Accessibility / Report Error

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

Abstract

Introduction

Microsporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization.

Methods

Larvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles.

Results

The molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon.

Conclusions

Samples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Microsporidae; Amblyospora spp; Polydispyrenia spp; Phylogenetic analysis


INTRODUCTION

Black flies (Diptera: Simuliidae) cause severe medical and veterinary problems worldwide. Simuliidae species are able to transmit parasites that can result in severe disease in humans and animals. In addition, their bites can cause allergic reactions and dermatitis in sensitized individuals, resulting in severe economic losses to tourism centers and negatively impacting animal production11. Shelley A, Hernandez L, Maia-Herzog M, Luna Dias A, Garritano D. The blackflies (Diptera: Simuliidae) of Brazil. In: Arias JR, Golovatch S, Wantzen KM, Dominguez E, editors. Aquatic biodiversity in Latin America (ABLA). Vol. 6. Pensoft:Sofia-Moscow; 2010. p. 821.33. Ribeiro do Amaral-Calvão AM, Maia-Herzog M. Adolpho Lutz's collection of black flies (Diptera: Simuliidae), its history and importance. Hist Cienc Saude Manguinhos 2003; 10:259-271.. Black fly control remains a major public health challenge. Microsporidia are unicellular, eukaryotic organisms that are obligate, intracellular parasites with public health relevance44. Vossbrinck CR, Maddox JB, Friedman S, Debrunner-Vossbrink BA, Woese CR. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987; 326: 411-414.. Several studies have suggested a new classification for microsporidia as fungi, but Ebersberger55. Ebersberger I, Simões RM, Kupczok A, Gube M, Kothe E, Voigt K, et al. A consistent phylogenetic backbone for the fungi. Mol Biol Evol 2012; 29:1319-1334. stated that phylogenetic analysis did not support fungal characterization for this group.

Microsporidia are the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood66. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191.,77. Ginarte CA, Andrade CFS, Gaona JC. Larvas de simulídeos (Diptera, Simuliidae) do Centro Oeste, Sudeste e Sul do Brasil, parasitadas por microsporídeos (Protozoa) e mermitídeos (Nematoda). Ser Zool 2003; 93:325-334.. Infections caused by this agent are often chronic and non-lethal, but they can cause sub-lethal host effects, such as reduced fecundity, decreased life span and general loss of vigor88. Castello-Branco A. Effects of Polydispyrenia simulii (Microspora; Duboscqiidae) on Development of the Gonads of Simulium pertinax (Diptera; Simuliidae). Mem Inst Oswaldo Cruz 1997; 94:421-424..

The objective of this study was to identify microsporidian species infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba City, on the north coast of State of São Paulo, by molecular and morphological characterization.

The city's economy greatly depends on tourism. Thus, the Simuliidae population plays an important role because black fly bites annoy visitors and have deleterious effects on the local economy. Monitoring and controlling black flies are essential to avoiding seasonal population outbreaks.

METHODS

Sampling and biological material processing

The sampling period was from May to August 2013, and the samples were collected from a stream in Caraguatatuba City, located on the north coast of the State of São Paulo, Brazil, which has a total area of 458,097km2 and had a population at that time of 100,84099. Online Archive of Instituto Brasileiro de Geografia e Estatística [Internet][cited 2013 May]. Available at: http://cod.ibge.gov.br/2335A.
http://cod.ibge.gov.br/2335A...
. All of the larvae were held in aerated containers with water from the breeding site until examination. Tissues showing evidence of infection (whitish abdomens or whitish digestive tracts) were dissected in NaCl 0.9% solution, and fat bodies and adjacent tissues were removed1010. Undeen A H, Vavra J. Research Methods for Entomopathogenic Protozoa In: Lacey LA. Manual of Techniques in Insect Pathology. Chapter 4. 1st ed. Great Britain: Academic Press; 1997. p. 115-151.. Processed samples were frozen in 1.5ml tubes with 30µl of diethylpyrocarbonate (DEPC) (Invitrogen® Life Technologies, Carlsbad, CA, USA). Fresh smears of fat bodies were made, fixed with methanol for 5min and stained with 10% Giemsa in 7.4 pH buffer for 20min. The slides were washed in water and dried at 25°C overnight1111. Becnel J J. Preparations of Entomopathogens. In: Lacey LA. Manual of Techniques in Insect Pathology. Chapter VIII-1. 1st ed. Great Britain: Academic Press; 1997; p. 337-353. for further morphological analysis of spores.

