JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2021-050 e2021-50 | Vol. 53 First detection of Gongylonema species in Geotrupes mutator in Europe Daniel Bravo-Barriga1, Manuel Martín-Pérez1,*, Jorge M. Lobo2, Ricardo Parreira3, Abstract 1 Juan Enrique Pérez-Martín The detection of three Gongylonema sp. infective larvae in two 1 and Eva Frontera specimens of the dung beetle Geotrupes mutator (Marsham, 1Parasitology Area, Department 1802) from western Spain is reported here for the first time of Animal Health, Veterinary in Europe. Scanning electron microscopy confirmed that the Faculty, University of Extremadura, analyzed specimens belong to the genus Gongylonema, but it Cáceres, Spain. was not possible to determine the species identity by the lack of 2 morphological information in the literature and because many of the Department of Biogeography and phenotypic characteristics had not yet fully developed at this juvenile Global Change, Museo Nacional de stage. Nevertheless, a phylogenetic analysis using amplified cox1 Ciencias Naturales, CSIC, Madrid, nucleotide sequences has revealed that the studied larvae could be Spain. clearly discriminated (< 89% identity) from all the other Gongylonema 3Global Health and Tropical cox1 sequences available in public genetic databases. While our Medicine (GHTM), Instituto de results are limited by the scarcity of genetic information available Higiene e Medicina Tropical (IHMT), for this genus, the possibility that the analyzed specimens might Universidade Nova de Lisboa correspond to a new species should not be ruled out, and more (UNL), Grupo de Virologia/Unidade studies are needed. The results provided in this report indicate that de Microbiología Médica, Lisbon, G. mutator is involved in the transmission cycle of Gongylonema sp. Portugal. to vertebrates in Europe. *E-mail: mmartinly@alumnos. unex.es Keywords Beetles, Geotrupes, Gongylonema, Host-parasitic relationship, Daniel Bravo-Barriga and Manuel Molecular biology, Nematodes, Spain. Martín-Pérez contributed equally to this work. This paper was edited by Zafar Ahmad Handoo. Received for publication October 07, 2020. Under the functional name of “dung beetles”, approxi- wide variety of pathogenic bacteria and/or viruses, mately 9,500 worldwide species have been grouped as well as eukaryotic parasites favouring their into the Aphodiinae, Scarabaeinae, and Geotrupinae dispersion and transmission (Nichols et al., 2017). subfamilies of the superfamily Scarabaeoidea (Löbl This is particularly true for different platyhelminthes and Smetana, 2016). Dung beetles are considered (flatworms) and nematode (roundworms) belonging the most important agents promoting the recycling to the genera Gongylonema, Spirocerca, Ascarops, of mammal faeces in terrestrial ecosystems. They Acanthocephalus, Macracanthorynchus, or Physo­ contribute to the nitrification and aeration of the cephalus (Poinar, 1975). These parasites use dung soil, seed dispersal, and the control of fly and cattle beetles as intermediate, incidental, or paratenic parasites (Nichols et al., 2008). However, as dung hosts (Mowlavi et al., 2009; Mukaratirwa et al., 2010; beetle species are in close contact with animal and Nichols and Gómez, 2014) affecting numerous spe- human faeces, they may also act as hosts for a cies of birds, other domestic or wild mammals, as © 2021 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Gongylonema detected in G. mutator: Bravo-Barriga et al. well as humans (Kinsella et al., 2016; Mukaratirwa were dehydrated through a graded series of etha- et al., 2010; Nichols and Gómez, 2014). nol from 70 to 100%, freeze-dried with liquid CO2 Gongylonema Molin, 1857 (Spirurida: Gongylo- according to the critical point method, and mounted nematidae) is a genus that includes approximately onto stubs. Mounting of the sample on the sample 50 species, which are known parasites of birds and holder was done using double-sided carbon adhesive mammals, including humans (Cordeiro et al., 2018; tape. Mounted specimens were then sputter-coated Kinsella et al., 2016). However, due to variations with a gold layer (20 nm ± 5%) and observed with an of critical phenotypic characters, morphological FE-SEM Hitachi S-4800 (FE by Field Emission, with identification of these species is difficult and their Field Emission Electron Gun). number may have been underestimated (Setsuda Genomic DNA (gDNA) was extracted from indivi- et al., 2018). Gongylonema infection is a neglected dual worms found in beetles using an NZY tissue gDNA zoonotic parasitic disease, reported in several kit (NZYTech) following the manufacturer’s instructions. countries, that is mainly caused by consumption of The cox1 genes were partially amplified using