Establishment of Liebermannia dichroplusae n. comb. on the Basis of MolecularCharacterization of Perezia dichroplusae Lange, 1987 (Microsporidia)

YULIYA Y. SOKOLOVA,a,b CARLOS E. LANGEc and JAMES R. FUXAa

aLaboratory of Insect Pathology, Department of Entomology, Louisiana State University Agricultural Center, 404 Life Sciences Bldg.,

Baton Rouge, Louisiana 70803-1710, USA, andbInstitute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 St. Petersburg, Russia, and

cComision de Investigaciones Cientıficas (CIC) de la provincia de Buenos Aires, Centro de Estudios Parasitologicos y de Vectores (CEPAVE),

UNLP—CONICET, Calle 2 # 584, La Plata (1900), Argentina

ABSTRACT. Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichropluselongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and withother microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and per-formed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) ofthe new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65%similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the mi-crosporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hier-archical level, the Liebermania–Orthosomella branch forms a clade with the Endoreticulatus–Cystosporogenus–Vittaforma group andwith Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We proposetransferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based bothon SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence ofelongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores ofvariable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperateregion of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic inL. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores fromthe revised diagnosis of the genus Liebermannia.

Key Words. Acrididae, Argentina, Dichroplus elongatus, grasshopper, Liebermannia, molecular taxonomy, nuclear cycle, Orthoptera,Perezia, ultrastructure.

A MICROSPORIDIUM Perezia dichroplusae Lange has beendescribed from epithelial cells of Malpighian tubules of the

Argentine grasshopper Dichroplus elongatus (Lange 1987). Itsassignment to the genus Perezia Leger & Duboscq, 1909, wasbased on the presence of meronts with diplokaryotic nuclei,polysporoblastic sporogony via multiple fission of moniliformsporogonial plasmodium, and spores with isolated nuclei. Amongthree known Perezia species, only P. dichroplusae parasitizesterrestrial invertebrates; the other two infect marine hosts. Thetype species, Perezia lankesteriae, is a hyperparasite of the greg-arine Lankesteria ascidiae (Apicomplexa: Eugregarinorida) in theascidian Ciona intestinales (Urochordata: Cionidae) (Ormieres,Loubes, and Maurand 1977). Perezia nelsoni is distributed world-wide, infecting muscles of a wide range of prawns of the familyPenaeidae (Crustacea, Decapoda) (Canning, Curry, and Over-street 2002). Questions arose about the relatedness of the micro-sporidium from D. elongatus to P. lankestariae and P. nelsoni andits placement into the genus Perezia, because it parasitizes unre-lated hosts with no ecological interface and exhibits differenttissue tropism and morphology.

In order to determine the relationships of the grasshopper mi-crosporidium with other Perezia species and with other micro-sporidian genera and species, we sequenced the small subunitribosomal RNA gene (SSU rDNA) of this microsporidium andperformed phylogenetic analysis of the novel sequence against17 microsporidian SSU rDNA sequences available in GenBank,using maximum-likelihood (ML), neighbor-joining (NJ), andmaximum-parsimony (MP) algorithms. We unambiguously placethe grasshopper microsporidium into the recently described genus

Liebermannia (Sokolova, Lange, and Fuxa 2006b) based on theresults of this analysis together with reevaluation of morpholog-ical and developmental characters.

MATERIALS AND METHODS

Microsporidium-infected D. elongatus were collected duringJanuary–March, 1985–1988, in pastures at the type locality of thepathogen, Brandsen, Buenos Aires Province, Argentina, where itnormally occurs enzootically as it does in other areas of thePampas region (Lange 1989, 2003). On the basis of light andelectron microscopy, the microsporidium was previously de-scribed as P. dichroplusae (Lange 1987).

DNA sequencing. Spore suspensions from infected Malpigh-ian tubules of the host, collected from 1985 to 1988, were pre-pared by homogenization, filtration, and centrifugation in doubledistilled water (Undeen and Vavra 1997) and stored at � 32 1Csince 1985–1988. DNA was extracted from these spores, ampli-fied, and directly sequenced as described previously (Vossbrinck,Andreadis, and Debrunner-Vossbrinck 1998; Vossbrinck et al.2004; Sokolova, Kryukova, and Glupov 2006a; Sokolova et al.2006b). Reactions were run in a Labsystems thermocycler: dena-turation at 94 1C for 3 min, followed by 35 cycles of 94 1C for 45 s,45 1C for 30 s, and 72 1C for 90 s, with a 5-min extension at 72 1C.PCR products were separated on an ethidium-bromide-stained 2%agarose gel, and the desired bands were extracted with the aid of aZimoclean Gel DNA Recovery Kit (Orange, CA). The isolatedDNA fragments were sequenced at the Molecular Medicine Lab-oratory at the Louisiana State University School of VeterinaryMedicine. The primers for PCR amplificaton were V1 (50-CACCAG GTT GAT TCT GCC TGA C-30) and 1492r (50-GGT TACCTT GTT ACG ACT T-30); the primers for sequencing wereV1, 530r (50-CCG CGG C(T/G)G CTG GCA C-30), 1061f (50-GGT GGT GCA TGG CCG-30), and 1492r (Vossbrinck et al.2004). These primers produced overlapping sequences that

