Ecology, systematics and evolutionary biology of frog blood parasites in northern KwaZulu-Natal

Blood parasites have been recorded in a variety of vertebrate and invertebrate hosts, inhabiting both aquatic and terrestrial environments. Until this study, only a few blood parasite surveys had been carried out on frogs in sub-Saharan Africa. Thus information on the diversity of these parasites remained limited. To increase our knowledge of frog blood parasites, a large multi-approach study on the diversity, evolutionary biology, and ecology of frog blood parasites was undertaken. The majority of the fieldwork took place in northern KwaZulu-Natal (KZN), South Africa, focussing specifically on the area adjacent to the Phongolo River and its associated floodplain. However, samples also included those collected from frogs in the southern regions of the Kruger National Park, South Africa, and from frogs in Belgium. The latter was fortuitous, as Europe is the type locality for many frog blood parasite species and genera. These samples provided essential data for phylogenetic comparisons between the African and European species. Presently this is the largest multi-species, generic and family amphibian blood parasite survey to be completed, including a total of 643 anurans of 38 species, 20 genera and 13 families. The study was divided into three main components for the collection, analysis and reporting of data. The first component was to determine the frog blood parasite diversity, the second to determine phylogenetic relationships in conjunction with the former component, and lastly the ecological and host-vector-parasite relationships. Blood samples were drawn from the femoral artery of each frog and thin blood smears prepared for screening and morphometrics; the remaining blood fixed in 70% molecular grade EtOH for later molecular analysis. Giemsa stained blood smears were screened microscopically for the presence of any blood inhabiting organisms. Positive infections were then further analysed according to the aims of the respective chapters. Analyses included both morphological and molecular aspects. Morphology was used for the description and identification of species, and molecular analyses were used to assist with the morphology-based descriptions, as well as to allow for phylogenetic relationship comparisons of the blood parasites with one another.

In the present study, three new species of Hepatozoon were described from hyperoliid frogs (Afrixalus fornasini, Hyperolius argus, and Hyperolius marmoratus), namely Hepatozoon involucrum Netherlands, Cook & Smit, 2018; Hepatozoon tenuis Netherlands, Cook & Smit, 2018; and Hepatozoon thori Netherlands, Cook & Smit, 2018. Phylogenetic relationships show that species of Hepatozoon isolated from African frogs form as a monophyletic group, separate from the species of Hepatozoon isolated from European and North American frogs. Two species of Dactylosoma Labbé, 1894, were found parasitising three species of frogs namely, Ptychadena anchietae and Sclerophrys gutturalis from South Africa, as well as Pelophylax lessonae from Belgium. Based on morphometrics and molecular findings a new dactylosomatid, Dactylosoma sp. 1, is described form Pty. anchietae and Scl. gutturalis. The species of Dactylosoma isolated from Pel. lessonae conforms morphologically with Dactylosoma splendens Labbé 1894, thus placing in question the validity of D. splendens synonymy with D. ranarum (Kruse, 1890). Phylogenetic analysis shows species of anuran Dactylosoma as a monophyletic group, separate from the other haemogregarine groups. Five species of frogs from South Africa and two from Belgium were found parasitised with haemococcidia. Based on morphological, morphometric and molecular findings Lankesterella minima (Chaussat 1850) is redescribed from Pelophylax kl. esculentus (Linnaeus, 1758) and Pel. lessonae (Camerano, 1882) from Belgium. Additionally, two new species of Lankesterella were described, namely Lankesterella sp. 1 in Pel. lessonae from Belgium, and Lankesterella sp. 2 in Afr. delicates and Afr. fornasini from South Africa. Furthermore, a new genus of haemococcidia, with a new species combination, is described from Phr. mababiensis, Pty. anchietae, and Pyx. edulis from South Africa; as well as a new species, haemococcidia sp. 2, described from Afr. fornasini from South Africa. This is the first study to provide molecular data for species of haemococcidia from African and European anurans.A new species of amphibian filarial nematode (Onchocercidae: Waltonellinae) was described from the toads Scl. gutturalis and Scl. garmani. The life history of this nematode was elucidated from its natural mosquito vectors Uranotaenia (Pseudoficalbia) mashonaensis and Uranotaenia (Pfc.) montana. All stages of development were characterised using morphological and molecular methods. This study is the first to elucidate the life history of an amphibian filarial nematode from southern Africa, and provide data on its phylogenetic placement within the Onchocercidae. In addition to the taxonomic and phylogenetic perspective of this study, this study also aimed at exploring the potential of frog blood parasites as indicators of environmental health. For this, blood parasites infecting grass frogs (Ptychadena Boulenger, 1917) from the Phongolo River system in South Africa were used as a case study. In general, findings indicate that frogs from more impacted sites harboured more blood parasites than from less-impacted sites.

In summary, this study explored the efficacy of a large multi-species, multi-approach survey on the diversity of frog blood parasites from northern KwaZulu-Natal, South Africa. Based on the results several new species of frog blood parasites from different taxa were discovered and described, greatly contributing to knowledge and species records on the overall diversity of frog blood parasites from South Africa. Furthermore, this study provides the first molecular data for species of Dactylosoma and Lankesterella for frogs from Africa, as well as the first molecular data for a filarial nematode for frogs from South Africa. The phylogenetic relationships of species of Hepatozoon, Dactylosoma, Lankesterella, and the filarial nematode were also characterised based on comparisons to other available molecular data. From an ecological perspective, blood parasites from this study adhere to several criteria of what is considered a good indicator and thus demonstrate potential as indicators for healthy ecosystems and intact food webs. The results of this study establish a foundation for future research into the blood parasite biodiversity in northern KZN, an area that this study has highlighted as not only rich in anuran diversity, but also rich in anuran blood parasite diversity. Furthermore, this study provides a baseline for future taxonomic and ecological studies on these parasite groups, not only in South Africa but globally as well.