Helicosporidium sp. was isolated originally from the blackfy Simulium jonesi Stone & Snoddy (Diptera: Simuliidae) and produced in Helicoverpa zea. Approximately 4e7 cysts suspended in 0.15 M NaCl were applied to a linear gradient of 1.00-1.3003 g Ludox HS40 ml-1 (DuPont). Helicosporidial cysts that banded at an estimated density of 1.17 g ml-1 were collected, diluted in 10 vols deionized water and washed free of residual Ludox by repeated low-speed centrifugation steps. The pellet, resuspended in 50 µl water, was extracted with the use of the Masterpure Yeast DNA extraction kit (Epicentre Technologies), following the manufacturer's protocol. Examination of the cells before and after lysis treatment revealed the presence of numerous, highly refractile cysts before treatment and, after incubation in the lysis buffer at 50°C, cysts appeared to dehisce, releasing the filamentous cells. However, no massive disruption of the ovoid cells or filamentous cells was observed in these preparations. Visible pellets were observed after RNase treatment, phenol/chloroform extraction and ethanol precipitation. The final pellet, suspended in molecular-biology-grade water, was frozen at -20°C. The DNA preparations, electrophoresed on a 0.8% agarose gel and stained with ethidium bromide, produced a single, discrete ~20 kb band diagnostic for genomic DNA. Mature cysts were also processed for electron microscopy according to previously published protocols (Boucias et al., 2001).
The ITS1ħ5.8SħITS2, 28S and 18S ribosomal regions of the helicosporidial DNA were amplified with a mixture of Taq DNA polymerase (Promega) and Pfu polymerase (Stratagene), using the primers TW81 and AB28 for the ITSħ5.8S (Curran et al., 1994) and NL-1 and NL-4 primers for the 28S (Kurtzman & Robnett, 1997). Two primer sets, designed from consensus regions of selected fungal, algal and protozoan sequences downloaded from GenBank, were used to amplify the 18S region. Several series of primers, also designed from consensus regions of selected fungal, algal and protozoan genes, were used to amplify partial sequences of the actin and β-tubulin genes by PCR. DNA was excised from agarose gels, extracted with the QiaxII gel extractionkit (Qiagen) and sent to the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for sequencing.
The helicosporidial 18S rDNA region sequence was aligned with 138 other sequences from representative eukaryotic taxa obtained from the Ribosomal Database Project (RDP; Maidak et al., 2000). Downloaded sequences were pre-aligned based on the secondary structure of the rDNA. An additional 18S sequence from the pathogenic alga Prototheca wickerhamii was downloaded from GenBank (accession no. X56099) and incorporated in the small-subunit (SSU) RNA dataset. Additionally, 28S sequences were downloaded from GenBank and aligned with the helicosporidial 28S sequence using clustal x (Thompson et al., 1997). Eventually, the SSU- and large subunit (LSU)-rDNA datasets were combined to infer one single ribosomal phylogeny. Both actin and β-tubulin sequences were aligned with homologous sequences downloaded from GenBank. Alignments were obtained using clustal x software with default parameters. All datasets were checked by eye before further analyses and are available upon request. They can also be obtained from TreeBase (Morell, 1996; http://www.treebase.org/treebase/) with the study accession number S604. The 18S algal alignment was kindly provided by V. A. R. Huss (University of Erlangen, Germany). Aligned datasets were subjected to a partition-homogeneity test using the program paup*, version 4.0b4a (Swoċord, 2000), in order to assess the extent of character incongruence between the datasets (Farris et al., 1994). Phylogenies were then reconstructed using neighbour-joining (NJ) as implemented in the paup* program version 4.0b4a. NJ analyses were based on the Paralinear/LogDet model of nucleotide substitution (Lockhart et al., 1994). This method allows for non-stationary changes in base composition and has been shown to reduce support for spurious resolutions, such as long-branch attraction. Monophyly of groups was assessed with the bootstrap method (100 replicates). Additionally, maximum-parsimony analyses, including jackknifing (100,000 replicates ; Farris et al., 1996), were also performed using paup*. We chose the latter, conservative approach for its ability to search rapidly a large amount of tree space and to estimate support for an unambiguously resolved group (Lipscomb et al., 1998).