Corresponding Author: Current address: Yuliya Y. Sokolova, Labo-ratory of Insect Pathology, Department of Entomology, Louisiana StateUniversity Agricultural Center, 404 Life Sciences Bldg., Baton Rouge,LA 70803-1710, USA—Telephone number: (225) 578 1849; FAX num-ber: (225) 578-1643; e-mail: jsokolova@lsu.edu

223

J. Eukaryot. Microbiol., 54(3), 2007 pp. 223–230r 2007 The Author(s)Journal compilation r 2007 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2007.00256.x

were assembled with ChromasPro 1.34 software (http://www.technelysium.com.au/ChromasPro.html). DNA isolation andPCR amplification were performed for two vouchers of sporescollected in 1985 and 1988; sequencing was performed at leasttwice in forward and reverse directions for each amplification toavoid sequence errors and to clarify ambiguities.

Phylogenetic analysis. Sequences of a SSU rDNA with max-imum similarity to our isolate were obtained from GenBank by aBLAST search. Sequences belonging to Liebermannia pat-agonica, Orthosomella operophtera, Vittaforma corneum, Cyst-osporogenes spp., and Endoreticulatus spp. produced the highestscores in the BLAST search. Other sequences were included in theanalyses in order to determine the relationships of the currentlystudied microsporidium with P. nelsoni and with the closely re-lated Ameson michaelis, as well as with the following micros-poridia from insects: Thelohania solenopsae, a microsporidiumfrom fire ants that share habitats with D. elongates (Briano, Pat-terson, and Cordo 1995); recently molecularly characterized Ann-caliia meligethi that represents a genus (formerly Brachiola) witha broad host range (Franzen et al. 2006b); and with the type spe-cies of the genera Nosema and Paranosema, Nosema bombycisand Paranosema grylli. We also included Nosema apis and Pa-ranosema whitei in the analyses to compare SSU rDNA-inferredpairwise distances separating congeneric species within the gen-era Cystosporogenes, Endoreticulatus, Nosema, Paranosema,Perezia, and Liebermannia. Hosts and accession no. of the se-quences of the 17 microsporidia used in the phylogenetic analysisare presented in Table 1.

Sequences were aligned with the CLUSTAL X program(Thompson et al. 1997) without additional changes and weretrimmed at the 30-end to a final length of 1,300 characters includ-ing gaps. A zygomycete fungus, Basidiobolus ranarum (Fungi:Zygomycetes) (Accession No. D29946), was selected as an out-group. The resultant alignment was analyzed by NJ, MP, and byan ML algorithms with PAUP�, version 4.0 (Swofford 2002). AGTR1G model of nucleotide substitution was suggested as a best-fit one by likelihood ratio tests and AIC criteria in Modeltest3.6 (Posada and Crandall 1998), and its settings were applied toML analyses. Bootstrap values for all tree-building methods wereobtained from 100 resamplings. Manipulations of trees were car-ried out with Tree-View, version 1.6.6. Sequence comparison inthe form of a data matrix was calculated by the Phylip DistanceMatrix Algorithm built in the CLUSTAL X program (Thompsonet al. 1997).

Electron microscopy. The embedding blocks from 1985,which were not examined by Lange (1987), were subjected toelectron microscopy. Ultrathin sections were cut on a Reichert-Jung ultramicrotome, stained with uranyl acetate and lead citrate,and viewed and photographed with JEOL JEM100 microscope.

RESULTS

Small subunit rRNA gene sequence and phylogenetic ana-lyses. An approximately 1,400 nucleotide DNA band was ampli-fied from genomic DNA preparations obtained from purifiedspores preserved in water solution at �32 1C since 1985–1988.The consensus SSU rRNA gene sequence is 1,242 base pairs with58.4% GC content (GenBank Assession No. EF016249). Thenovel sequence showed 96% identity with the SSU rDNA se-quence of L. patagonica; 82% with the Orthosomella operophterasequence; 73%–74% with those of the genera Cystosporogenes,Vittaforma, Enterocytozoon, and Paranosema; and low similarity(62%–65%) to other microsporidian sequences including P. nel-soni. The similarity of the new sequence with the SSU rDNA ofBasilobolus ranarum (outgroup) was calculated as 59% (Table 2).

