Sequence and phylogenetic analysis of SSU rRNA gene of five microsporidia.

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.

Introduction

Microsporidia are obligate intracellular parasites that infect a broad range of invertebrates and humans, they were recognized as a causative agent for many hosts including insects, fishes, rodents, and primates. In 1857, the first microsporidian species—N. bombycis was described by Nageli [1]. Until now, more than 1,200 microsporidia species belonging to ~150 genera have been reported [2, 3].

The adaptation of microsporidia to a parasitic lifestyle precipitates a number of profound changes which lead to a seemingly paradoxical mixture of characteristics [4]: on one hand, parasites may evolve extremely complex and sophisticated mechanisms to invade their host and evade its defenses, while on the other hand, they may also appear more “simple” by dispensing with characteristics they no longer need as they increasingly depend on host metabolism for nutrients and energy. Although microsporidia were considered to be eukaryotes, they lack some typical eukaryotic characteristics including mitochondria, peroxisomes, classical stacked Golgi apparatus, and 80S ribosomes. The microsporidia contain 70S ribosomes, ribosomal subunits (30S and 50S), and rRNAs (16S and 23S) with prokaryotic size, and they have no separate 5.8S rRNA. They also lack flagella, pilus and 9 + 2 microtubular structure [5]. Up to now, the genome size has been determined for numerous microsporidian species, and they range from 19.5 Mbp in Glugea atherinae to just 2.3 Mbp in Encephalitozoon intestinalis [6].

For a long time, microsporidia were generally regarded as primitively simple eukaryotes and classified as an early branch from prokaryotes to eukaryotes [7], and they were universally thought to be classified as Protista, Protozoa, Microspora in five kingdom system. This conclusion was supported by early molecular phylogenic evidence, but more and more recent trees constructed from the conserved protein genes provide further evidence for the argument that microsporidia should properly be classified as fungi. Thus, in 1998 Cavalier-Smith [8] classified the microsporidia into the kingdom Fungi. And in 2002 NCBI concluded a description of microsporidia as Cellular Organisms, Eukaryote, Fungi/Metazoa group, Fungi, Microsporidia. For the Society of Classification has not approved the description of microsporidia, there is not universal agreement on the evolution and classification of microsporidia.

There has been increasing interest in studying large subunit rRNA (LSU rRNA), SSU rRNA, internal transcribed spacers (ITS), translation elongation factors (EF), heat shock protein (HSP), and beta-tubulin of microsporidium at the molecular level, which provided important evidence for the phylogenic analysis of microsporidia. As a conserved gene, the SSU rRNA universally existed in eukaryotic and prokaryotic organisms, varied with constant rate, and can be used as the molecular clock of evolution. For its relatively highly variable regions provides important information for the relationship among species, the SSU rRNA gene also can be used as the biological identification label. And the molecular characterization of different microsporidia has widely relied on sequencing and analyzing of SSU rRNA genes [9], which also can be used as a molecular marker for estimating relationships among microsporidia [10]. Therefore, the SSU rRNA gene plays an important role in the phylogenetic research.

Traditional taxonomic studies and species classification of the microsporidia are mainly based on biological characters such as morphology and development of spore, microsporidium–host relationships, life cycle, parasitic site as well as transmission way. However, for some basic biological problems, taxonomic determinations based on these criteria alone can sometimes be problematic. With the development of molecular biotechnology, many Nosema species were proven to be distant from N. bombycis in molecular phylogeny, and divided into other genus [11, 12]. So, phylogenetic analysis of SSU rRNA gene is an important tool for the classification of microsporidian species, which helps to construct more valuable classification system for the microsporidia.

In this paper, we cloned the complete SSU rRNA sequences of five microsporidia, compared them with the corresponding sequences of other 94 microsporidia for which data were available, and have preliminarily determined the taxonomic status of the four novel microsporidian isolates by constructing and analyzing of phylogenetic tree, and comparing the length and G+C content of SSU rRNA sequences.

