Scanning Electron Microscopic Observation on Adult Gnathostoma doloresi Worms and the Phylogenetic Analysis of G. doloresi Based on ITS2 and COX1 Gene Sequences

Objective To observe the ultrastructure of adult Gnathostoma doloresi worms isolated from wild boar by using scanning electron microscope(SEM), and analyze its phylogenetic relationships based on ITS2 and COX1 gene sequences. Methods Two adult G. doloresi worms were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the ITS2 and COX1 genes were performed following the extraction of total genomic DNA. Sequence analysis was performed based on multiple alignments and phylogenetic analysis was made by Neighbor-Joining method using MEGA 6.0. Results The bottle-shaped adult worm covered with numerous small spines. The cervical groove connected head bulb and body without spines. There was obvious distinction in body spines which surround cervical papillae and swollen area in the middle part of the body. The fragments of ITS2(418 bp) and COX1(381 bp) gene were obtained by PCR combined with sequencing, and were registered to the Gen Bank database with the accession No. of JN408329 and JN408299, respectively. The BLAST results showed that, two sequences had 99% and 98% consistency with G. doloresi ITS2(Gen Bank accession No. AB181156)and COX1(No. AB180100) gene sequences, respectively. The phylogenetic tree indicated that the two G. doloresi worms were at the same clade with a bootstrap value at 100% and 85% based on the ITS2 and COX1 sequences, respectively.G. doloresi and G. hispidum were also clustered together. Conclusion The results provide adequate information for theSEM morphological data of adult G. doloresi worms, and its phylogenetic relationship.