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ISEP XIV |
1 Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611-0620, USA
2 Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, FL 32604, USA
3 Microbiology & Molecular Biology Department, Brigham Young University, Provo, UT 84602-5253, USA
Correspondence
Aurélien Tartar
aurelien{at}ufl.edu
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ABSTRACT |
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This paper was presented at the XIVth meeting of the International Society for Evolutionary Protistology in Vancouver, Canada, 19–24 June 2002.
Published online ahead of print on 28 March 2003 as DOI 10.1099/ijs.0.02559-0.
The GenBank/EMBL/DDBJ accession numbers for the sequences of the plastid 16S rRNA genes of the black fly Helicosporidium and the weevil Helicosporidium are respectively AF538864 and AF538865.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sayre & Clark, 1978; Purrini, 1984; Avery & Undeen, 1987; Pekkarin, 1993). These pathogens have been found in Europe, South America, North America, Asia and Africa (Keilin, 1921; Weiser, 1970; Kellen & Lindegren, 1973; Hembree, 1979; Seif & Rifaat, 2001). Although Helicosporidium spp. seem to be ubiquitous, they have been studied so little that their occurrence and their importance as invertebrate pathogens are unclear. Significantly, their taxonomic position has remained incertae sedis. After the first description of a Helicosporidium sp. by Keilin (1921), this organism has been thought to be either a protozoan or a fungus, but it has never been clearly associated with any other known protist.
Recently, a Helicosporidium sp. was isolated from larvae of the black fly Simulium jonesii Stone and Snoddy collected in Florida (Boucias et al., 2001). Microscopic observation of the vegetative growth of Helicosporidium sp. under in vivo and in vitro conditions led Boucias et al. (2001) to associate this protist with green algae, particularly the unicellular, non-photosynthetic and pathogenic algae belonging to the genus Prototheca. Boucias et al. (2001) noticed that, as protothecans, the vegetative cells of Helicosporidium sp. undergo one or two cell divisions within a pellicle. This pellicle eventually splits open and releases either two or four daughter cells. This association between Helicosporidium and Prototheca was surprising, but was later confirmed by molecular sequence comparisons (Tartar et al., 2002). Phylogenetic analyses of several Helicosporidium sp. genes (rDNA, actin and 
-tubulin) all identified this organism as a member of the green algae clade (Chlorophyta). Moreover, a nuclear 18S rDNA phylogeny of the Chlorophyta depicted Helicosporidium sp. as a close relative of Prototheca wickerhamii and Prototheca zopfii, within the class Trebouxiophyceae. Based on both morphological and molecular evidence, the transfer of the genus Helicosporidium to Chlorophyta, Trebouxiophyceae was proposed.
Prototheca spp. have been shown to be closely related to the photoautotrophic genus Chlorella (Chlorophyta, Trebouxiophyceae), based on phylogenetic analyses inferred from the nuclear 18S rRNA and the plastid 16S rRNA genes (Huss et al., 1999; Nedelcu, 2001). The plastid 16S rRNA gene (rrn16) is a chloroplast gene. Despite having lost their photosynthetic abilities, non-photosynthetic green algae such as protothecans have been found to retain vestigial, degenerate chloroplasts called leucoplasts. The presence of such plastids has been demonstrated extensively in the non-photosynthetic green algae of the genus Polytoma (Lang, 1963; Siu et al., 1976), which are closely related to Chlamydomonas (Chlorophyta, Chlorophyceae). To our knowledge, there are no records of microscopic observations of a leucoplast in a Prototheca sp. cell. However, the plastid genome of Prototheca wickerhamii has recently been isolated and partially sequenced (Knauf & Hachtel, 2002). Similar to the situation described previously for plastid genomes in non-photosynthetic plants (reviewed by Hachtel, 1996), this genome is highly reduced in size but is believed to be functional.
