Hepatozoon species (Adeleorina: Hepatozoidae) of African bufonids, with morphological description and molecular diagnosis of Hepatozoon ixoxo sp. nov. parasitising three Amietophrynus species (Anura: Bufonidae)

Abstract

BackgroundHaemogregarines comprise a large group of apicomplexan blood parasites. In 1996 all anuran haemogregarines still in the genus Haemogregarina Danilewsky, 1885 were reassigned to the genus Hepatozoon Miller, 1908. Most (11/15, 73%) African anuran Hepatozoon species have been recorded from the family Bufonidae, however, all these are recorded from only two host species, Amietophrynus mauritanicus (Schlegel, 1841) and Amietophrynus regularis (Reuss, 1833) from Northern and central Africa. To the authors’ knowledge the only description of an anuran haemogregarine from South Africa is Hepatozoon theileri (Laveran, 1905), parasitising Amietia quecketti (Boulenger, 1895).MethodsThin blood smears for morphometrics and whole blood for molecular work, were collected from 32 Amietophrynus garmani (Meek, 1897), 12 Amietophrynus gutturalis (Power, 1927), and nine Amietophrynus maculatus (Hallowell, 1854), in Ndumo Game Reserve and Kwa Nyamazane Conservancy, KwaZulu-Natal, South Africa. Smears were Giemsa-stained, screened for haemogregarines, parasite stages measured, compared to each other and to other described African bufonid haemogregarines. Parasite 18S rDNA was amplified using two apicomplexan-specific primer sets, HepF300/HepR900 and 4558/2733. Resulting sequences of the haemogregarine isolates from the three Amietophrynus species were compared with each other and to comparative haemogregarine sequences selected from GenBank.ResultsMorphological characteristics of parasite stages, in particular characteristically capped mature gamont stages, and molecular findings, supported all three haemogregarine isolates from all three Amietophrynus species to be the same, a species of Hepatozoon, and furthermore different morphologically from other previously recorded bufonid Hepatozoon species. The haemogregarine fell within a clade comprising other anuran Hepatozoon species and furthermore, within a monophyletic sub-clade along with H. theileri and are described as Hepatozoon ixoxo sp. nov.ConclusionsThis is the first morphological and molecular account of Hepatozoon species within the family Bufonidae from South Africa, a study hoped to encourage the redescription and molecular analysis of those Hepatozoon species described in the past from Amietophrynus species, as well as to promote the use of both morphological and molecular characteristics in Hepatozoon species descriptions. This will aid in comprehensive Hepatozoon descriptions, which along with the use of phylogenetic analysis will give a better indication of these parasites possible vectors and life cycle dynamics.

FirstHemoliviafrom southern Africa: reassigning chelonianHaemogregarina parvulaDias, 1953 (Adeleorina: Haemogregarinidae) toHemolivia(Adeleorina: Karyolysidae)

“…The phylogenetic analyses performed here concur with the findings of Barta et al (2012), Haklová-Kočíková et al (2014), Kvičerová et al (2014) and Netherlands et al (2014aNetherlands et al ( , 2014b, but conflict with those of Harris et al (2013) in that Hemolivia is seen as a sister taxon to Hepatozoon. A similar problem is evident for classification of species of Karyolysus Labbé, 1894, which also fall within the Hepatozoon clade (see Haklová-Kočíková et al 2014).…”

“…A second PCR was carried out using the apicomplexan-specific parasite primer set 4558 (5′-GCTAATACATGAGCAAAATCTCAA-3′) and 2733 (5′-CGGAATTAACCAGACAAAT-3′) (see Mathew et al 2000), targeting a longer fragment (approximately 1 120 bp) of the 18S rDNA. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 40 cycles, entailing 94 °C denaturation for 1 min, annealing at 55 °C for 2 min with an end extension at 72 °C for 2 min, and following the cycles a final extension of 72 °C for 10 min (see Mathew et al 2000;Netherlands et al 2014b). …”

Hemoparasites of the Lake Frog Pelophylax ridibundus (Pallas, 1771) (Ranidae, Anura) Inhabiting Reservoirs of the North-Western Ciscaucasia

“…sp. found in toads in three populations from Africa also showed significant between-population differences in morphology of gamonts (Netherlands et al, 2014).…”

“…In ad dition, the trophozoite and gamont infection intensity is higher in frogs from Krasny Kut reservoir ( = 3.02 and 2.04, respectively). In South Africa intensity across frog species ( = 17.683, = 0.028), across families (χ = 11.717, = 0.006), and across the different habitat types (χ = 7.227, = 0.007) showed a significant difference (Netherlands et al, 2015).…”

Diversity of haemoprotozoan parasites infecting the wildlife of South Africa

“…Most other assessments of apicomplexan parasites in southern Africa have targeted specific species and parasites (e.g. Dubey et al 2014, Netherlands et al 2014, Cook et al 2016, which means that presence of parasites in many species remains either completely unknown or is based only on older microscopic screening surveys (e.g. Basson et al 1967).…”