Materials and Methods

The Origin of Microsporidia

The five species of microsporidia studied in this paper were isolated from diverse geographic regions and different hosts. The names of isolates, host insects, geographic regions, and accession number for them are described in Table 1. The five microsporidian species were isolated by Professor Shen ZhongYuan in 2006–2007 and kept in Pathology Department of Sericulture Research Institute of Chinese Academy of Agricultural Sciences. Additionally, the four novel microsporidian isolates which were separately isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori were unclassified.

Table 1

The host insects, geographic regions and accession of five microsporidian species

No.	Microsporidia	Host insects	Geographic regions	Accession
1	Nosema sp. PR	Pieris rapae	Zhenjiang, Jiangsu, China	EU864531
2	Nosema sp. PA	Phyllobrotica armta	Mianyang, Sichuan, China	EU864528
3	N. heliothidis	Helicoverpa armigera	Baoding, Heibei, China	EU864529
4	Nosema sp. HA	Hemerophila atrilineata	Zhenjiang, Jiangsu, China	EU864527
5	Endoreticulatus sp. Zhenjiang	Bombyx mori	Zhenjiang, Jiangsu, China	EU864530

Preparation of Genomic DNA of Microsporidia

To prepare the genomic DNA of microsporidia, 400 μl suspension of purified spores (1010 spores/ml) was mixed with 40 μl KOH (2 mol/l) in a 1.5 ml Eppendorf tube and incubated at 27°C for 1 h, TEK buffer (1 mmol/l Tris–HCl, 10 mmol/l EDTA, 0.17 mol/l KCl, pH 8.0) was added and continued incubating at 27°C for 1 h. Adjusted pH to 8.0 with 1 mol/l HCl, added 10% SDS in order to attain 0.5% in the mixture and kept it in ice-bath for 15 min. 20 mg/ml proteinase K (TaKaRa Biotechnology Co. LTD) was added in order to attain 200 μg/ml in the mixture and incubated at 50°C for 4 h. Subsequently, isovolumetric tris–phenol extraction was performed twice, and washed with chloroform: isoamyl alcohol (24:1 v/v). 10% NaOAC was added to the recovered aqueous phase after centrifuging (10,000 r/min, 5 min), and then the DNA was precipitated by 2.5 times volume of cold ethanol at −20°C for 40 min before centrifuging (12,000 r/min, 10 min). In addition, the precipitated DNA was rinsed twice with 500 μl cold 70% ethanol before drying at 37°C for 5–10 min. Then, the extracted DNA was stored at −20°C after dissolution in 50 μl TE buffer at 65°C for 10 min.

Amplification, Cloning and Sequencing of SSU rRNA Gene

To amplify the SSU rRNA genes, primer sets were designed as Huang et al. [13] described, forward primer: 5′-CACCAGGTTGATTCTGCC-3′; reverse primer: 5′-TTATGATCCTGCTAATGGTTC-3′. The amplification was performed with following condition: after initial denaturation of DNA at 94°C for 8 min, 28 cycles were run—94°C for 1 min, 43°C for 1 min 40 s, and 72°C for 1 min 40 s—with a 10 min 72°C extension.

The PCR amplified products were cloned into pGEM-T vector and then sequenced. All the sequences were submitted to GenBank, and the accession number were shown in Table 1.

Construction of Phylogenetic Tree and Analysis of SSU rRNA Gene Sequences

The SSU rRNA gene sequences were aligned using the “ClustalX 1.83” program. Phylogenetic trees based on the resultant alignments were, respectively, constructed using the Neighbor-Joining algorithm (NJ) of Phylip software with Kimura-2 parameter and UPGMA algorithm of MEGA 4.0 software with Maximum Composite Likelihood, and one thousand replications were generated to test the robustness of the tree. Subsequently, the length and GC content of the complete SSU rRNA sequences of 22 microsporidia were analyzed with the Editseq program of DNAStar software, and the identity and divergence of the sequences of 26 microsporidia were analyzed with the MegAlign program of DNAStar software.