Maximum likelihood, MP, and NJ analyses applied to the SSUrDNA sequence alignment produced similar general treetopologies with bootstrap support for all nodes more than 70%,except for the position of the genus Paranosema (Fig. 1). In theML tree, the Paranosema branch derived independently as anoutgroup to the Nosema–Enterocytozoon–Endoreticulatus–Orthosomella–Liebermannia superclade (Fig. 1). In the MP tree,the Paranosema branch fell within one clade with Liebermanniato form a sister group with Orthosomella (Fig. 2). When the NJalgorithm was applied, Paranosema appeared as a sister group tothe Orthosomella–Liebermannia branch (Fig. 3). The bootstrapsupport for the Paranosema node was as low as 53% and 64% forMP and NJ trees, respectively. It is interesting that removingsequences from or adding them into the alignment resulted insporadic placement of Paranosema as a sister group to Lie-bermannia in ML analyses without changing the topology of therest of the tree (data not shown). Thus, the relationships betweenthe genera Liebermannia and Paranosema, which both containparasites of orthopteran insects, remain unresolved.

In all trees, sequences of P. dichroplusae and L. patagonicawere sister taxa that clustered with Orthosomella operophterae.At the higher hierarchical level, the Liebermania–Orthosomellabranch formed a clade with the Endoreticulatus–Cystosporoge-nus–Vittaforma group and with Enterocytozoon bieneusi. Perezianelsoni fell into another large clade together with Nosema andAmeson species (Fig. 1).

General morphology and ultrastructure. All stages of P.dichroplusae develop in direct contact with host-cell cytoplasm.

Table 1. Hosts and GenBank accession numbers for the SSU rRNA se-quences of 17 microsporidia species used in the phylogenetic analyses.

Microsporidium Host AccessionNo.

Ameson michaelis Callinectes sapidus (Crustacea,Decapoda)

L15741

Anncaliia meligethi Meligethes aeneus (Insecta,Coleoptera)

AY894423

Cystosporogeneslegeri

Lobesia botrana (Insecta,Lepidoptera)

AY233131

Cystosporogenesoperophterae

Operophtera brumata (Insecta,Lepidoptera)

AJ303320

Endoreticulatusschubergi

Choristoneura fumiferana (Insecta,Lepidoptera)

L39109

Endoreticulatusbombycis

Bombyx mori (Insecta, Lepidoptera) AY009115

Endoreticulatus sp. Lymantria dispar (Insecta,Lepidoptera)

AY502945

Liebermanniapatagonica

Tristira magellanica (Insecta,Orthoptera)

DQ 239917

Nosema apis Apis mellifera (Insecta, Hymenoptera) U97150Nosema bombycis Bombyx mori (Insecta, Lepidoptera) AB125662Orthosomella

operophteraeOperophtera brumata (Insecta,Lepidoptera)

AJ 302317

Paranosema grylli Gryllus bimaculatus (Insecta,Orthoptera)

AY305325

Paranosema whitei Tribolium sp. (Insecta, Coleoptera) AY305323Perezia

dichroplusaeDichroplus elongatus (Insecta,Orthoptera)

EF016249

Perezia nelsoni� Litopenaeus setiferus (Crustacea,Decapoda)

AJ252959

Thelohaniasolenopsae

Solenopsis invictae (Insecta,Hymenoptera)

AF134205

Vittaforma corneae Homo sapiens (Mammalia,Primates)

U11046

�Cited in GenBank as Pleistophora sp. (LS); identified as Perezia nel-soni by Canning et al. (2002).

224 J. EUKARYOT. MICROBIOL., VOL. 54, NO. 3, MAY–JUNE 2007

Diplokaryotic meronts undergo several rounds of mitotic divis-ions before the parasite switches to sporogony (Fig. 4). The trans-itional stage from merogony to sporogony has a large diplokaryonwith a thick dense zone of contact between the two adjacent nu-clei; in this region individual membranes of the nuclear envelopeare becoming indistinguishable (Fig. 5). Presence of spindleplaques in each counterpart of the diplokaryon suggests that thecell has entered the chromosome-replication stage (Fig. 5). Cyto-plasm in the meront/sporont transitional stage looks lucid pre-sumably due to the formation of numerous vacuoles, themembranes of which apparently were not fixed adequately. Dis-sociation of nuclei of the diplokaryon apparently occurs next andcorresponds with deposition of patches of dense material outsidethe parasite (Fig. 6, 7). Dense thread-like structures resemblingsynaptonemal complexes are occasionally noticeable inside dis-sociating nuclei, indicating the occurrence of meiosis (Fig. 6).

During sporogony, each nucleus divides at least once, and thecytoplasm of the parasite undergoes reorganization and no longerappears lucid (Fig. 8, 9). The cell eventually elongates and trans-forms into a multinuclear sporogonial plasmodium enveloped by acontinuous dense layer (Fig. 9). Sporoblasts with initial signs ofmorphogenetic activity at the Golgi zone (Fig. 10) and those at amore advanced stage of morphogenesis (Fig. 11) reside separatelyinside the cytoplasm of the host cell, indicating that the plas-modium divides into uninucleate sporoblasts before sporogenesisbegins. Sporoblasts mature into spores after considerable shrink-age (Fig. 12, 13). The number of polar tube coils observed in ma-ture spores (i.e. spores with well-developed exo- and endospores)varies from three to eight. The presence of a tubular network at theposterior end of the spore and the variable number of polar fila-ment coils indicate that growth of the polar filament might takeplace after formation of the spore wall (Fig. 12, 13). Numerousmitochondria and cisternae of endoplasmic reticulum regularlyoccurred in the host-cell cytoplasm adjacent to all stages of P.dichroplusae, but we never observed continuous layers of hostendoplasmoic reticulum around parasites.