In order to confirm Helicosporidium sp. as a green alga and as a close relative of the genus Prototheca, the presence of plastid DNA in helicosporidial cells was investigated. Herein, we report the PCR amplification and sequencing of plastid 16S rDNA homologues from two different isolates of Helicosporidium. This gene was targeted because it has been previously sequenced for numerous photosynthetic and non-photosynthetic algal species (Nedelcu, 2001), thereby allowing us to infer a plastid 16S rDNA phylogeny of the Chlorophyta that includes the genus Helicosporidium.
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METHODS |
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). A second Helicosporidium sp. has since been isolated from the weevil Cyrtobagous salviniae (Coleoptera: Curculionidae). This insect is a biological control agent for the aquatic weed Salvinia molesta (Goolsby et al., 2000). Both isolates were successfully amplified in Helicoverpa zea larvae, as described previously (Boucias et al., 2001). Cysts produced in Helicoverpa zea larvae were purified by gradient centrifugation on Ludox and grown in artificial medium (TNM-FH insect medium, supplemented with gentamicin and 5 % fetal bovine serum; Sigma-Aldrich) before harvest and DNA extraction. The two isolates will be referred to as weevil Helicosporidium and black fly Helicosporidium.
DNA extraction and amplification.
Helicosporidial DNA was extracted according to Boucias et al. (2001) using the Masterpure Yeast DNA extraction kit from Epicentre Technologies. Cellular DNA was used as a template for the PCR amplification of rrn16 using chloroplast 16S rRNA gene-specific primers ms-5' and ms-3' listed by Nedelcu (2001). PCR products were gel-purified with the QiaxII gel extraction kit (Qiagen) and cloned in pGEM-T vectors using the pGEM-T easy vector systems (Promega). Positive clones were sent to the Interdisciplinary Core for Biotechnology Research (ICBR) at the University of Florida for sequencing.
Phylogenetic analyses.
The two plastid 16S rDNA sequences from Helicosporidium spp. were aligned with homologous sequences available in GenBank. The alignment was obtained using CLUSTAL X software with default parameters (Thompson et al., 1997) and optimized manually. Analyses of the aligned sequences were performed in PAUP* version 4.0 beta 10 (Swofford, 2000), using maximum-parsimony (MP), maximum-likelihood (ML) and neighbour-joining (NJ) methods. The statistical model of DNA substitution used in ML analyses was determined by likelihood ratio tests as implemented in MODELTEST (Posada & Crandall, 1998). This program identified the General Time Reversible model (GTR+I+G) as being the most appropriate for our data. MP analyses were performed using the default parameters in PAUP*. NJ analyses were based on the two-parameter method of Kimura, but other models including the likelihood model used for ML analyses and HK85 were also used. Branch support for MP and NJ analyses was assessed by bootstrapping (100 replicates). Relative rate tests were performed using Tajima's test (Tajima, 1993), as implemented in MEGA version 2.1 (Kumar et al., 2001). Our alignment, as well as the resulting MP and ML trees, can be obtained from TreeBase (Morell, 1996; http://www.treebase.org), with the study accession number S819.
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RESULTS |
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-tubulin fragments sequenced from the two isolates also showed some differences in nucleotide sequences (data not shown). The helicosporidial plastid 16S rRNA genes are very AT-rich: 60·7 % for both rrn16 sequences. Such a deviation from homogeneity is common in non-photosynthetic alga genes; for example, the A+T content of the Prototheca zopfii plastid 16S rDNA is 63·1 % (Nedelcu, 2001).
The two plastid 16S rRNA gene sequences were compared with 21 homologous sequences from algal species belonging to two major classes of Chlorophyta, Trebouxiophyceae and Chlorophyceae. Both classes include some non-photosynthetic species. Phylogenetic reconstructions using NJ and MP methods produced the same tree, presented in Fig. 1. Likelihood analyses resulted in a similar tree, with one topological difference (discussed below).