Results

Analysis of Phylogenetic Tree of Microsporidia

The complete SSU rRNA gene sequences of the five microsporidia were aligned with the homologs from other 94 species of various microsporidian genera, such as Nosema, Amblyospora, Endoreticulatus, Vairimorpha, Pleistophora, Encephalitozoon, and Glugea, available in public databases (Table 2). And Beauveria bassiana was used as an out-group (Table 2).

Table 2

The taxon, accession number, author, and published year of microsporidia and Beauveria bassiana which were aligned for homology

No.	Taxon	Accession	No.	Taxon	Accession	No.	Taxon	Accession
1	Amblyospora californica	U68473	33	Hazardia milleri	*AY090067	65	Pleistophora typicalis	AJ252956
2	Amblyospora canadensis	*AY090056	34	Heterosporis anguillarum	AF387331	66	Polydispyrenia simulii	*AJ252960
3	Amblyospora cinerei	*AY090060	35	Hyalinocysta chapmani	*AF483838	67	Pseudoloma neurophilia	*AF322654
4	Amblyospora connecticus	*AF025685	36	Ichthyosporidium sp.	L39110	68	Pseudonosema cristatellae	AF484694
5	Amblyospora crenifera	*AY090061	37	Intrapredatorus barri	*AY013359	69	Schroedera plumatellae	*AY135024
6	Amblyospora excrucii	*AY090044	38	Janacekia debaisieuxi	AJ252950	70	Spraguea lophii	*AF104086
7	Amblyospora ferocious	*AY090062	39	Kabatana takedai	*AF356222	71	Tetramicra brevifilum	*AF364303
8	Amblyospora indicola	*AY090051	40	Larssonia obtusa	AF394527	72	Thelohania solenopsae	AF134205
9	Amblyospora khaliulini	*AY090047	41	Loma embiotocia	*AF320310	73	Trachipleistophora hominis	AJ002605
10	Amblyospora opacita	*AY090052	42	Microgemma caulleryi	*AY033054	74	Trichonosema pectinatellae	*AF484695
11	Amblyospora stictici	*AY090049	43	Microsporidium prosopium	*AF151529	75	Vairimorpha necatrix	Y00266
12	Amblyospora stimuli	*AY090050	44	Nadelspora canceri	*AF305706	76	Vairimorpha lymantriae	AF033315
13	Amblyospora weiseri	*AY090048	45	Oligosporidium occidentalis	*AF495379	77	Vavraia culicis	AJ252961
14	Ameson michaelis	L15741	46	Tubulinosema acridophagus	AF024658	78	Vavraia oncoperae	X74112
15	Antonospora scoticae	AF024655	47	Nosema apis	U97150	79	Vittaforma corneae	U11046
16	Bacillidium sp.	*AF104087	48	Nosema bombi	AY008373	80	Weiseria palustris	AF132544
17	Berwaldia schaefernai	*AY090042	49	Nosema carpocapsae	*AF426104	81	Antonospora locustae	AY376351
18	Brachiola algerae	AM422905	50	Nosema ceranae	DQ486027	82	Glugea plecoglossi	*AJ295326
19	Bryonosema plumatellae	*AF484692	51	Nosema furnacalis	U26532	83	Glugea sp. GS1	*AJ295325
20	Culicosporella lunata	AF027683	52	Nosema granulosis	*AJ011833	84	Tubulinosema kingi	*DQ019419
21	Cystosporogenes operophterae	AJ302320	53	Nosema oulemae	U27359	85	Nosema plutellae	AY960987
22	Encephalitozoon cuniculi	Z19563	54	Nosema portugal	AF033316	86	Nosema sp. C01	AY383655
23	Encephalitozoon lacertae	*AF067144	55	Nosema tyriae	AJ012606	87	Nosema sp. PX1	AY960986
24	Endoreticulatus bombycis	*AY009115	56	Nosema vespula	U11047	88	Nosema spodopterae	AY747307
25	Endoreticulatus schubergi	L39109	57	Ordospora colligata	AF394529	89	Pleistophora mulleri	*EF119339
26	Nucleospora salmonis	*AF186003	58	Orthosomella operophterae	AJ302317	90	Endoreticulatus sp. CHW-2004 Bulgaria	AY502945
27	Flabelliforma magnivora	AJ302318	59	Ovipleistophora mirandellae	*AF356223	91	Endoreticulatus sp. CHW-2004 Taiwan	AY502944
28	Glugea anomala	AF044391	60	Parathelohania anophelis	AF027682	92	Endoreticulatus sp. CHW-2008 Austria	*EU260046
29	Glugea atherinae	U15987	61	Parathelohania obesa	*AY090065	93	Nosema bombycis	AY259631
30	Gurleya daphniae	*AF439320	62	Pleistophora hippoglossoideos	AJ252953	94	Nosema antheraeae	DQ073396
31	Marssoniella elegans	*AY090041	63	Pleistophora mirandellae	AJ252954	95	Beauveria bassiana	*EU334679
32	Gurleya vavrai	AF394526	64	Pleistophora ovariae	AJ252955