DISCUSSION

Relationships of Perezia dichroplusae with other micro-sporidia. Phylogenetic analysis suggests that P. dichroplusae isunrelated to P. nelsoni and by no means supports placement of

these two species into the same genus. The genus Perezia waserected for P. lankesteriae (type species), a hyperparasite of thegregarine Lankesteria sp. which parasitizes the ascidian C.intestinales. Another Perezia was initially described from striat-ed muscles of the penaeid shrimp Farfantepenaeus aztecus asNosema nelsoni (Sprague 1950). Later it was transferred first tothe genus Ameson (Loubes et al. 1977) and finally to Perezia(Vivares and Sprague 1979) because of structural and develop-mental similarity to P. lankesteriae (Canning et al. 2002; Or-mieres et al. 1977; Vivares and Sprague 1979). Gregarines of thegenus Lankesteria are broadly distributed parasites of a widerange of marine invertebrates, including both ascidians and deca-pods (Leander et al. 2006), so it is probable that P. nelsoni is re-lated to the type species, P. lankesteria, a conclusion awaitingsupport by molecular characterization of the type species. TheSSU rDNA-based phylogenetic analyses placed P. nelsoni as asister taxon to A. michaelis, a parasite of the crab Callinectes sp.(Crustacea, Decapoda) (Refardt et al. 2002), which was consistentwith assignment of both species to the family Pereziidae based onsimilar morphology and developmental patterns (Sprague 1977;Sprague, Becnel, and Hazard 1992).

It is noteworthy that Thelohania butleri from Pandalus jordani(Crustacea, Decapoda), the only ‘‘true’’ (Hazard and Oldacre1975) Thelohania from marine decapods, when characterized bySSU rDNA sequence, fell into one clade with A. michaelis and P.nelsoni (Brown and Adamson 2006) within a fish-parasitic super-taxon (Vossbrinck and Debrunner-Vossbrinck 2005). The unre-latedness of P. dichroplusae from the Argentine grasshopper to P.nelsoni and A. michaelis from marine decapods demonstrated inour study was not unexpected, just as T. solenopsae from fire antswas distant from T. butleri from the crustacean P. jordani (Brownand Adamson 2006). Both studies support the viewpoint that hostgroup and host habitat are basic indicators of relatedness for mostmicrosporidia genera and higher taxa (Baker et al. 1995, 1997;Vossbrinck and Debrunner-Vossbrinck 2005).

Adaptive divergence of obligate parasites generally follows di-vergent evolution of their hosts and reflects their mutual evolu-tionary history (Roberts and Janovy 2005); this rule also appearsto apply to microsporidia (Vossbrinck and Debrunner-Vossbrinck2005; Vossbrinck et al. 2004). Another factor that influences thepattern of association among taxa of hosts and their parasites iscolonization (‘‘host switching or host capture’’), which mirrors

Table 2. Comparison of SSU-rDNA sequences: Percentage of identity (top diagonal) and pairwise distance (bottom diagonal) (Phylip distance matrix).

