Computational protocol: Ultrastructural and Molecular Characterisation of an Heterosporis-Like Microsporidian in Australian Sea Snakes (Hydrophiinae)

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Protocol publication

[…] A 25 mg piece of fresh-frozen tissue from each of the three individuals (HM1, HM2 and HE1) was processed for DNA extraction using a DNEasy Blood and Tissue kit (Qiagen, Chadstone, Victoria), as per manufacturer directions. ssrRNA gene sequences were amplified by PCR using the universal microsporidial primers 18f (5′-CAC CAG GTT GAT TCT GCC-3′) and 1492r (5′-GGT TAC CTT GTT ACG ACT T-3`) for amplifying unknown rRNA genes in novel microsporidia species []. PCR was performed in a 25 μL reaction volume, comprising 6 μL of DNA, 2.5 μL 10x PCR buffer (Qiagen, Chadstone, Victoria), 4 μL of dNTP at 1.25 mM (Qiagen, Chadstone, Victoria), 2.5 μL each of 18f and 1492r primer at 10 mM, 1.25 units of HotStarTaq (Qiagen, Chadstone, Victoria) and the remaining volume made of nuclease-free water. A negative control sample containing nuclease free water in place of DNA was run in parallel. Amplification was carried out in a Biorad C1000 thermal cycler. After initial denaturation for 4 minutes at 94°C, 35 cycles were completed of the following: denaturation at 94°C for 50 seconds, annealing at 56°C for 50 seconds and extension at 72°C for 80 seconds. A final extension of 7 minutes at 72°C was completed before holding at 4°C. The resulting PCR amplification product was analysed by gel electrophoresis prior to sequencing. The PCR product was then purified and sequenced by the Animal Genetics Laboratory (AGL) within the School of Veterinary Science, The University of Queensland. Chromatographs were read and analysed using the software program Finch TV v1.4.0 (Geospiza Inc., Seattle, WA) and contigs assembled by combining corresponding forward and reverse sequences. Sea snake sequences were aligned using the ClustalW accessory application within BioEdit c1.0.9.0 []. A BLAST search was completed to determine similarity with other sequences in GenBank. All sequences with greater than 92% sequence similarity and greater than 90% query cover were included in phylogenetic analysis. To assist with determining identity and phylogenetic placement, at least one representative from each major microsporidian group (as per Vossbrinck, 2005) was also included in phylogenetic analysis. Sequences selected represented species from freshwater, terrestrial and marine hosts and from the classes Aquasporidia, Marinosporidia and Terresporidia. Non-microsporidian outgroups for phylogenetic analysis included a representative from the neighbouring Aphelidia, Cryptomycota, Chytridiomycota and Zygomycota phyla []. Partial ssRNA sequences were used to generate phylogeny. All sequences were aligned using Muscle [] to a final alignment length of 1920 positions (the inclusion of non-microsporidian outgroups resulted in increased dataset length—Data in ). The maximum likelihood tree was constructed using PhyML [] with 1000 bootstrap replications. Bootstrap values were expressed as percentages and only values above 50 were shown in the final tree. The TIM3 + G + I substitution model was specified based the results of a jModelTest 2.0 analysis of the alignment. MrBayes 3.2.4 [] was used to construct a Bayesian inference tree using 3 million generations, sample frequency of 100 and a burn-in value of 10%. As TIM3 is not supported by MrBayes, the GTR + G + I model was specified as the closest over-parameterised model []. Convergence and burn-in values were assessed using Tracer []. Only posterior probabilities above 50 were shown in the final tree. Sea snake microsporidia sequences were submitted to GenBank with the accession numbers KT380106, KT380107 and KT380108. […]

Pipeline specifications

Software tools Clustal W, BioEdit, MUSCLE, PhyML, jModelTest, MrBayes
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Anguilla japonica
Diseases Infection, Fractures, Bone