3.2. SSU rRNA gene fragment analysis
PCR using primers based on conserved regions of microsporidian
SSU rRNA sequences listed at GenBank and DNA template from
microsporidian-infected P. monodon hepatopancreatic tissue
yielded an amplicon of 886 bp which was in the expected amplicon
range of approximately 900–1000 bp. After cloning and sequencing,
a fragment of 848 bp (excluding the primer sequences) was
subjected to a general BLASTn search that yielded hits only for
microsporidian sequence records. Top hits included Nucleospora
salmonis (AF185998) at 87% identity and E. bieneusi (GenBank
AF023245) at 86% identity. By contrast, sequence identity for A. penaei
(the causative agent of ‘cotton shrimp’ reported from P. monodon
in Thailand) was only 71%. In addition, the sequence identity
match between N. salmonis and E. bieneusi (from different genera in
the family Enterocytozoonidae) was 85%. These results indicated
that the 848 bp sequence from P. monodon was novel and approximately
equidistant from N. salmonis and E. bieneusi in terms of
identity difference. A subsequent CLUSTAL W alignment was carried
out and a phylogenetic tree was constructed with the optimal
criteria set for distance and using SSU rRNA gene sequences of
available microsporidians in the public database. The un-rooted