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) was rooted with the plastid 16S rDNA sequence of Nephroselmis olivacea, a member of the class Prasinophyceae, which is thought to include descendants of the earliest-diverging green algae (Turmel et al., 1999). The relationships among green algal taxa depicted in Fig. 1 are consistent with affiliations suggested previously by other phylogenetic studies (Bhattacharya & Medlin, 1998; Huss et al., 1999; Nedelcu, 2001; Tartar et al., 2002). First, both classes (Trebouxiophyceae and Chlorophyceae) appear monophyletic. Within the Chlorophyceae, two non-photosynthetic clades can be identified (Fig. 1): Polytoma uvella, Polytoma obtusum and Polytoma mirum are monophyletic and are sister taxa to Chlamydomonas applanata, whereas Polytoma oviforme is more closely related to Chlamydomonas moewusii. A paraphyletic Polytoma has been demonstrated previously by Nedelcu (2001) based on nuclear 18S rDNA and plastid 16S rDNA phylogenies. Only one non-photosynthetic clade exists among the Trebouxiophyceae (as identified by Nedelcu, 2001). This clade is strongly supported by bootstrap values and it includes Helicosporidium spp., Prototheca spp. and Chlorella protothecoides, an auxotrophic, mesotrophic, but photosynthetic species. The genus Prototheca appears paraphyletic, as shown previously by nuclear 18S rDNA and plastid 16S rDNA phylogenies (Huss et al., 1999; Nedelcu, 2001). In our tree (Fig. 1), the two Helicosporidium isolates are depicted as being monophyletic, sister taxa to Prototheca zopfii, and this relationship is supported by maximal bootstrap values. This is consistent with a previously published nuclear 18S rDNA phylogeny (Tartar et al., 2002), which associated the black fly Helicosporidium and Prototheca zopfii.
Likelihood analyses depicted the same relationships, except for Prototheca wickerhamii strain 1533. In the ML tree (not shown; available at http://www.treebase.org), the two strains of Prototheca wickerhamii appeared monophyletic, sister taxa to Chlorella protothecoides. Kishino–Hasegawa tests (Hasegawa et al., 1985) showed that the ML tree is significantly different from the MP/NJ tree, using ML or MP parameters and optimality criteria (data not shown). However, it should be noted that the lack of stability of the Prototheca taxa has already been reported (Nedelcu, 2001) and that it did not have any influence on the position of the Helicosporidium spp. in our analyses. As shown in Fig. 1, Helicosporidium spp. always appear closely related to Prototheca zopfii, within the Prototheca clade (sensu Nedelcu, 2001).
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DISCUSSION |
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introduced the hypothesis that there may be more than one species of Helicosporidium. However, to date, there is only one named species (Helicosporidium parasiticum Keilin 1921), and few comparative analyses have occurred. Here, we report the discovery of two Helicosporidium isolates, from different hosts. These isolates exhibit some polymorphism in all known nucleotide sequences (nuclear 18S rDNA, actin, -tubulin and plastid 16S rDNA), suggesting that they can be differentiated at a molecular level. However, it remains unclear whether these nucleotide differences are significant and sufficient to propose that our two isolates represent different strains or species. A thorough characterization of these two isolates is currently under way.
The presence of plastid genes suggests strongly that Helicosporidium cells may contain a degenerate plastid (leucoplast) and plastid DNA. By itself, the existence of a degenerate chloroplast in Helicosporidium cells provides strong arguments in favour of Helicosporidia being non-photosynthetic green algae, but is not sufficient. Indeed, some protozoans (Apicomplexa) have also been shown to possess a degenerate, vestigial chloroplast (apicoplast) with a functional genome (Wilson, 2002). This plastid has been proposed to derive from an endosymbiotic interaction with a red alga (secondary symbiosis). However, the algal nature of Helicosporidium has already been suggested by morphological observations (Boucias et al., 2001) and strongly supported by phylogenetic analyses inferred from several nuclear genes (Tartar et al., 2002). Therefore, helicosporidial cells are likely to possess a plastid similar to other non-photosynthetic Chlorophyta, derived from a primary endosymbiosis.
