Bulinus tropicus (Krauss, 1848)

Hammoud, Cyril, Kayenbergh, Annelies, Tumusiime, Julius, Verschuren, Dirk, Albrecht, Christian, Huyse, Tine & Bocxlaer, Bert Van, 2022, Trematode infection affects shell shape and size in Bulinus tropicus, International Journal for Parasitology: Parasites and Wildlife 18, pp. 300-311 : 301-302

2.2. Genetic characterisation of Bulinus tropicus

To examine snail population structure, we genetically characterized all snails in our study. Snails were first visually inspected and measured using a ruler, to exclude specimens that are potentially juvenile (<7 mm) or with damaged shells from further analyses. All soft tissue was

301

removed from each snail and the shell was kept for subsequent phenotypic characterization. The tissues were placed on absorbing paper to remove traces of ethanol and subsequently they were homogenized using a sterilized scalpel. DNA was extracted using the E. Z.N.A. Mollusc DNA Kit (OMEGA Biotek, Norcross, GA, USA) and final extracts were diluted in ultrapure water in a 1:10 concentration. Shells and DNA extracts are deposited in the RMCA (specimen codes BE_RMCA_- MOL_DNA.000548 to BE_RMCA_MOL_DNA.000966). We barcoded each snail with a fragment of the gene cytochrome c oxidase subunit 1 (COX1) using primers of Folmer et al. (1994). We performed Polymerase Chain Reaction (PCR) using the Qiagen™ Taq DNA polymerase kit containing 1.5 mM PCR buffer (Qiagen™), 0.6 mM dNTP mix (Qiagen™), 1.5 mM MgCl2, 0.45 units of Taq Polymerase (Qiagen™), 0.8 mM μM HCO primer and 0.8 μM LCO primer. PCR was performed in a Tprofessional Thermocycler (Biometra™) with initial denaturation at 95 ◦ C for 5 min, followed by 39 cycles of 95 ◦ C for 45 s, 50 ◦ C for 45 s and 72 ◦ C for 45 s and a final elongation step at 72 ◦ C for 10 min. PCR products were subjected to gel electrophoresis on a 1.2% agarose gel with Midori Green Direct® staining at 120 V for 30 min, followed by visualization under UV light. All PCR products were purified using the ExoSAP (Fermentas™) kit and sequenced by Macrogen™. For each sample, the HCO and LCO sequences were assembled into a consensus sequence in Geneious v. 2020.2.4. The resulting chromatograms were checked for ambiguities to optimize sequence quality. All COX1 sequences were then aligned in Geneious using MUSCLE ( Edgar, 2004). Aligned DNA sequences were translated to amino acids to avoid inclusion of nuclear mitochondrial DNA (NUMT, Lopez et al., 1994). We assessed genetic diversity by computing the genetic distances among all specimens and illustrated population genetic structure with a TCS haplotype network ( Clement et al., 2002) using PopART v. 1.7 ( Leigh and Bryant, 2015).