Morphological analysis

The Nis Elements F 3.0 NIKON H550S software, with phase III objective scale 100X settings, was used for spore measurement. Morphological characterization was performed according to Sprague1212. Sprague V, Becnel JJ, Hazard EI. Taxonomy of Phylum Microspora. Crit Rev Microbiol 1992; 18:285-395..

Molecular assay

Molecular assays were performed with frozen tissues from infected larvae, and Aedes aegypti larvae infected with Edhazardia aedis were used as positive controls.

DNA extraction

Larvae exhibiting symptoms of infection had deoxyribonucleic acid (DNA) extracted using a viral DNA kit (QIAamp® viral RNA, Qiagen, Inc, Hilden, Germany). Healthy larvae (Figure 1A) were discarded. Tissue samples were processed with a proteinase K kit, incubated at 56°C for 2h and mixed every 20min. The supernatants were used to amplify the r16S ribosomal gene1313. Adler PH, Becnel JJ, Moser B. Molecular charaterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black flies (Diptera: Simuliidae), with host-derived relationships of the North American caudosporids. J Invertebr Pathol 2000; 75:133-143..

FIGURE 1-
Simulium pertinax larvae. Healthy larvae with normal coloration of the integument (A). Larvae with symptoms of microsporidian infection in the fat bodies (B).

Small subunit ribosomal gene (SSUrDNA) PCR (r16S)

Polymerase chain reaction (PCR) amplification was performed with 18f (CAC CAG GTT GAT TCT GCC) and 1492r (GGT TAC CTT GTT ACG ACT T), according to Vossbrinck et al.1414. Vossbrinck CR, Andreadis TG, Vavra J, Debrunner-Vossbrink BA. Verification of intermediate hosts in the life cycles of Microsporidia by small subunit rDNA sequencing. J Eukaryot Microbiol 1998; 45:290-292..

The amplification products were visualized on 2% agarose gels, with positive and negative controls and a 100 bps ladder (Invitrogen® Life Technologies, Carlsbad, CA, USA), following electrophoresis.

Nucleotide sequencing

PCR products were purified with the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Limited, Little Chalfont, Buckinghamshire, UK) and were quantified with 2% agarose gel ethidium bromide staining, according to the Low DNA Mass Ladder (Invitrogen®) protocol. The products were sequenced using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), following the standard manufacturer protocols. The data were analyzed with the phred/phrap software, and the contigs were assembled with the cap3 software1515. Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res 1999; 9:868-877..

Phylogenetic analysis

The analyses were performed using the Seaview software1616. Gouy M, Guindon S, Gascuel O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 2010; 27:221-224.. A phylogenetic tree was constructed, with reference sequences3232. Vossbrinck CR, Andreadis TG, Vavra J, Becnel JJ. Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae). J Eukaryot Microbiol 2004; 51:88-95.4646. Andreadis TG, Vossbrinck CR. Life cycle, ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae). J Eukaryot Microbiol 2002; 49:350-364. from Table 1(supplementary file), using the maximum likelihood method with the general time reversible (GTR) model of nucleotide substitution and gamma distribution (G) (GTR + G)1717. Waddell PJ, Steel MA. General time-reversible distances with unequal rates across sites: mixing gamma and inverse Gaussian distributions with invariant sites. Mol Phylogenet Evol 1997; 8:398-414.. The model was selected by the Modeltest software, version 3.0.61818. Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics 1998; 14:817-818., and was optimized by the Seaview software. We calculated the bootstrap values with 1,000 replications to support the verification of branches in the topologies of the trees obtained, and bootstrap values greater than 70 were considered significant.

