关键词: 串珠蛇舌状虫, 若虫, 超微结构, 18S rRNA, 种系发育树

Abstract:   Objective  To observe the ultrastructure of nymphal Armillifer sp. isolated from Macaca fascicularis by using scanning electron microscope (SEM), and analyze the phylogenetic relationships based on 18S rRNA gene sequences.  Methods  The parasite samples stored in 70% alcohol were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the 18S rRNA gene were performed following the extraction of total genome DNA. Sequence analysis was performed based on multiple alignment using ClustalX1.83, while phylogenetic analysis was made by Neighbor-Joining method using MEGA4.0.  Results  The nymphs were in cylindrical shape, the body slightly claviform tapering to posterior end. Abdominal annuli were gradually widened from anterior to posterior parts, the 12th-13th abdominal annuli of which were similar in width. The annuli ranged closer in the front half body, whereas in the latter part there were certain gaps between them. The circular-shaped mouth located in the middle of head ventrally. Folds were seen in inner margin of the mouth with a pair of curved hooks on both sides above it which practically disposed in a straight line. Two pairs of large sensory papillae were observed symmetrically over the last thoracic annulus of cephalothoraxs lying below the outer hook, and the first abdominal annulus was near the median ventral line. The number of abdominal annuli was 29, not including 2 incomplete terminal annuli. Rounded sensory papillae were fully distributed on the body surface, except the dorsal side of head and the ventral part of the terminal annulus. Agglomerate-like anus opening was observed at the end of ventral abdominal annuli and distinctly sub-terminal. These morphological features demonstrated that the nymphs were highly similar with that of Armillifer moniliformis Diesing, 1835. A fragment of 18S rRNA gene (1 836 bp)sequences was obtained by PCR combined with sequencing, and was registered to the Gen-eBank database with an accession number HM048870. The phylogenetic tree indicated that A. moniliformis, A. agkistrodontis and A. armillatus were at the same clade with a bootstrap value at 95%, and A. moniliformis and A. agkistrodontis were solo at a clade with a bootstrap value of 75%.  Conclusion  The nymphs isolated from Macaca fascicularis are identified as A. moniliformis temporarily.

Key words: Armillifer moniliformis, Nymph, Ultrastructure, 18S rRNA, Phylogenetic analysis