Pd� Lp Oo Pg Pw Esp Eb Cl Co Vc Eb Anm Ts Br Na Nb Amm Pn

Pd 0 95.8 82.0 73.1 73.7 75.9 76.5 75.4 74.2 75.8 73.8 63.4 62.0 59.0 65.8 63.8 60.9 64.9Lp 0.042 0 80.9 72.0 72.6 75.3 75.4 74.3 73.3 74.4 72.9 61.8 60.1 58.6 65.0 62.6 60.0 64.0Oo 0.180 0.191 0 74.2 74.6 79.0 78.9 77.1 77.0 77.3 77.5 64.5 62.7 60.2 66.9 65.2 59.2 64.1Pg 0.269 0.280 0.258 0 96.9 69.5 69.5 66.8 66.8 66.7 67.0 64.0 62.7 59.4 62.4 58.4 57.3 63.1Pw 0.263 0.274 0.254 0.031 0 70.2 70.2 67.4 67.3 67.0 67.2 64.3 62.6 59.6 62.0 58.1 57.7 63.0Esp 0.241 0.247 0.210 0.305 0.298 0 99.3 88.9 88.3 88.8 73.1 65.2 64.5 63.0 67.9 65.9 59.9 63.5Eb 0.235 0.246 0.211 0.305 0.298 0.007 0 89.5 88.4 89.1 74.2 65.8 65.2 62.2 68.5 66.5 61.2 64.4Cl 0.246 0.257 0.229 0.332 0.326 0.111 0.105 0 97.9 91.4 74.6 65.6 65.7 62.2 70.2 68.8 60.7 63.7Co 0.258 0.267 0.230 0.332 0.327 0.117 0.116 0.021 0 90.9 74.0 65.2 64.8 61.8 69.9 68.9 60.9 63.5Vc 0.242 0.256 0.227 0.333 0.330 0.112 0.109 0.086 0.091 0 75.0 65.3 65.6 61.5 69.3 67.5 61.3 63.0Eb 0.262 0.271 0.225 0.330 0.328 0.269 0.258 0.254 0.260 0.250 0 62.4 62.9 60.8 66.5 65.2 60.8 63.8Anm 0.366 0.382 0.355 0.360 0.357 0.348 0.342 0.344 0.348 0.347 0.376 0 86.9 59.7 63.8 63.2 57.5 61.4Ts 0.380 0.399 0.373 0.373 0.374 0.355 0.348 0.343 0.352 0.344 0.371 0.131 0 59.9 64.0 63.5 58.3 60.1Br�� 0.410 0.414 0.398 0.406 0.404 0.370 0.378 0.378 0.382 0.385 0.392 0.403 0.401 0 59.8 57.5 55.7 57.9Na 0.342 0.350 0.331 0.376 0.380 0.321 0.315 0.298 0.301 0.307 0.335 0.362 0.360 0.402 0 84.7 66.3 66.8Nb 0.362 0.374 0.348 0.416 0.419 0.341 0.335 0.312 0.311 0.325 0.348 0.368 0.365 0.425 0.153 0 65.9 66.5Amm 0.391 0.400 0.408 0.427 0.423 0.401 0.388 0.393 0.391 0.387 0.392 0.425 0.417 0.443 0.337 0.341 0 73.0Pn 0.351 0.360 0.359 0.369 0.370 0.365 0.356 0.363 0.365 0.370 0.362 0.386 0.399 0.421 0.332 0.335 0.273 0

�For full species names see Table 1.��Basdiobolus ranarum.

225SOKOLOVA ET AL.—LIEBERMANNIA DICHROPLUSAE N. COMB. (MICROSPORIDIA)

ecological relations between partners of host-parasite systems(Roberts and Janovy 2005). In fact, many cases of unexpectedclustering of microsporidia that parasitize distantly related hosts,such as mammals (including humans) and insects, mammals andfish, insects and bryozoans, insects and crustaceans, or crusta-ceans and fish, can be explained by potential ecological bondsbetween distantly related ancestral hosts, such as common foodchains, symbionts, ecological niches, or yet unrevealed interrela-tions, that would have facilitated expansion of the parasite hostrange (Baker et al. 1995; Cheney, Lafranchi-Tristem, andCanning 2000; Cheney et al. 2001; Freeman, Bell, and Sommer-ville 2003; Sokolova et al. 2006a; Vossbrinck and Debrunner-Vossbrinck 2005; Weiss and Vossbrinck 1999). Given the highlevel of divergence of microsporidian species and genera in in-sects and in crustaceans, it is unlikely that they would be para-sitized by microsporidia of the same genus. On the other hand,marine decapods and terrestrial insects could hardly share an en-vironment in the recent past, ever since a hypothetical pancrusta-

cean ancestor from the Silurian gave rise to hexapods andcrustaceans about 300 million years ago (Grimaldi 2003; Grim-aldi and Engel 2005).

The percentage of sequence similarity has proved to be a reli-able criterion for assessing taxonomic relationships among mi-crosporidian species (Baker et al. 1994; Franzen et al. 2006b;Sokolova et al. 2003). Indeed, P. dichroplusae should be trans-ferred to the genus Liebermannia, based on its having only 4.2%of SSU rDNA sequence divergence from L. patagonica, its closestrelative on the phylogenetic tree (Fig. 1). Interspecific distanceswithin most genera of insect microsporidia subjected to SSUrDNA-based sequence analyses range from 0.7% to 5.0%: 0.7%between Endoreticulatus sp. and E. bombycis; 2.1% betweenCystosporogenes legeri and Cystosporogenes operophterae (Ta-ble 2); 3.1%–5.0% among Paranosema spp. (Sokolova et al.2003); 1%–2% among Tubulinosema spp.; and 1.4%–3.3%among Anncaliia spp. (Franzen et al. 2005, 2006a, b). On thecontrary, pairwise distances of approximately 10% or more sep-arate sequences of representatives of closely related genera. Forexample, Paranosema spp. differ by 9.6%–10.7% from Anton-ospora scoticae (Sokolova et al. 2003, 2005), while Cystosporo-genes spp. are differentiated from Endoreticulatus spp. by 10.5%–11.1% SSU rDNA sequence divergence, and Vittaforma corneaefrom Cystosporiogenes spp. by 8.6%–9.1% (Table 2). As much as15.3% divergence between N. apis and N. bombycis indicated thatthese species are not congeneric, which is in agreement with pre-vious placement of N. apis in one clade with Vairimorpha necatrix(Baker et al. 1994; Johny et al. 2006; Vossbrinck and Debrunner-Vossbrinck 2005). Interspecific distances among Nosema speciesfrom lepidopteran hosts (‘‘true nosemas’’) ranged from 0% to 5%(Baker et al. 1994). We suggest that SSU rDNA sequence diver-gence between any new species of the genus and the type speciesL. patagonica Sokolova, Lange & Fuxa (2006b) (GeneBank Ac-cession No. DQ239917), should be less than 10%, depending onmorphological and ecological considerations.