TABLE 1-
Sequences and accession numbers used for phylogenetic analysis.

Nucleotide sequences and accession numbers

The nucleotide sequences obtained in this work were submitted to the GenBank nucleotide sequences databank under the following accession numbers GenBank: KC855552-KC855557 (L1_L6); and GenBank: KC855558 (L2).

RESULTS

A total of 1,574 S. pertinax larvae were examined. Eight larvae exhibited symptoms of microsporidian infection localized to the fat body (Figure 1B).

Morphological characterization indicated Polydispyreniaspp. infections in 7 larvae (Figure 2A), representing 87.5% of the infected larvae. Amblyospora sp. infection was observed in one larva (12.5% of the infected larvae) (Figure 2B). The prevalence of microsporidia parasitizing larvae of S. pertinax was 0.51%.

FIGURE 2-
Phase-contrast microscopy of smear slides of Simulium pertinax infected by microsporidia. Sporophorous vesicle of Polydispyrenia sp. containing 32 mononuclear spores (A). Octospores of Amblyospora spp. containing 8 uninucleate spores each (B).

Polydispyrenia spp. infections were characterized by the presence of at least 32 mononuclear spores contained within a persistent sporophorous vesicle, with the spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm (n = 23). Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores contained within a sporophorous vesicle, with the spores measuring 4.5 × 3.5µm (n = 12).

The PCR products targeting the 16S region and electrophoresis agarose gel analysis confirmed the presence of microsporidian DNA in 8 samples.

Six samples (Brazilian larvae) were found to be related to, but in a separate cluster (Figure 3) than, the Polydispyrenia simulii [GenBank: AY090069] and Caudospora palustris [GenBank: AF132544] reference sequences (with 100% bootstrapping). One sample (L2) was clustered with Amblyospora spp. [GenBank: AJ252949] with 100% bootstrapping. The Edhazardia aedis positive control (CONT+) taxon was clustered with Edhazardia aedis [GenBank: AF027684] with 100% bootstrapping.

FIGURE 3-
Phylogenetic tree generated for microsporidia. Unrooted tree constructed with the maximum likelihood method using the general time reversible model of nucleotide substitution and gamma distribution (GTR + G), using Seaview software. The robustness of the phylogenetic groups was evaluated using 1,000 bootstrap replicates, and bootstrap values greater than 70 were considered significant.

DISCUSSION

Herein, we reported microsporidia parasitizing S. pertinax larvae in the State of São Paulo, with a prevalence of 0.51%. Araújo-Coutinho66. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191. previously reported a 0.5-2.0% prevalence of microsporidia in S. pertinax in State of Rio de Janeiro. Our study showed a similar prevalence to that previously reported by Crosskey1919. Crosskey RW. The Natural History of Black flies. 1st ed. West Sussex, England: Wiley; 1990. in other populations of black flies, with rates of up to 1%. Polydispyrenia spp. were the most prevalent parasitic species in S. pertinax from Caraguatatuba/SP in this study, while Amblyospora spp. showed a higher prevalence in Rio de Janeiro66. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191.. This difference could be explained by the small sample size, which prevented further analysis of the species population dynamics between S. pertinax from Rio de Janeiro and Caraguatatuba.

In this study, spores of the Polydispyrenia spp. measured 6.9 ± 1.0µm in length × 5.0 ± 0.7µm in width. Araújo-Coutinho66. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191.reported spores of a similar size for a Polydispyrenia sp. from S. pertinax that was ovocylindrical and measured 7.0 ± 0.6 × 4.9 ± 0.8µm. However, Castello-Branco and Andrade2020. Castello-Branco A, Andrade CFS. Studies on Polydispyrenia simulii (Microspora; Pleistophoridae) in Simulium pertinax (Diptera; Simulidae) in Brazil. Mem Inst Oswaldo Cruz 1993; 88:167. reported larger-sized spores measuring 8.3µm in length × 6.3µm in width for P. simulii from S. pertinax collected in State of São Paulo, Brazil. Sprague1212. Sprague V, Becnel JJ, Hazard EI. Taxonomy of Phylum Microspora. Crit Rev Microbiol 1992; 18:285-395. stated that the spore dimensions were 4.5 to 5.5µm × 2.5 to 3.5µm for P. simulii with the hosts listed as S. pertinax and S. perflavum from Brazil.