Note: The SSU rRNA gene noted with ‘*’ is partial sequence

Both the two phylogenetic trees of SSU rRNA genes (Figs. 1, 2) constructed by Neighbor-Joining and UPGMA revealed that, besides Nosema microsporidia, the microsporidia of other genera such as Amblyospora, Endoreticulatus, Vairimorpha, Pleistophora, Encephalitozoon, Glugea clustered in each clades, which is consistent to early published classifications of microsporidia. Consequently, the phylogenetic tree we constructed is valuable.

Fig. 1

Phylogenetic tree constructed by the Neighbor-Joining method for the SSU rRNA sequences of microsporidia. Beauveria bassiana is included as an out-group. Neighbor-Joining consensus tree used 1000 bootstrap replicates. The number represents the percentage of bootstrap values

Fig. 2

Phylogenetic tree constructed by the UPGMA method for the SSU rRNA sequences of microsporidia. Beauveria bassiana is included as an out-group. The UPGMA tree used 1000 bootstrap replicates. The number represents the percentage of bootstrap values, and the evolutionary distance was shown below

Three novel microsporidian isolates isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata separately, were grouped in the Nosema clade, indicating that this three novel microsporidian isolates belong to the genus Nosema. Therefore, we temporarily name them as Nosema sp. PA, Nosema sp. PR and Nosema sp. HA, respectively. Another novel microsporidium isolated from Bombyx mori got clustered with the Endoreticulatus clade. So, we propose to classify it into the genus Endoreticulatus, with temporary designation Endoreticulatus sp. Zhenjiang. These two phylogenetic trees based on the SSU rRNA sequences suggest that Nosema sp. PR is closely related to N. bombycis, while Nosema sp. PA, Nosema sp. PR, and Nosema sp. HA have close relationship with each other.

Further analysis of two SSU rRNA phylogenetic trees manifests that Vairimorpha lymantriae, Vairimopha necatrix, Oligosporidium occidentalis, and 20 Nosema species form a large cluster which can be classified into two parts. Part one is composed of four Nosema species cloned in this experiment and other nine Nosema species, while part two is composed of Vairimorpha lymantriae, Vairimopha necatrix, Oligosporidium occidentalis, and seven Nosema species. Consequently, we divide 20 Nosema species into two groups for further analysis. Group I comprise 13 Nosema species and group II comprise other seven Nosema species.

Analysis of Length and GC Content of SSU rRNA Gene of Microsporidia

We analyzed the complete SSU rRNA sequences of 22 microsporidia and found that the lengths of complete SSU rRNA gene sequences of Nosema species ranged from 1231 to 1393 bp, and that of Endoreticulatus species ranged from 1252 to 1254 bp (Table 3). For the four Nosema species cloned in this experiment, the lengths of complete SSU rRNA gene sequences of Nosema heliothidis, Nosema sp. PA, Nosema sp. PR, and Nosema sp. HA are 1232 and 1233 bp, respectively, and that of Endoreticulatus sp. Zhenjiang is 1254 bp. These results provide further molecular information for the four novel microsporidian species in our study.