General morphology and ultrastructure of Perezia dichro-plusae in comparison with Perezia nelsoni and Liebermanniapatagonica. Previous extensive study of P. dichroplusae cytolo-gy by light and electron microscopy revealed characteristics con-sistent with placement of this microsporidium into the genusPerezia, including diplokaryotic meronts, sporonts, and sporogo-nial plasmodia with isolated nuclei, polysporous sporogony, pro-duction of uninucleated sporoblasts, and spores from moniliformmultinucleate sporonts (Lange 1987). In our study, the SSU rDNAsequence, which directly reflects the unique evolutionary historyof species, revealed a close relationship between P. dichroplusaeand L. patagonica. Based on this new perspective, we reevaluatedthe literature on structural and developmental characters of P.nelsoni, P. dichroplusae, and L. patagonica (Table 3). These datasuggest greater similarity between P. dichroplusae and L. pat-agonica than between P. dichroplusae and P. nelsoni, except forthe diplokaryotic versus monokaryotic arrangement of nuclei insporoblasts and spores. In contrast to microsporidia from grass-hoppers, P. nelsoni infects muscle cells of decapod hosts, and itselongated sporogonial plasmodia divide directly into uninucleateyoung spores, often separated by cytoplasmic bridges until com-plete maturation (Canning et al. 2002), a process similar to that inthe type species P. lankesteriae (Ormieres et al. 1977). As a resultof such sporogony, perfectly ovoid spores are produced in P. nel-soni and P. lankesteriae (Canning et al. 2002; Sprague and Ver-nick 1969) (Fig. 14). Comparison of spores of P. dichroplusae andP. nelsoni supports the idea that spore shape is an important mor-phological character reflecting phylogeny of the species and is ofprime significance for microsporidian taxonomy (Larsson 1999;Sokolova et al. 2005). In contrast, the diplokaryotic versus mono-karyotic arrangement of the nucleus at a certain stage of the

Fig. 1–3. Phylogenetic relationships of Perezia dichroplusae withother Microsporidia inferred from small subunit rDNA (SSU rDNA) genesequences. 1. In maximum-likelihood (ML) tree, sequences of P. dichro-plusae and Liebermania patagonica are sister taxa that clustered withOrthosomella operophterae. At the higher hierarchical level, theLiebermania–Orthosomella branch forms a clade with the End-oreticulatus–Cystosporogenes–Vittaforma group and with Enterocytozoonbieneusi. Perezia nelsoni fell into another large clade together withNosema and Ameson species. Phylogenetic trees based on neighbor-joining (NJ) and maximum-parsimony (MP) algorithms displayed identi-cal topologies, except for the position of the genus Paranosema. The first,second, and third numbers at nodes are bootstrap values for the node sup-port in ML, NJ, and MP analyses, respectively, all in 100 replicates. 2, 3.Position of the branch P. grilli–P. whitei on the phylogenetic trees derivedfrom NJ (2) and MP (3) analyses.

226 J. EUKARYOT. MICROBIOL., VOL. 54, NO. 3, MAY–JUNE 2007

Fig. 4–13. Electron microscopy of Perezia dichroplusae. 4. Meront with two dividing diplokarya (DK). Spindle plaques are indicated by arrows. 5.Meront/sporont transitional stage with a large diplokaryon and lucid cytoplasm. Notice thick dense zone of contact between two adjacent nuclei; in thiszone individual membranes of the nuclear envelope are undistinguishable (insert). Presence of spindle plaques in each counterpart of the diplokaryon(arrows) suggests that the cell has entered the stage of chromosome replication. 6. Two nuclei formerly comprising a diplokaryon are in the process ofdissociation. This process corresponds in time with deposition of patches of dense material outside the parasite (arrowheads). Dense thread-like structuresresembling synaptonemal complexes (arrows) are noticeable inside dissociating nuclei. 7. Meront/sporont transitional stage with two recently dissociatednuclei (N). Golgi complex (G) and a few vacuoles (V) are the only organelles distinguishable in this poorly preserved cytoplasm. The cell surface iscovered with dense layer (arrowheads). 8. Sporont with two nuclei (N). Presence of chromosomes (arrows) suggests mitosis. 9. Multinuclear sporogonialplasmodium in which constrictions of cytoplasm (arrows) indicate the sites of division into sporoblasts. 10. Appearance of dense deposits in the Golgizone (arrows) marks initiation of morphogenetic activity in the sporoblast. 11. Sporoblast undergoing the advanced stage of morphogenesis. Precursors ofpolar filament (PF), polar disk (PD), and polaroplast (Pp) are easily distinguishable. 12. Mature spore displays only three polar filament coils and a tubularnetwork at the posterior end of the spore. 13. Mature spores with variable numbers of polar filament coils. Bar 5 1mm.

