Monophyly of the species of Hepatozoon (Adeleorina: Hepatozoidae) parasitizing (African) anurans, with the description of three new species from hyperoliid frogs in South Africa

Abstract

Haemogregarines (Apicomplexa: Adeleiorina) are a diverse group of haemoparasites reported from almost all vertebrate classes. The most commonly recorded haemogregarines to parasitize anurans are species of Hepatozoon Miller, 1908. To date 16 Hepatozoon species have been described from anurans in Africa, with only a single species, Hepatozoon hyperolli (Hoare, 1932), infecting a member of the Hyperoliidae. Furthermore, only two Hepatozoon species are known from South African anurans, namely Hepatozoon theileri (Laveran, 1905) and Hepatozoon ixoxo Netherlands, Cook and Smit, 2014, from Amietia delalandii (syn. Amietia quecketti) and three Sclerophrys species, respectively. Blood samples were collected from a total of 225 individuals representing nine hyperoliid species from several localities throughout northern KwaZulu-Natal, South Africa. Twenty frogs from three species were found positive for haemogregarines, namely Afrixalus fornasinii (6/14), Hyperolius argus (2/39), and Hyperolius marmoratus (12/74). Based on morphological characteristics, morphometrics and molecular findings three new haemogregarine species, Hepatozoon involucrum Netherlands, Cook and Smit n. sp., Hepatozoon tenuis Netherlands, Cook and Smit n. sp. and Hepatozoon thori Netherlands, Cook and Smit n. sp., are described from hyperoliid hosts. Furthermore, molecular analyses show anuran Hepatozoon species to be a separate monophyletic group, with species isolated from African hosts forming a monophyletic clade within this cluster.

Molecular characterisation and morphological description of two new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina: Hepatozoidae) infecting leukocytes of African leopards Panthera pardus pardus (L.)

“…Prior to the 21st century, classification of species of Hepatozoon was based on their life history, host identity and morphological characteristics. However, with recent advances in molecular techniques, phylogenetic analyses on the relationships between species have become possible [13][14][15][16]. Morphological characteristics used to distinguish between species of Hepatozoon include gamont and nucleus dimensions; position of the nucleus within the gamont; number and arrangement of vacuoles and staining properties [17,18], as well as characteristics of other developmental stages [19,20].…”

“…Due to constant improvement of molecular techniques, the number of studies on haemogregarines has systematically increased, with several studies relying solely on these methods to detect species of Hepatozoon in their hosts [11,[28][29][30][31][32]. Recent research has shown that a difference in p-distance of between 1-2% of the 18S rRNA gene is sufficient to distinguish between species of haemogregarines if supported by morphological data [16,18,33,34]. Other studies, such as Metzger et al [35], incorporate both molecular and some degree of morphological investigations, showing the importance of utilizing a more holistic approach when distinguishing among species of Hepatozoon.…”

A paradise for parasites? Seven new haemogregarine species infecting lizards from the Canary Islands

“…(2018) used the pair of primers HepF300 and HepR900 (Ujvari et al ., 2004) to amplify a 600 bp fragment of the 18S rRNA. In the current work we tested two other primers, which combined with the previous pair produced an amplicon of around 1700 bp, the majority of the 18S rRNA (Netherlands et al ., 2018). The set of primers HAMF (Criado-Fornelio et al ., 2006) and HepF300 produced a 900 bp fragment, providing 300 extra bases to the 5′ end of the 600 bp fragment previously obtained, while 2868 (Medlin et al ., 1988) and HepR900 amplified a 1400 bp product, adding 800 bp to the 3′ end of the previous fragment.…”

Two new species of Hepatozoon (Apicomplexa: Hepatozoidae) parasitising species of Philothamnus (Ophidia: Colubridae) from South Africa

“…Conditions for PCR of both fragments were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles, entailing a 95 °C denaturation for 30 s, annealing at 61 °C for 30 s with an end extension at 72 °C for 2 min, and following the cycles a final extension of 72 °C for 10 min (Netherlands et al 2018).…”