Table 3

The length and GC content of complete SSU rRNA sequences of 22 microsporidian species

No.	Taxon	Lengths (bp)	GC content (%)	No.	Taxon	Lengths (bp)	GC content (%)
1	Nosema sp. HA	1232	34.09	12	N. tyriae	1233	34.39
2	Nosema sp. PA	1233	33.98	13	N. apis	1242	38.73
3	Nosema sp. PR	1232	34.25	14	N. bombi	1247	38.09
4	N. antheraeae	1237	34.28	15	N. ceranae	1259	36.46
5	N. bombycis	1232	34.17	16	N. oulemae	1251	36.85
6	N. furnacalis	1232	33.93	17	N. portugal	1393	35.75
7	N. heliothidis	1232	34.09	18	N. vespula	1245	36.79
8	N. plutellae	1231	34.93	19	E. schubergi	1252	51.04
9	Nosema sp. C01	1236	34.22	20	Endoreticulatus sp. CHW-2004 Bulgaria	1254	51.04
10	Nosema sp. PX1	1232	34.09	21	Endoreticulatus sp. CHW-2004 Taiwan	1254	51.12
11	N. spodopterae	1232	34.09	22	Endoreticulatus sp. Zhenjiang	1254	50.96

It is found as well that the GC content of the Nosema sp. PR, Nosema sp. PA, N. heliothidis, and Nosema sp. HA varied from 33.98 to 34.25%, and the GC content of Endoreticulatus sp. Zhenjiang is 50.96% (Table 3). The GC content of the SSU rRNA gene in Nosema species ranged from 33.93 to 38.73%, and that of Endoreticulatus species ranged from 50.96 to 51.12% (Table 3), which is significantly higher than that Nosema species. Based on the GC content, the nucleotide acid length and phylogenetic analysis for the SSU rRNA gene, we suggested that the four novel microsporidian species, Nosema sp. PA, Nosema sp. PR and Nosema sp. HA, belong to genus Nosema, while Endoreticulatus sp. Zhenjiang belongs to the genus Endoreticulatus.

Additionally, we analyzed the lengths and GC content of the two Nosema groups. Nosema group I, which contains 1231–1237 bp with GC content of 33.93–34.93%, is shorter and lower than that of Nosema group II, which contains 1242–1393 bp with GC content of 35.75–38.73%.

Analysis of Identity and Divergence of the SSU rRNA Sequences of Microsporidia

The identities and divergence of SSU rRNA gene among 20 Nosema species varied from 83 to 100%, and 0–0.193, while that among six Endoreticulatus species varied from 98.3 to 99.7%, and 0.003–0.017, respectively. The identities and divergence of SSU rRNA gene between Nosema and six Endoreticulatus varied from 66.0 to 71.0%, and 0.369–0.457. Consequently, the genus Endoreticulatus is markedly different from the genus Nosema, and the relationship among Endoreticulatus species is closer than that of Nosema species. Additionally, the identities and divergence of SSU rRNA gene among Nosema species in group I varied from 96.3 to 100% and 0–0.038, while that among Nosema species in group II varied from 94.8 to 99.8%, and 0.002–0.054, indicating that there was higher degree of similarity of SSU rRNA gene sequence among the group I species than that among group II species in Nosema.

It is clear that the SSU rRNA genes of microsporidia have high identities and less divergence among species in same genus and have lower identities and higher divergence between different genera. Consequently, it is limited to measure the close phylogenetic relationships between various species in the genus based on the identities and divergence of the SSU rRNA gene, while the identities and divergence of the SSU rRNA gene are more suitable for the analysis of phylogenetic relationships between different genera and the determination of taxonomical status on the genus level.