227SOKOLOVA ET AL.—LIEBERMANNIA DICHROPLUSAE N. COMB. (MICROSPORIDIA)

microsporidian life cycle cannot be considered a robust taxonomicor evolutionary criterion of relationships between species, be-cause of the remarkable ‘‘plasticity of nuclear transition’’ char-acteristic of Microsporidia (Flegel and Pasharawipas 1995).

Perezia dichroplusae also resembles Orthosomella spp. by pa-rasitizing epithelial cells of the insect alimentary tract and by itsproduction of elongated multinuclear plasmodia and ovocylindri-cal uninuclear spores (Andreadis, Maier, and Lemmon 1996; Can-ning et al. 1985). This correlates with the clustering of

P. dichroplusae–L. patagonica and O. operophterae together onthe SSU rDNA phylogenetic tree. It is noteworthy that the lifecycle of O. operophterae does not include diplokaryotic stages atall, unlike P. dichroplusae and L. patagonica.

Ultrastructural reexamination of P. dichroplusae confirmed theresults of the previous study (Lange 1987) and provided additionalevidence of structural resemblance of this species to L. patagonica(Sokolova et al. 2006b). In addition to having the same type ofsporogony and spore shape, the two species displayed similar finestructure of diplokaryotic meronts, meront/sporont transitionalstages, and spores. In spores of P. dichroplusae and L. patagonica,unlike many other microsporidia, the posterior vacuole is not de-veloped; instead, a ‘‘tubular network’’—the microsporidian lateGolgi compartment—is located at the posterior end of the spore.Perezia dichroplusae spores average more polar filament coils (upto eight) than L. patagonica, but some have only three, as does L.patagonica. The major differences between the two species is theoccurrence of nuclear dissociation in the meront/sporont transi-tional stage of P. dichroplusae and the presence of a singe nucleusin all subsequent stages of sporogony, although the diplokaryoticarrangement of the nucleus of L. patagonica is preserved duringsporogony as well. The elimination of the sexual process andhaplophase in L. patagonica along with other traits of life-cycle

Table 3. Comparative characteristics of Perezia dichroplusae, Liebermannia patagonica, and Perezia nelsoni�.

Characters P. dichroplusae L. patagonica P. nelsoni

Habitat, area of distribution Grasslands in Buenos Aires Province,Argentina

Steppes in southern Patagonia,Argentina

Coastal waters worldwide

Host Dichroplus elongatesAcrididae,OrthopteraInsecta

Tristira magellanicaTristiridae,Orthoptera,Insecta

Penaeus spp., other prawns of thefamily Penaeidae, Decapoda,Malacostraca

Tissue tropism Malpighian tubules epithelium Midgut and gastric caecumepithelium

Striated muscles

Merogony By binary fission of round or slightlyelongate cells with two to four nucleiarranged in DKs

Transition to sporogony Vacuolized round or elongate cellswith one to two DKs undergoingnuclei dissociation, or with two to fourMKs, patches of electron-densematerial on the cell surface

Vacuolized round or elongate cellswith one to two DKs with patchesof electron-dense material on thecell surface, loosely surrounded byhost RER

Vacuolized round or elongate cellswith two or more DKs undergoingnuclei dissociation, or with four ormore MKs, with patches of electrondense material on the cell surface

Sporonts (sporogonialplasmodia)

Elongate cells surrounded by electron-dense layer, with nuclei in the processof mitosisContains two to four MKs Contains two to four DKs.Cells are

wrapped in RERContains two or more DKs

Cytoplasm constrictions mark thelimits between individual cells, whichtransform into sporoblasts afterbudding off the sporogonialplasmodium

Sporoblasts Monokaryotic Diplokaryotic MonokaryoticSporoblasts are produced by multiplefission of moniliform sporogonialplasmodiaSegregation of individual sporoblastscompletes before sporogenesis

Young spores are often connectedby cytoplasmic bridges

Spore: shape Ovocylindrical Ovocylindrical OvoidLength/width 2.33 2.41 1.75Size, mm 1.6–6.7 � 1.0–2.7 1.9–3.9 � 0.8–1.6 1.4–2.8 � 0.9–1.9Nucleus MK DK MKPF Six to eight coils, isofilar Three coils, isofilar Eight to 11 coils, isofilarPolaroplast Lamellar, uniform Lamellar, uniform Central lamellar part and outer region

of compact membranes

DK, diplokaryon; MK, monokaryon; RER, host rough endoplasmic reticulum; PF, polar filament.�Data on P. dichroplusae from Lange (1987), on L. patagonica from Sokolova et al. (2006b), on P. nelsoni from Canning et al. (2002), Sprague (1950),

Sprague and Vernick (1969).

Fig. 14. Scheme for segregation of sporoblasts from sporogonialplasmodia and the resultant spore shape in (a) Perezia lankesteria andPerezia nelsoni and in (b) Liebermannia patagonica, Perezia dichro-plusae, and Orthosomella operophterae.

