Morphometric and molecular analyses of Tylodelphys sp. metacercariae (Digenea: Diplostomidae) from the vitreous humour of four fish species from Lake Naivasha, Kenya

Abstract

Even in the relatively well-characterized faunas of the developed world, it is difficult to discriminate species of metacercariae in the Diplostomidae using morphology, infection site or host use. The taxonomy, diversity and ecology of diplostomids infecting freshwater fishes in the African continent are particularly poorly known, but recent morphometric and genetic studies have revealed four species of diplostomids in the eyes and brains of siluriform fishes. In the present study, diplostomid metacercariae were collected from the eyes of 288 fish comprising two species within the Cyprinidae (Cyprinus carpio, n = 145, and Barbus paludinosus, n = 67), two Cichlidae (Oreochromis leucostictus, n = 56, and Tilapia zillii, n = 18) and one Centrarchidae (Micropterus salmoides, n = 2) caught in Lake Naivasha, Kenya. Morphometric (14 characters and 8 indices in 111 specimens) and molecular (sequences from the barcode region of the cytochrome c oxidase 1 gene in 11 specimens) data were used to discriminate species. All fish species except B. paludinosus were infected with Tylodelphys metacercariae that were initially separated into two types differing mainly in body length. However, this morphological distinction received only intermediate support in quantitative morphological analysis and molecular data indicated that both morphotypes were conspecific. All the specimens therefore are inferred to belong to a single unidentified species of Tylodelphys, which is not conspecific with any other diplostomid for which comparable molecular data are available, including four diplostomid species known from siluriform fish in Nigeria and Tanzania.

Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes

“…S5). Data are from the present study and Galazzo et al (2002), Niewiadomska and Laskowski (2002), Moszczynska et al (2009), Locke et al (2010a, Rellstab et al (2011), Cavaleiro et al (2012, Behrmann-Godel (2013), Chibwana et al (2013), Georgieva et al (2013), Haarder et al (2013), Blasco-Costa et al (2014), Pérez-del-Olmo et al (2014), Kuhn et al (2015), Otachi et al (2015) and Kania and others (unpublished, GenBank accession numbers KJ888995-013). studies (disagreement in the boundaries of at least nine species).…”

“…These were analysed with 1167 sequences generated by Moszczynska et al (2009), Locke et al (2010a, Désilets et al (2013), which are published herein, and 336 sequences published by Moszczynska et al (2009) Kuhn et al (2015), and Otachi et al (2015); details are in Supplementary Table S1. Alignment of these 2026 CO1 sequences from Diplostomum, Tylodelphys and Austrodiplostomum was unambiguous (Supplementary Data S1; GUIDANCE confidence scores of all sequences and all 621 positions P0.993) and 99.4% of the Kimura 2-parameter (K2P) distances were either less than 2.0% or greater than 9.0% (Fig.…”

“…In addition, species were delineated based on gaps in sequence divergence detected using Automatic Barcode Gap Discovery (ABGD, Puillandre et al, 2012). The primary criterion for selecting outcomes was consistency of the two approaches (BINs, ABGD) with the conclusions of prior studies, particularly Galazzo et al (2002), Locke et al (2010a), Chibwana et al (2013), Georgieva et al (2013), Blasco-Costa et al (2014, Pérez-del-Olmo et al (2014), O'Hear et al (2014) and Otachi et al (2015). After establishing consistency, the methods were used to extrapolate for unknown species.…”

“…Aside from their importance in fish health, eye-dwelling diplostomids are frequent subjects of evolutionary and ecological studies (e.g., Ballabeni and Ward, 1993;Kalbe and Kurtz, 2006). However, diplostomid metacercariae are difficult to identify morphologically and molecular methods are often used to distinguish species (Locke et al, 2010a,b;Rellstab et al, 2011;Cavaleiro et al, 2012;Behrmann-Godel, 2013;Chibwana et al, 2013;Désilets et al, 2013;Georgieva et al, 2013;Ndeda et al, 2013;Blasco-Costa et al, 2014;Pérez-del-Olmo et al, 2014;Kuhn et al, 2015;García-Varela et al, 2015;Mateos-Gonzalez et al, 2015;Otachi et al, 2015). Consequently, DNA sequences from diplostomids are accumulating from all over the world, particularly from Diplostomum spp.…”

“…Consequently, DNA sequences from diplostomids are accumulating from all over the world, particularly from Diplostomum spp. Sequences can clarify whether morphological heterogeneity represents interspecific (BlascoCosta et al, 2014;Faltý nková et al, 2014;Pérez-del-Olmo et al, 2014) or intraspecific variation among metacercariae (Cavaleiro et al, 2012;Otachi et al, 2015). Molecular links to adults from avian definitive hosts are usually needed to identify, rather than merely discriminate, species of metacercariae (Galazzo et al, 2002;Locke et al, 2010aLocke et al, , 2011Georgieva et al, 2013;Pérez-delOlmo et al, 2014).…”