First report of Hexametra angusticaecoides Chabaud & Brygoo, 1960 (Nematoda: Ascarididae) in a population of captive central bearded dragons, Pogona vitticeps Ahl (Squamata: Agamidae)

Syst Parasitol (2025) 102:6 https://doi.org/10.1007/s11230-024-10202-y First report of Hexametra angusticaecoides Chabaud & Brygoo, 1960 (Nematoda: Ascarididae) in a population of captive central bearded dragons, Pogona vitticeps Ahl (Squamata: Agamidae) Laura Hernández‑Hurtado · Jacinto Gomes · Luisa Pereira · Maria João Vila‑Viçosa · Carlos Gutiérrez‑Gutiérrez Received: 28 May 2024 / Accepted: 25 October 2024 © The Author(s) 2024 Abstract Between 2022 and 2023 ascaridoid nematodes of the genus Hexametra were recovered from two captive central bearded dragons (Pogona vitticeps Ahl) in the Extremadura region of Spain. These nematodes were identified by an integrative approach combining morpho-anatomical and molecular data. We used two rRNA regions (D2–D3 expansion segments of the 28S, and ITS fragments), and the partial region of the cytochrome oxidase I gene (COI Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s11230-024-10202-y. L. Hernández‑Hurtado · J. Gomes · L. Pereira Polytechnic Institute of Portalegre, Biosciences School of Elvas, 7350‑092 Elvas, Portugal L. Hernández‑Hurtado · J. Gomes VALORIZA—Research Centre for Endogenous Resource Valorization, 7300‑555 Portalegre, Portugal M. J. Vila‑Viçosa Department of Veterinary Medicine, Victor Caeiro Parasitology Laboratory, School of Science and Technology & MED—Mediterranean Institute for Agriculture, Environment and Development, University of Évora, Pólo da Mitra, Ap. 94, 7006‑554 Évora, Portugal C. Gutiérrez‑Gutiérrez (*) NemaLab/MED—Mediterranean Institute for Agriculture, Environment and Development & CHANGE—Global Change and Sustainability Institute, Instituto de Investigação e Formação Avançada, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006‑554 Évora, Portugal e-mail: mtDNA) sequences. Hexametra angusticaecoides Chabaud & Brygoo, 1960 (Ascarididae) was morphologically characterized and illustrated using light and scanning electron microscopy. Bayesian inference (BI) and Maximum Likelihood (ML) phylogenetic trees for these genetic markers established relationships that highlight the importance of using molecular and phylogenetic data for accurate species identification within the genus Hexametra. Phylogenetic trees indicated that the Spanish isolates of H. angusticaecoides were clustered in a well-supported clade together with other isolates from Central Europe of the same species. Molecular phylogenetic findings also reflect that there was a higher intraspecific variation for ITS genetic markers at the intraspecific level. Phylogenetic results based on 28S rRNA suggest that Ascarididea was divided into three monophyletic major clades. To our knowledge, this is the first report of this species parasitizing bearded dragons, extending the host range of this species. These findings represent the first formal record of H. angusticaecoides in Spain. Introduction Ascarididae Baird, 1853 is one of the most diverse and biologically versatile families within the phylum Nematoda paraziting animals (Okulewicz et al., 2002). They are one of the largest groups in terms of the number of taxonomically recognized genera Vol.: (0123456789) 6 Page 2 of 16 (Hodda, 2022) including gastrointestinal parasites of reptiles, amphibians, and other vertebrates. Notably, it includes some clinical and socio-economically important helminth parasites of humans (Gazzinelli et al., 2012). The genus Hexametra Travassos, 1920 has seven valid species, subdivided into two groups based on morphological characters and host specificity (Sprent, 1978; Bowman, 1984; Baker, 1987): (i) Hexametra species occurring in lizards, such as H. hexametra (Gedoelst, 1916) Travassos, 1920; H. applanata Linstow, 1899; H. angusticaecoides Chabaud & Brygoo, 1960; and H. rotundicauda (Linstow, 1904) Mozgovoy, 1953, and (ii) Hexametra species occurring in snakes, such as H. boddaertii (Baird, 1860) Kreis, 1944; H. quadricornis (Wedl, 1861) Kreis, 1944; and H. leidyi Bowman, 1984. Overall, female Hexametra spp. are characterized by six uterine branches, inter-labia absent, and an intestinal caecum that can be present or absent (Sprent, 1978; Baker, 1987). Likewise, the shape and length of the lips in females and the shape and length of the copulatory spicules in males, however, differs between the subgroups (Sprent, 1978). Furthermore, the systematic position of several species of this genus and the genus itself has been questioned (Travassos, 1920; Baylis, 1921, 1936; Kreis, 1944; Yorke & Maspletone, 1926; Skrjabin et al., 1951; Mozgovoy, 1953; Hartwich, 1957, 1974; Yamaguti, 1961; Kutzer & Grünberg, 1965; Araujo, 1969; Sprent, 1978; Baker, 1987; Barton et al., 2020; Hodda, 2022). To date, there is a limited range of host species known for each species of Hexametra (Brygoo, 1963; Chabaud et al., 1962; Caballero, 1968; Sprent, 1978; Bowman, 1984; Bursey et al., 1995; Dias et al., 2005; Pinto et al., 2020; McAllister et al., 2011; Morton & Krysko, 2012; Santoro et al., 2013; Peichoto et al., 2016; Carbajal-Márquez et al., 2018; Stets, 2019). Among Hexametra spp., H. angusticaecoides has been most widely reported in lizards, mainly in the family Chamaeleonidae, such as Furcifer oustaleti Mocquard (see McAllister et al., 2011), F. pardalis Cuvier (see Stets, 2019; Reitl et al., 2020), Chamaeleo calyptratus Duméril & Bibron (see Jacobson, 2007; Rataj et al., 2011) and the family Diplodactylidae, such as Correlophus ciliatus Guichenot (see Barton et al., 2020). Despite the fact that four Hexametra species have been reported as paraziting lizards (Caballero, 1968; Sprent, 1978; Bowman, 1984; Baker, 1987; McAllister et al., 2011; Stets, Vol:. (1234567890) Syst Parasitol (2025) 102:6 2019; Barton et al., 2020; Reitl et al., 2020), only H. angusticaecoides has been reported in countries in Central and Eastern Europe (Rataj et al., 2011; Stets, 2019; Reitl et al., 2020). Integrative taxonomy is still considered the most efficient approach for accurate diagnosis and identification (e.g., Zhu et al. 2000; Tokiwa et al., 2014; González-Solís et al., 2019; Barton et al., 2020; Chen & Li, 2023). Until recently, identifications of Hexametra spp. were exclusively based on morphology (Chabaud et al., 1962; Sprent, 1978; Vicente et al., 1993). However, morphological approaches present considerable limitations in the differentiation of closely related species (Barton et al., 2020); the large body size makes handling specimens a challenge, and their high inter-specific variability and intra-specific plasticity in morphological traits make species identification within the genus Hexametra a complex and time-consuming task (Barton et al., 2020). Sequencing of RNA-based markers presents a powerful approach for species-level taxonomic identification (e.g., Barton et al., 2020; Reitl et al., 2020; Sharifdini et al., 2021; Barrera et al., 2022; Chen & Li, 2023). Recently, molecular markers based on ribosomal RNA ( (...truncated)