Comparative analyses of the plastid gene sequences confirm that Helicosporidia are closely related to non-photosynthetic algae in the class Trebouxiophyceae (Chlorophyta). In all our phylogenetic trees, Helicosporidium spp. appear as members of the Prototheca clade (as defined by Nedelcu, 2001), sister taxa to Prototheca zopfii. The positions of Helicosporidium spp. are identical in phylogenies based on nuclear 18S rRNA genes (Tartar et al., 2002). Similar to the situation observed in the 18S rDNA phylogeny, the branch leading to the Helicosporidium+Prototheca zopfii clade is the longest of the tree, suggesting that this association could be an artifact due to long-branch attraction. However, it should be noted that Helicosporidium spp. are depicted in exactly the same position even if Prototheca zopfii is removed from the sequence alignment and that their relationship with Prototheca wickerhamii is still very strongly supported (data not shown). Therefore, this relationship is probably not an artifact.
Based on our phylogenetic analyses (Tartar et al., 2002; this study), we propose that the Helicosporidia should be included in the Prototheca clade defined by Nedelcu (2001). The clade is consistently and strongly supported by resampling tests, suggesting that Helicosporidium spp., Prototheca spp. and Chlorella protothecoides may have arisen from a common ancestor. Within the clade, the relationships are less robust: the genus Prototheca has always appeared paraphyletic, and Chlorella protothecoides, despite being proposed to be the closest green relative of Prototheca spp., has never appeared in a basal position (Huss et al., 1999; Nedelcu, 2001; Tartar et al., 2002). In our trees (Fig. 1), these ambiguities remain. However, additional resolution may be obtained inside the Prototheca clade by adding more taxa and/or by using other genes, such as protein-encoding genes, which are likely to exhibit a lower rate of nucleotide substitution. For the plastid 16S rRNA genes, Tajima's tests (Tajima, 1993) showed that the substitution rates inside the Prototheca clade are heterogeneous and are significantly different from the photosynthetic clade rates (data not shown). These differences in evolutionary rate may explain why the relative position of Prototheca wickerhamii strain 1533 changes with the type of method used (Nedelcu, 2001; this study).
Phylogenetic affinities, and the presence of a plastid gene, suggest that Helicosporidium spp. are likely to possess a plastid genome similar to Prototheca wickerhamii. In this non-photosynthetic alga, the size of the chloroplast (leucoplast) genome has been estimated to be 54 100 bp, which is much smaller than the 150 kb chloroplast DNA of the photosynthetic relative Chlorella vulgaris (Knauf & Hachtel, 2002). This decrease in size is common in all secondarily non-photosynthetic green plants and algae (Hachtel, 1996) and has been explained by the loss of most of the plastid genes that were involved in photosynthesis. However, some plastid genes have been selectively retained, suggesting that they may encode essential protein products. In Prototheca, the functions of these proteins are not known (Knauf & Hachtel, 2002). In Apicomplexa, retained plastid ORFs have been associated with the apicoplast's hypothetical primary functions: fatty acid and isoprenoid biosynthesis (reviewed by Wilson, 2002).
Additionally, Prototheca wickerhamii is also known to possess a very characteristic mitochondrial genome. As reviewed by Nedelcu et al. (2000), the Prototheca-like mitochondrial genome represents an ancestral type among green algae that features (among other characteristics) a larger size (45–55 kb) and a more complex set of protein-coding genes than the derived, Chlamydomonas-like mitochondrial genome. Having shown that the Helicosporidia are non-photosynthetic green algae and close relatives of the genus Prototheca, our hypothesis is that Helicosporidium spp. possess Prototheca wickerhamii-like organelles and organelle genomes, i.e. a highly reduced plastid genome and an ancestral type of mitochondrial genome.
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ACKNOWLEDGEMENTS |
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