Computational protocol: Intermediate hosts of Protostrongylus pulmonalis (Frölich, 1802) and P. oryctolagi Baboš, 1955 under natural conditions in France

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Protocol publication

[…] Gastropods collected under planks or on plot, were examined for lungworm larvae. All were stored in separate labeled plastic boxes with ventilation openings and refrigerated (4°C) for a maximum of three weeks. Except for the operculated snails the gastropods were drowned in an airtight jar full of water to kill them and involved the output of the foot out of the snail shell. Foots were recovered and crushed between two heavy glass slides to spread the snail tissue for observation with a stereo-microscope [,].Larvae were observed in some snails in the musculature of the foot. After pre-identification on simple morphological criteria (size, sheath, darkened cuticle) as described by Boev (1984) [], some larvae were extracted out of muscle, which was easy after dissection or pressing between slide and cover slip. Then they were preserved in 90° ethanol for molecular analysis (Table ).After rinsing larvae in distilled water, then drying in an oven at 37°C, DNA extraction was performed with a Qiamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Sequencing of the D2 domain of the 28S was used for the identification of nematodes as described previously [,] (Table ). PCR products were directly sequenced in both directions with the primers used for DNA amplification (Genoscreen, France). Sequence alignment was performed using the ClustalW routine included in the MEGA version 5 software and checked by eye.Haplotypes of the larvae were compared to sequences of Protostrongylinae available in GenBank: P. pulmonalis (EU595590), P. pulmonalis (KJ450993-1018), P. oryctolagi (KJ450993-1018), P. boughtoni (EU595595), P. rushi (EU595598), P. stilesi (EU595599), P. rufescens (EU595600) and P. rupicaprae (EU595601). […]

Pipeline specifications

Software tools MUSCLE, Clustal W, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Diseases Lung Diseases, Nematode Infections