228 J. EUKARYOT. MICROBIOL., VOL. 54, NO. 3, MAY–JUNE 2007

simplification and shortening might reflect specific adaptations ofthis microsporidium to survival and multiplication in short-livedmidgut cells (Sokolova et al. 2006b). The question about meiosisin P. dichroplusae remains open. Further research may indicatewhether nuclear dissociation and subsequent divisions of the dip-lokaryon counterparts in P. dichroplusae are analogous to first andsecond meiotic divisions. Another distinguishing feature of P.dichroplusae is the absence of a noticeable association betweenthe parasite cells and the host endoplasmic reticulum, whereas thesporonts of L. patagonica, the most abundant stage of the life cy-cle, are wrapped in an envelope composed of flattened cisternae ofhost-cell ER, which might be interpreted as the parasite’s defenseagainst the relatively active phago-lysosomal system of the host’smidgut cells (Sokolova et al. 2006b).

Thus, the morphology and development of P. dichroplusae inD. elongatus justify transferring this species to the genus Lie-bermannia. On the basis of both molecular and structural evidencewe establish here a new combination Liebermannia dichroplusaen. comb. for P. dichroplusae Lange 1987.

TAXONOMIC SUMMARY

Phylum: Microsporidia Balbiani 1882Family: incertae sedisGenus: Liebermannia

Refined diagnosis for the genus Liebermannia Sokolova,Lange & Fuxa 2006.

Nuclei in diplokaryotic arrangement at least at the merontstage. Development in direct contact with the host-cell cytoplasm.Sporoblasts produced sequentially from an elongated sporogonialplasmodium. Ovocylindrical diplokaryotic or uninucleate spores.Mushroom-like anchoring disk, polar sac, and lamellar bipartitepolaroplast. Infect primarily epithelial cells of insect hosts.

Synopsis of the genus Liebermannia1. Liebermannia patagonica Sokolova, Lange & Fuxa (Soko-

lova et al. 2006b), type species2. Liebermannia dichroplusae n. comb. (syn. Perezia dichro-

plusae Lange 1987)

Refined diagnosis for Liebermannia dichroplusae n. comb.Type host. Dichroplus elongatus Tos (Orthoptera, Acrididae,

Melanoplinae).Type locality. Brandsen, Buenos Aires Province, Argentina.Site of infection. Epithelial cells of Malpighian tubules.Interfacial envelopes. All stages in direct contact with host-

cell cytoplasm.Merogony. Rounded and elongated diplokaryotic cells.Sporogony. Polysporous. Moniliform plasmodia with isolated

nuclei undergo multiple fission to form uninucleate sporoblasts.Spore. Free and uninucleate. Ovocylindrical in shape and vari-

able in size (1.6–6.7 mm � 1.0–2.7 mm). Polar tube with eight orfewer coils.

We include SSU rDNA gene partial sequence under NCBIGenBank Assession no. EF016249 in the species diagnosis.

Type material. Giemsa-stained smears and EM blocks havebeen deposited in the Center for Parasitological Studies and Vec-tors (CEPAVE), La Plata National University, Argentina, and inthe International Protozoan Type Slide Collection, SmithsonianInstitution, Washington, DC.

Differential diagnosis of Liebermannia dichroplusae.Liebermannia dichroplusae differs from the type species L.

patagonica as follows: (1) they parasitize grasshoppers belongingto different families of the order Orthoptera, L. dichroplusae in D.elongatus (Acrididae) and L. patagonica in Tristira magellanica

(Tristiridae); (2) L. dichroplusae infects epithelial cells ofMalpighian tubules, whereas L. patagonica infects enterocytesof the midgut epithelium and gastric caecum; (3) dissociation ofthe diplokaryon of the meront/sporont transitional stage takesplace at the onset of sporogony in L. dichroplusae, and all sub-sequent sporogonial stages, including spores, are monokaryoticwhile dissociation of nuclei was not observed in L. patagonica,and all sporogonial stages, including spores, are diplokaryotic; (4)sporonts of L. dichroplusae are not specifically associated withhost-cell ER, whereas sporonts of L. patagonica are enclosed in anenvelope composed of flattened host-cell ER cisternae; (5) mostspores of L. dichroplusae have longer polar filaments arranged inthree to eight coils, whereas the number of polar filament coils inL. patagonica never exceeded 3; (6) SSU rDNA sequence diver-gence between L. dichroplusae and L. patagonica is 4.2%.

ACKNOWLEDGMENTS

The work was supported partly by ‘‘Consejo Nacional de In-vestigaciones Cientificas y Tecnicas’’ (CONICET) (Grant PIP #2062) and ‘‘Comision de Investigaciones Cientıficas (CIC)’’ ofBuenos Aires province, and partly by the Russian Foundation forBasic Research (Grant RFBR # 04-04-49314). This paper wasapproved for publication by the director of the Louisiana Agri-cultural Experiment Station as Manuscript No. 06-26-0664.

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Received: 10/30/06, 01/22/07; accepted: 01/29/07

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