In this study, for Amblyospora spp. from Caraguatatuba, the spore measurement was 4.5µm in length × 3.5µm in width, similar to that found by Araújo-Coutinho66. Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191. for Amblyospora spp. infecting S. pertinax in the State of Rio de Janeiro. Both of these results were similar to those from Amblyospora bracteata and Amblyospora varians, described in black flies in North America and Europe2121. Vávra J, Undeen AH. Microsporidia (Microspora: Microsporida) from Newfoundland blakflies (Diptera: Simuliidae). Can J Zool 1981; 59:1431-1446.. According to Sprague1212. Sprague V, Becnel JJ, Hazard EI. Taxonomy of Phylum Microspora. Crit Rev Microbiol 1992; 18:285-395., the morphological similarity between species of microsporidia, particularly the spore measurements, makes identification difficult, and other methods are needed for identification. Such evidence indicates that spore dimension diversity is too variable; thus, molecular analysis could help in species identification.

Our sample, identified morphologically as Polydispyreniaspp., was grouped with the P. simulii and C. palustris clusters. This identification corroborated previous results2222. Lom J, Nilsen F, Dykova I. Thelohania contejeani Henneguy, 1892: dimorphic life cycle and taxonomic affinities, as indicated by ultrastrutural and molecular study. Parasitol Res 2001; 87:860-872.2626. Dong S, Shen Z, Xu L, Zhu F. Sequence and phylogenetic analisys of SSU rRNA gene of five Microsporidia. Curr Microbiol 2010; 60:30-37. regarding the phylogeny of these parasites.

The genera Parathelohania, Hazardia, Marsoniella, Gurleya, Larssonia, Berwaldia, Varimorpha, Amblyospora and the Amblyospora sp. from S. pertinax in this study form a separate group from the main Amblyospora cluster (Figure 3). Excluding the Varimorpha sp., which was characterized in an ant species, Solenopsis richteri (Forel, 1909), all genera in this group are parasites of aquatics hosts2727. Baker MD, Vossbrinck CR, Becnel JJ, Andreadis TG. Phylogeny of Amblyospora (Microsporida: Amblyosporidae) and related genera based on small subunit ribosomal DNA data: A possible example of host parasite cospeciation. J Invertebr Pathol 1998; 71:199-206.3030. Refardt D, Canning EU, Mathis A, Cheney SA, Lafranchi-Tristem NJ, Ebert D. Small subunit ribosomal DNA phylogeny of microsporidia that infect Daphnia (Crustacea: Cladocera). Parasitol 2002; 124:381-389..

Because the Amblyospora group is divided into two clades, corresponding to the hosts (Culex or Aedes/Ochlerotatus)2828. Moser BA, Becnel JJ, Maruniak J, Patterson RS. Analysis of the ribosomal DNA sequences of the microsporidia Thelohania and Vairimorpha of fire ants. J Invertebr Pathol 1998; 72:154-159., the aquatic group also demonstrated distinct phylogenetic characteristics according to the host. The genera that infect both Culex quinquefasciatus (Say, 1823) and crustaceans (Hazardia, Marsoniella, Gurleya, Larssonia and Berwaldia) are the main members of this clade. The genera that infect anopheline mosquitoes (Parathelohania), simulids (Amblyospora spp 3 in this study) and a species of ant (Varimorpha sp.), are more closely related to the aquatic group than to the main Amblyospora group. The Amblyospora spp. in this study were clustered with Amblyospora sp. (AJ252949) from Simulium spp. from the Paleartic2929. Cheney SA, Lafranchi-Tristem NJ, Canning EU. Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of Trachipleistophora hominis infections. J Eukaryot Microbiol 2000; 47:280-287.,; confirming the morphological and molecular similarities between these 2 species.

