Study of Diplostomum (Digenea: diplostomoidea) in South Africa: diversity and effect of metacercariae on fish behaviour
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A large and widely distributed group of parasites within the genus Diplostomum (Digenea: Diplostomoidea) utilises a complex life cycle with life stages that parasitise freshwater snails, fish (intermediate hosts) and piscivorous birds (definitive hosts). Metacercariae of Diplostomum infecting the eyes (lens, vitreous humour, and retina) and brains of fish, have a well-known reputation for their pathogenicity in aquaculture fish farms. In taxonomy, the genus Diplostomum have been a controversial topic for many years because identification of most of the nominal species currently known have been based solely on morphological characteristics of the life stages. To date, almost 80 nominal species of Diplostomum have been reported worldwide; with the majority of the species recorded from the Palearctic region. However, most of the morphology-based identifications of species within this genus require critical revision due to difficulties in identifying larval stages based on their simple morphology and disagreements among parasitologists of the validity of some of the reported species. The application of molecular methods based on multiple genetic markers has increased available knowledge on the species diversity within Diplostomum in the last decade, making accurate identification of cryptic species possible (by primary use of mitochondrial markers). So far, based on the development of the molecular approach, eight species and 38 unidentified species-level genetic lineages have been reported globally. In Africa, only eight species of Diplostomum were described based on morphology and only one species from Nigeria has been identified based on molecular evidence. One of the major challenges in Africa is the lack of baseline data for the diversity and distribution of Diplostomum parasitising freshwater fishes and is mainly due to a lack of knowledge, expertise, sampling effort and funding in the field of parasitology. Numerous experimental studies exploring the effect of metacercariae on fish behaviour, predominantly done in Europe, found that metacercariae of Diplostomum have an effect on the escape response, feeding- and swimming behaviour as well as habitat selection of their intermediate hosts; thus facilitating transmission to the definitive hosts. In contrast, no published data on the influence of metacercariae of Diplostomum on fish behaviour in Africa exists. Thus, the aims of the present study were: (i) to determine the diversity of Diplostomum in South African fishes by applying molecular and traditional morphological methods, and (ii) to determine the effect of Diplostomum infections on fish behaviour using the Plain squeaker Synodontis zambezensis Peters, 1852 as model species. To achieve this aim, a total of 160 fishes belonging to 17 species were collected and the eyes and brains were examined for the presence of Diplostomum and analysed along with specimens from the Water Research Group (WRG) collection that were collected during previous sampling expeditions in the Phongolo (2016, 2017, 2018), Riet (2017), Usuthu (2017) and Mooi Rivers (2019). Metacercariae were recovered from the eye lenses of 38 fishes belonging to five species of the families Anguillidae, Cichilidae and Mochokidae, with an overall low prevalence of infection (18%). Representative metacercariae were subjected to morphological analysis and molecular sequencing including partial mitochondrial cox1 and ribosomal 28S rDNA genes as well as the ribosomal ITS1-5.8S-ITS2 region. The presence of three species of Diplostomum was discovered. The three species matched those previously reported from Nigeria, Iraq and China, therefore those from Tilapia sparrmanii Smith, 1840 and S. zambezensis were identified as Diplostomum sp.; those from Anguilla labiata (Peters, 1852), Oreochromis mossambicus (Peters, 1852) and S. zambezensis were named Diplostomum sp. 14; and those from Pseudocrenilabrus philander (Weber, 1897) were named Diplostomum sp. 16. Ten S. zambezensis previously collected from the Ndumo Game Reserve (NGR) (2017) and 22 S. zambezensis (NGR, 2018) were used in the laboratory and field-based quantitative behavioural experiments. Analyses of video recordings and statistical data applying unpaired Welsch’s t-tests and One-Way ANOVA revealed a significant difference in behaviour between infected and uninfected fish during acclimation and attacks based on the time spent in top and bottom zones, frequency of zone alternations, minimum and maximum acceleration and mobility state (immobile to highly mobile). During attack trials only, which was not found during the acclimation period, a significant difference was found in distance moved and swimming speed between infected and uninfected fish. This study is the first dedicated assessment of Diplostomum applying both molecular and morphological approaches in freshwater fishes in South Africa. The first morphological and molecular evidence provided for Diplostomum sp., Diplostomum sp. 14 and Diplostomum sp. 16 as well as statistical evidence of significant effects of metacercariae of Diplostomum on the behaviour of S. zambezensis, contributes to the elucidation of the life cycle of Diplostomum, expands our knowledge on the geographical distribution of species within this genus and provides baseline data for future behavioural studies of fish infected with diplostomids in Africa.