A Specific DNA Probe to Identify the Intermediate Host of a Common Microsporidian Parasite of Penaeus merguiensis and P. monodon

Abstract

The intermediate host required for transmission or a common microsporidian (Agmasoma) parasite of the penaeid shrimp, Penaeus merguiensis and P. monodon is still unknown, so cross­infection studies and studies to develop preventive or therapeutic measures are not possible. The purpose of this study was to develop a specific nucleic acid probe to aid in finding the intermedi­ate host, and in tracing the life cycle of the parasite. To prepare the probe, DNA was extracted from purified microsporidian spores derived from infections in P. merguiensis and P. monodon. The prepared genomic DNAs were then incompletely digested with Sau3AI and fragments were ligated to the pGEM7Zf+ vector before being transformed to Escherichia coli JM109. Candidate probes were screened by in situ colony hybridization, slot blot hybridization and Southern blot hybridization using dATP32P labelled Agmasoma DNA. All the candidate probes from each Agmasoma source cross-hybridized with whole Agmasoma DNA digest from the other source and with roughly the same quality of signal. Finally, a probe (I-mo) derived from Agmasoma of P. monodon gave the best sensitivity because it was found to be derived from a multicopy sequence and it did not cross-hybridize with other organisms from shrimp ponds (i.e., both shrimp species, Artemia, mixed protozoa, E. coli and Vibrio parahaemolyticus). DNA extracts were prepared from 22 animal species (including fish, crustaceans, molluscs and mixed plankton) present in seawater canals where microsporidian-infected shrimp were present. These were probed with digoxigenin (DIG) labelled 1-mo probe. Two fish species, Priacanthus tayenus and Scatophagus argus, gave positive hybridization signals.