Phylogenetic analysis with the 16S gene showed considerable distance between the Amblyospora spp., which infect simulids, and the main group of Amblyospora spp., which infects mosquitoes, indicating that these groups are not congeneric. The differences between taxonomic relationships, based on phylogenetic placement and classical morphological characteristics, could probably be explained by the possibility that some of these characteristics (diplokaryon, sporophorous vesicles, and meiosis) appear to have multiple origins3131. Baker MD, Vossbrinck CR, Didier ES, Maddox JV, Shadduck JA. Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. J Eukaryot Microbiol 1995; 42:564-570.. Thus, molecular analysis of other genomic regions could improve the phylogenetic understanding of microsporidia. This work contributes to the phylogenetic analysis of microsporidia because it provides two genus sequences from these parasites.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FINANCIAL SUPPORT

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2012/23947-0

REFERENCES

  • 1
    Shelley A, Hernandez L, Maia-Herzog M, Luna Dias A, Garritano D. The blackflies (Diptera: Simuliidae) of Brazil. In: Arias JR, Golovatch S, Wantzen KM, Dominguez E, editors. Aquatic biodiversity in Latin America (ABLA). Vol. 6. Pensoft:Sofia-Moscow; 2010. p. 821.
  • 2
    Coscarón S, Coscarón-Arias CL. Neotropical Simuliidae (Diptera: Insecta). In: Adis J, Arias JR, Rueda-Delgado G, Wantzen KM, editors. Aquatic biodiversity in Latim America (ABLA). Vol. 3. Pensoft: Sofia-Moscow; 2007. p. 685.
  • 3
    Ribeiro do Amaral-Calvão AM, Maia-Herzog M. Adolpho Lutz's collection of black flies (Diptera: Simuliidae), its history and importance. Hist Cienc Saude Manguinhos 2003; 10:259-271.
  • 4
    Vossbrinck CR, Maddox JB, Friedman S, Debrunner-Vossbrink BA, Woese CR. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987; 326: 411-414.
  • 5
    Ebersberger I, Simões RM, Kupczok A, Gube M, Kothe E, Voigt K, et al. A consistent phylogenetic backbone for the fungi. Mol Biol Evol 2012; 29:1319-1334.
  • 6
    Araújo-Coutinho CJ, Nascimento ES, Figueiro R, Becnel JJ. Seasonality and prevalence rates of microsporidia in Simulium pertinax (Diptera: Simuliidae) larvae in the region of Serra dos Órgãos, Rio de Janeiro, Brasil. J Invertebr Pathol 2004; 85:188-191.
  • 7
    Ginarte CA, Andrade CFS, Gaona JC. Larvas de simulídeos (Diptera, Simuliidae) do Centro Oeste, Sudeste e Sul do Brasil, parasitadas por microsporídeos (Protozoa) e mermitídeos (Nematoda). Ser Zool 2003; 93:325-334.
  • 8
    Castello-Branco A. Effects of Polydispyrenia simulii (Microspora; Duboscqiidae) on Development of the Gonads of Simulium pertinax (Diptera; Simuliidae). Mem Inst Oswaldo Cruz 1997; 94:421-424.
  • 9
    Online Archive of Instituto Brasileiro de Geografia e Estatística [Internet][cited 2013 May]. Available at: http://cod.ibge.gov.br/2335A.
    » http://cod.ibge.gov.br/2335A
  • 10
    Undeen A H, Vavra J. Research Methods for Entomopathogenic Protozoa In: Lacey LA. Manual of Techniques in Insect Pathology. Chapter 4. 1st ed. Great Britain: Academic Press; 1997. p. 115-151.
  • 11
    Becnel J J. Preparations of Entomopathogens. In: Lacey LA. Manual of Techniques in Insect Pathology. Chapter VIII-1. 1st ed. Great Britain: Academic Press; 1997; p. 337-353.