Discussion

Ribosomal gene sequences of microsporidia are frequently used for diagnostic tests, phylogenetic studies, epidemiological investigations of the host range, and the geographic distribution and pathogenetic studies of fish pathogens [14]. Baker et al. [15] analyzed a short fragment of LSU rRNA gene of eight Nosema species, and found the N. bombycis shared high homologies with other Nosema species isolated from Lepidoptera insects, low homologies with Vairimorpha species, and very low homologies with Amblyospora species. Slamovits [11] proposed transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb. by constructing a phylogenetic tree based on the SSU rRNA gene and with the ultrastructure of mature N. locustae spores.

In this study, three novel microsporidia, isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata, were classified into genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA, respectively. And the novel microsporidium isolated from the silkworm in countryside in Zhenjiang, Jiangsu Province, China, was temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The first report of Endoreticulatus species which infected Bombyx mori was in Mianyang Silkworm Eggs Production Farm and Santai Silkworm Eggs Production Farm in Sichuan Province, China. Subsequently, the Endoreticulatus species were successively found in silkworm eggs production in China. For their rather small size, they were usually called “mini-spore”. In 1995, Wan Yongji classified a “mini-spore” isolated from the silkworm in Sichuan Province into genus Endoreticulatus by studying its morphological character, ultrastructure, and life cycle [16].

Further analysis of two SSU rRNA phylogenetic trees manifests that the 20 Nosema species can be divided into two groups. Group I comprise 13 Nosema species, while group II comprise other seven Nosema species. The comparison of the SSU rRNA genes revealed that the phylogenetic relationship among 13 Nosema species in group I is closer than that of seven Nosema species in group II. For the significant differences between Nosema group I and Nosema group II, the phylogenetic trees based on SSU rRNA gene also provide further evidence for the argument that the genus Nosema should properly be separated into two groups [10, 15, 17, 18]. And SSU rRNA gene can be used to explore the differences among Nosema species, study the microsporidian phylogenetic relationship, and provide more evidence for the classification of microsporidia.

The complete SSU rRNA gene sequence of the N. heliothidis had 100% identity with the previously reported homologous sequences of Nosema sp. PX1 (isolated from Plutella xylostella) and N. spodopterae (isolated from Spodoptera litura). But the other ribosomal RNA sequences of these three microsporidian species, such as LSU, ITS, IGS and 5S sequences, did not have such high identity (data not shown). So the SSU rRNA sequence may not be a suitable marker for identifying closely related microsporidia [18, 19]. In 2007, Ku [20] constructed phylogenetic trees of the LSU rRNA, SSU rRNA, alpha-tubulin, beta-tubulin, and RPB1 (DNA dependent RNA polymerase II largest subunit) sequences of Nosema sp. PX1, Nosema sp. PX2, N. spodopterae and N. bombycis with four kinds of algorithm, found that PX2 was a novel species of microsporidia, and PX1 was closer to N. bombycis and probably a sub-species of N. bombycis. Recently, the ITS gene of ribosome was frequently used for the identification of parasite species and strain, and the molecular diagnosis for its inter-specific divergence. Consequently, SSU rRNA gene is suitable for the phylogenetic relationship analysis between genera and can be used as the taxonomical status at the genus level, while ITS gene of ribosome is better than SSU rRNA gene in the analysis of phylogenetic relationship between different species in the same genus and can be used for the classification of microsporidium at the species level. Therefore, phylogenetic analysis based on the combination of SSU rRNA gene and ITS gene of ribosome will provide more molecular evidence for the classification and evolution study of microsporidia.

Microsporidia are obligate intracellular parasites. They are different from independent organisms, and they live in the environments controlled by the genomes of the hosts with coevolution [21]. Since many microsporidia are considered to have a wide host range [22], the cross infection of microsporidia often occurs between hosts, sometimes, microsporidia can infect new hosts through the transition of intermediate host, generate and evolve in this procedure. So, the gene sequences of microsporidia have some variations through the long evolutionary process. Phylogenetic trees constructed from the SSU rRNA sequences of microsporidia indicated that microsporidia formed complicated phylogenetic relationship with each other in the evolutionary process. Therefore, the research of phylogenetic relationship based on microsporidian SSU rRNA gene and the epidemiological investigation of the host range have practical value on the prevention of microsporidiosis.