the contaminated water, raw food, or accidental ingestion primer set NTF and NTR following a PCR protocol of host insects (Xiaodan et al., 2018). Gongylonema previously described for the analysis of Spirurida species follow a terrestrial indirect life cycle involving (Casiraghi et al., 2001), and the product was sent for approximately 50 species of insects as intermediate sequencing to STABvida (https://www.stabvida.com/ hosts, such as dung beetles and cockroaches es). Sequences were edited in Chromas Lite 2.1.1 (Mowlavi et al., 2009; Mukaratirwa et al., 2010). How- (Technelysium Pty Ltd) and consensus sequences ever, the paucity of available observations prevents for each forward/reverse pair were created in BioEdit estimating the network of relationships between Sequence Alignment Editor (version 7.2.5, Carlsbad, these parasites and dung beetles (Nichols et al., CA. USA). Finally, the nucleotide sequences obtained 2017). In the present study, we aim to improve the (> 652 pb in length) were deposited at the DNA Data knowledge about these relationships by describing, Bank of Japan (DDBJ: accession numbers LC577505 for the first time, a Gongylonema species hosted by a and LC612845). In addition to the specimens collected European Geotrupinae species, and also comparing in the course of this study, a DNA extract prepared the genetic relationships of the specimens found with using a formerly unidentifiedGongylonema specimen, other nematode taxa by analyzing the mitochondrial obtained from Otus scops, Spain, and analyzed in a cox1 genetic sequences. previous study (Esperón et al., 2013), was provided by its authors in order to obtain its corresponding Materials and methods sequence for the cox1 barcode section (DDBJ: accession numbers LC620542). In October 2017, a sampling of beetles captured Phylogenetic analyses were carried out based on by 27 pitfall traps was carried out according to a the construction of multiple alignments of nucleo- standardized sampling procedure (Lobo et al., 1988). tide (nt) sequences either comprising a wide range The collections were carried out in three localities of of nematodes (including Cylicospirura, Dirofilaria, western Spain, in the provinces of Salamanca and Gongylonema, Mastophorus, Onchocerca, Physalo­ Zamora. Three types of excrements (pig, bovine, ptera, Setaria, Spirocerca, and Thelazia; n = 31) or canine) were used as bait or attractant at each restricted to the Gongylonema genus (n = 18). These location. The captures of each trap were collected were constructed using the iterative G-INS-I method 65−80 h after trap placements and immediately as implemented in MAFFT vs. 7 (https://mafft.cbrc. deposited in 10 × 10 × 10 cm plastic containers con- jp/alignment/server/) followed by their edition using taining soil and stored at 15°C during transport to GBlocks (http://molevol.cmima.csic.es/castresana/ the laboratory. The collected adult specimens with Gblocks_server.html). Phylogenetic trees were built infective larvae were identified using morphological using the Maximum Likelihood (ML) optimization identification keys provided by Martín-Piera and criterium and the Mega X software (Kumar et al., López-Colón (2000). The beetles were dissected 2018), and their topological stability was assessed by under a stereoscope for the detection of nematode bootstrapping with 1,000 re-samplings of the aligned larvae. In case of infection, larvae were placed in sequence data. Alternatively, a Bayesian approach 70% ethanol until molecular analyses could be was also used for phylogenetic reconstruction, taking performed, with the exception of one worm selected advantage of the BEASTv1.10.4 software (Suchard for optical (× 100−200; Nikon eclipse 80i and digital et al., 2018). For both situations, the best-fitting evolu- photomicrography system, DXM1200F) and scanning tionary model used was GTR + Γ + I. Bayesian analyses electron (SEM) microscopy. For SEM, specimens included two independent Markov chain Monte-Carlo 2 JOURNAL OF NEMATOLOGY (MCMC) runs carried out assuming a lognormal- externally, a circle of cephalic papillae constituted by relaxed molecular clock (as suggested by Mega X four large papillae could also be observed (Fig. 1.5). Molecular Clock test), and a constant population Two large amphids were located lateral to the mouth coalescent prior. These runs were executed until and two lateral depressions. Two lateral alae extend 1 × 108 states were sampled (10% of which were throughout the body length (Fig. 1.6). Finally, the discarded as burn-in), and the final tree sample was caudal region of the worm was adorned with a tuft of summarized as a maximum clade credibility (MCC) a dozen cuticular fingerings (Fig. 1.7). tree. Regarding molecular