buccal capsule from the posterior. The prevalence, intensity, mean intensity, and mean abundance was 52.91%, 2-38, 12.37 and 6.54, respectively. 18S rDNA fragments were amplified, sequenced, and compared to other camallanid taxa, and 18S data confirmed the identity of the newly obtained sequences (MN396556) as that of P. cyathopharynx, being identical to sequence DQ813445 from Tanzania. This represents the first geographical record of P. cyathopharynx in Kenya.

Gyrodactylus magadiensis n. sp. (Monogenea, Gyrodactylidae) parasitising the gills of Alcolapia grahami (Perciformes, Cichlidae), a fish inhabiting the extreme environment of Lake Magadi, Kenya.

A new species of Gyrodactylus von Nordmann, 1832 is described from the gills of Alcolapia grahami, a tilapian fish endemic to Lake Magadi. This alkaline soda lake in the Rift Valley in Kenya is an extreme environment with pH as high as 11, temperatures up to 42 °C, and diurnal fluctuation between hyperoxia and virtual anoxia. Nevertheless, gyrodactylid monogeneans able to survive these hostile conditions were detected from the gills the Magadi tilapia. The worms were studied using light microscopy, isolated sclerites observed using scanning electron microscopy, and molecular techniques used to genetically characterize the specimens. The gyrodactylid was described as Gyrodactylus magadiensis n. sp. and could be distinguished from other Gyrodactylus species infecting African cichlid fish based on the comparatively long and narrow hamuli, a ventral bar with small rounded anterolateral processes and a tongue-shaped posterior membrane, and marginal hooks with slender sickles which are angled forward, a trapezoid to square toe, rounded heel, a long bridge prior to reaching marginal sickle shaft, and a long lateral edge of the toe. The species is also distinct from all other Gyrodactylus taxa based on the ITS region of rDNA (ITS1-5.8s-ITS2), strongly supporting the designation of a new species. These findings represent the second record of Gyrodactylus from Kenya, with the description of G. magadiensis bringing the total number of Gyrodactylus species described from African cichlids to 18.

TITLE: Gyrodactylus magadiensis n. sp. (Monogenea, Gyrodactylidae), parasite des branchies d'Alcolapia grahami (Perciformes, Cichlidae), un poisson habitant l'environnement extrême du lac Magadi au Kenya. ABSTRACT: Une nouvelle espèce de Gyrodactylus von Nordmann, 1832 est décrite à partir des branchies d'Alcolapia grahami, un tilapia endémique du lac Magadi. Ce lac de soude alcaline dans la vallée du Rift au Kenya est un environnement extrême avec un pH allant jusqu'à 11, des températures allant jusqu'à 42 °C et des fluctuations diurnes entre l'hyperoxie et l'anoxie virtuelle. Néanmoins, des Monogènes Gyrodactylidae capables de survivre dans ces conditions hostiles ont été détectés sur les branchies du tilapia de Magadi. Les vers ont été étudiés par microscopie optique, les sclérites isolés ont été observés au microscope électronique à balayage et des techniques moléculaires ont été utilisées pour caractériser génétiquement les spécimens. Le gyrodactylidé est décrit comme étant Gyrodactylus magadiensis n. sp. et se distingue des autres espèces de Gyrodactylus infectant les cichlidés d'Afrique grâce à ses hamuli relativement longs et étroits, à une barre ventrale avec de petits processus antérolatéraux arrondis et à une membrane postérieure en forme de langue, ainsi qu'à des crochets marginaux à faucilles minces inclinées vers l'avant, un trapèze à bout carré, un talon arrondi, un long pont avant d'atteindre la faucille marginale et un long bord latéral de l'extrémité. L'espèce est également distincte de tous les autres taxons de Gyrodactylus sur la base sur la région ITS de l'ADNr (ITS1­5.8s­ITS2), ce qui soutient fortement la désignation d'une nouvelle espèce. Ces découvertes représentent la seconde mention d'un Gyrodactylus au Kenya, et la description de G. magadiensis amène à 18 le nombre total d'espèces de Gyrodactylus décrites parmi les cichlidés d'Afrique.

Masenia nkomatiensis n. sp. (Digenea: Cephalogonimidae) from Clarias gariepinus (Burchell) (Clariidae) in Incomati Basin, Mozambique.

A new species of Masenia Chatterji, 1933 is described based on material from the intestine of Clarias gariepinus (Burchell) in the Incomati River, Mozambique. The combination of morphological characteristics and analysis of 18S and 28S rDNA sequences delineated the specimens found in the present study as a distinct species. The new form is distinguished from other Masenia spp. in having a large reniform seminal receptacle, a cirrus-sac ending anterior to the ventral sucker, intestinal caeca extending into the hindbody to the level of the posterior testis, and the vitelline fields extending anteriorly to the ventral sucker and posteriorly to the middle of the ovary. Notably, the new form is the only record of African species having a sac-shaped excretory vesicle. Analysis of 28S rDNA sequence data supported its placement in the Cephalogonimidae Looss, 1899. 18S analyses also supported its placement in this family but showed it was not closely related to Masenia bangweulensis (Beverley-Burton, 1962), the sole other African species for which genetic data is currently accessible. The total pairwise differences for 18S and 28S sequences showed the new form differing from other cephalogonimids. Thus, both morphological and genetic characteristics indicate that the present form represents a distinct species, here described as Masenia nkomatiensis n. sp. An updated key to African Masenia spp., now five, is provided.

Using scanning electron microscopy (SEM) to study morphology and morphometry of the isolated haptoral sclerites of three distinct diplozoid species.

Diplozoidae infects the gills of cyprinid fishes in Africa, Europe, and Asia. Traditionally the hardened internal structures, crucial for identification of diplozoid species, are studied using light microscopy. Recently, the sclerotised haptoral structures of an African diplozoid, Paradiplozoon vaalense, were successfully isolated and visualised using scanning electron microscopy (SEM). In this paper, the haptoral sclerites of three diplozoid species are compared using SEM for the first time. Paradiplozoon ichthyoxanthon and Paradiplozoon vaalense occur on Labeobarbus and Labeo species, respectively, in the Vaal River system, South Africa, while Diplozoon paradoxum is widely-distributed in Europe and Asia, infecting several host species. Diplozoon paradoxum is a well-studied species, as well as being the type species of the family and ideal for inclusion in an exploratory study for comparative purposes. SEM study of D. paradoxum and P. ichthyoxanthon provided valuable information regarding surface morphology of the attachment structures hitherto not observed. Elaborate morphometric study of the haptoral hooks were incorporated, adding 12 point-to-point measurements to current morphometric characteristics. The results were analysed statistically, and significant differences support absolute separation (100.00%) of the three species following discriminant analysis. These point-to-point measurements could also be used for light microscopical study in the future and aid species delimitation within the Diplozoidae.