131 Loads of trematodes: discovering hidden diversity of paramphistomoids in Kenyan ruminants MARTINA R. LAIDEMITT1*, EVA T. ZAWADZKI1, SARA V. BRANT1, MARTIN W. MUTUKU2, GERALD M. MKOJI2 and ERIC S. LOKER1 1 Department of Biology, Center for Evolutionary and Theoretical Immunology, Parasite Division Museum of Southwestern Biology, University of New Mexico, 167 Castetter MSCO3 2020 Albuquerque, New Mexico 87131, USA 2 Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O. Box 54840- 00200, Nairobi, Kenya (Received 23 May 2016; revised 24 August 2016; accepted 7 September 2016; first published online 20 October 2016) SUMMARY Paramphistomoids are ubiquitous and widespread digeneans that infect a diverse range of definitive hosts, being particu- larly speciose in ruminants. We collected adult worms from cattle, goats and sheep from slaughterhouses, and cercariae from freshwater snails from ten localities in Central and West Kenya. We sequenced cox1 (690 bp) and internal transcribed region 2 (ITS2) (385 bp) genes from a small piece of 79 different adult worms and stained and mounted the remaining worm bodies for comparisons with available descriptions. We also sequenced cox1 and ITS2 from 41 cercariae/rediae samples collected from four different genera of planorbid snails. Combining morphological observations, host use infor- mation, genetic distance values and phylogenetic methods, we delineated 16 distinct clades of paramphistomoids. For four of the 16 clades, sequences from adult worms and cercariae/rediae matched, providing an independent assessment for their life cycles. Much work is yet to be done to resolve fully the relationships among paramphistomoids, but some correspond- ence between sequence- and anatomically based classifications were noted. Paramphistomoids of domestic ruminants provide one of the most abundant sources of parasitic flatworm biomass, and because of the predilection of several species use Bulinus and Biomphalaria snail hosts, have interesting linkages with the biology of animal and human schisto- somes to in Africa. Key words: Paramphistomoidea, biodiversity, DNA barcode, host specificity, Schistosoma. INTRODUCTION on cercariae collected from snails and adult worms from domestic animals from abattoirs. The Superfamily Paramphistomoidea is a prominent Paramphistomoids are of interest to parasitologists group of digeneans where adults are characterized by in several contexts. They are diverse in number of the absence of an oral sucker and the presence of an species and provide an understudied model group acetabulum at or near the posterior end of the body. for those focused on revealing patterns and mechan- The systematics of this group of digeneans is a work isms of diversity. Of the 12 recognized paramphisto- in progress. Sey (1991) concluded it is comprised of moid families recognized by Jones (2005a), eight families, whereas Jones (2005a) concluded representatives of nine occur in Africa. The diversity there are 12 families. Paramphistomoids are often of paramphistomoids in Africa reflects the presence of called rumen flukes because many of the best- many species of terrestrial mammals, including ele- known representatives live in this habitat in domes- phants, rhinoceroses, hippopotami and a rich diver- tic ruminants. However, many species also inhabit sity of wild and domestic ruminants. Three families the intestines of fish, amphibians, reptiles, birds in particular (Paramphistomidae, Gastrodiscidae and non-ruminant mammals. They feature a life and Gastrothylacidae) are speciose in Africa. The dis- cycle in which cercariae produced in rediae emerge tribution of diversity in rumen hosts can partly be from snails and encyst on vegetation as metacercar- explained by characters (e.g. regressed pharyngeal iae, which are later ingested by the definitive host appendages) that are apomorphic, which have (Jones, 2005a). As part of a larger study to determine allowed them to colonize the forestomach (Sey how digenean community diversity influences the 1991). The three families comprise over 40% of all transmission of schistosomes in Kenya, we provide known paramphistomoids, the majority of which new results regarding the overall diversity and host use ruminants as their definitive hosts (Sey, 1991). relationships of paramphistomoids in Kenya, based Paramphistomoids have thick bodies, which make detailed morphological characterization of adult fea- * Corresponding author: Center for Evolutionary and tures and species identification challenging (Horak, Theoretical Immunology, University of New Mexico, Department of Biology, 167 Castetter MSCO3 2020, 1971; Jones, 1991; Mage et al. 2002; Rinaldi et al. Albuquerque, New Mexico 87131, USA. E-mail: 2005). The bodies of paramphistomoid cercariae [email protected] are also relatively thick and typically filled with Parasitology (2017), 144, 131–147. © Cambridge University Press 2016. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Downloaded fromdoi:10.1017/S0031182016001827 https://www.cambridge.org/core. IP address: 170.106.39.137, on 04 Oct 2021 at 14:28:23, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182016001827 Martina R. Laidemitt and others 132 cystogenous material or pigment, also rendering We collected cercariae and adult worms from ten identification difficult. Nonetheless, a meticulous localities in Kenya. We provide stained whole framework for paramphistomoid identification and mounts and provisional identification of adults that classification has been developed (see reviews by are linked to sequence data for cytochrome oxidase Sey, 1991; Jones, 2005a). Given the inherent 1(cox1) and the internal transcribed region 2 difficulties in identification, coupled with a (ITS2). In some cases, we provide matches with growing list of studies from other digenean groups sequences obtained from cercariae and adult worms documenting the presence of cryptic species thus providing probable life cycle linkages. We also (Detwiler et al. 2012; Herrmann et al. 2014; provide new hypotheses for phylogenetic relation- McNamara et al. 2014), paramphistomoids are ships among the paramphistomoids that include ideal for studies attempting to meld traditional mor- available sequences from NCBI GenBank, which phological identification with sequence data charac- show that some species of paramphistomoids are terization provided by molecular approaches. The geographically widespread throughout Africa. Data number of studies that use molecular techniques to presented here will contribute to an increased under- provide assessments of the diversity of paramphisto- standing of the superfamily Paramphistomoidea, in- moids have in general been limited, especially so for cluding providing greater clarification for how these African species (Lotfy et al. 2010; Mansour et al. worms are distributed among hosts, their potential 2014; Sibula et al. 2014; Titi et al. 2014; Dube roles if any in causing disease in domestic or wild et al. 2015). animals, and their interactions with other digeneans, In addition to being speciose, paramphistomoids including schistosomes. are often remarkably abundant (Horak, 1971; Cheruiyot and Wamae, 1988; Rolfe et al. 1994; Sanabria and Romero, 2008). In fact, one might be MATERIALS AND METHODS fi hard pressed to nd a larger source of sheer digenean Sampling biomass than is presented routinely at abattoirs by ru- minant paramphistomoids. Given the large worm We collected larval and adult paramphistomoids from populations that can occur in individual cattle, goats ten different localities in central and especially western or sheep, vast numbers of paramphistomoid eggs are Kenya between 2005 and 2015 (Table 1). All species regularly passed into the environment. In rural West of field-collected aquatic snails were brought to the la- Kenya, we can routinely collect 10 000 paramphisto- boratory at Kisian, near Kisumu, Kenya. The snails moid eggs from a single cow dung sample. As domes- were cleaned and then placed individually into 12- tic ruminants regularly seek water from natural well tissue culture plates in 3 mL of aged tap water. habitats, it is not surprising that many paramphisto- The tissue culture plates were placed in natural light moid eggs enter freshwater, creating the potential for 2 h to induce shedding of cercariae. Snails shed- for high levels of infection in their snail hosts ding cercariae were identified using keys and informa- (Chingwena et al. 2002a; Mohammed et al. 2016). tion in Brown and Kristensen (1989)andBrown A review of the East African paramphistomoid lit- (1994), and cercariae were preliminarily identified erature reveals that many of the described species are using keys (Frandsen and Christensen, 1984; Schell, transmitted by Biomphalaria and Bulinus, the snail 1985) and by reference to regional monographs (e.g. genera also of concern with respect to their role in Fain, 1953). All cercariae designated as paramphisto- transmission of human schistosomiasis in Africa moids were confirmed as such according to Sey (Dinnik, 1954; Dinnik and Dinnik, 1957; Dinnik, (1991). Snails were either dissected at the time of col- 1961; Eduardo, 1983; Brown, 1994; Chingwena lection to procure rediae, or re-shed two and four et