  • 12
    Sprague V, Becnel JJ, Hazard EI. Taxonomy of Phylum Microspora. Crit Rev Microbiol 1992; 18:285-395.
  • 13
    Adler PH, Becnel JJ, Moser B. Molecular charaterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black flies (Diptera: Simuliidae), with host-derived relationships of the North American caudosporids. J Invertebr Pathol 2000; 75:133-143.
  • 14
    Vossbrinck CR, Andreadis TG, Vavra J, Debrunner-Vossbrink BA. Verification of intermediate hosts in the life cycles of Microsporidia by small subunit rDNA sequencing. J Eukaryot Microbiol 1998; 45:290-292.
  • 15
    Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res 1999; 9:868-877.
  • 16
    Gouy M, Guindon S, Gascuel O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 2010; 27:221-224.
  • 17
    Waddell PJ, Steel MA. General time-reversible distances with unequal rates across sites: mixing gamma and inverse Gaussian distributions with invariant sites. Mol Phylogenet Evol 1997; 8:398-414.
  • 18
    Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics 1998; 14:817-818.
  • 19
    Crosskey RW. The Natural History of Black flies. 1st ed. West Sussex, England: Wiley; 1990.
  • 20
    Castello-Branco A, Andrade CFS. Studies on Polydispyrenia simulii (Microspora; Pleistophoridae) in Simulium pertinax (Diptera; Simulidae) in Brazil. Mem Inst Oswaldo Cruz 1993; 88:167.
  • 21
    Vávra J, Undeen AH. Microsporidia (Microspora: Microsporida) from Newfoundland blakflies (Diptera: Simuliidae). Can J Zool 1981; 59:1431-1446.
  • 22
    Lom J, Nilsen F, Dykova I. Thelohania contejeani Henneguy, 1892: dimorphic life cycle and taxonomic affinities, as indicated by ultrastrutural and molecular study. Parasitol Res 2001; 87:860-872.
  • 23
    Terry RS, Smith JE, Sharpe RG, Rigaud T, Littlewood TDJ, Ironside JE, et al. Widespread vertical transmission and associated host sex-ratio distortion within eukaryotic Phylum Microspora. Proc R Soc Lond 2004; 271:1783-1789.
  • 24
    Vossbrinck CR, Debrunner-Vossbrinck BA. Molecular Phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitol 2005; 52:131-142.
  • 25
    Smith JE. The ecology and evolution of microsporidian parasites. Parasitol 2009; 136:1901-1914.
  • 26
    Dong S, Shen Z, Xu L, Zhu F. Sequence and phylogenetic analisys of SSU rRNA gene of five Microsporidia. Curr Microbiol 2010; 60:30-37.
  • 27
    Baker MD, Vossbrinck CR, Becnel JJ, Andreadis TG. Phylogeny of Amblyospora (Microsporida: Amblyosporidae) and related genera based on small subunit ribosomal DNA data: A possible example of host parasite cospeciation. J Invertebr Pathol 1998; 71:199-206.
  • 28
    Moser BA, Becnel JJ, Maruniak J, Patterson RS. Analysis of the ribosomal DNA sequences of the microsporidia Thelohania and Vairimorpha of fire ants. J Invertebr Pathol 1998; 72:154-159.
  • 29
    Cheney SA, Lafranchi-Tristem NJ, Canning EU. Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of Trachipleistophora hominis infections. J Eukaryot Microbiol 2000; 47:280-287.
  • 30
    Refardt D, Canning EU, Mathis A, Cheney SA, Lafranchi-Tristem NJ, Ebert D. Small subunit ribosomal DNA phylogeny of microsporidia that infect Daphnia (Crustacea: Cladocera). Parasitol 2002; 124:381-389.
  • 31
    Baker MD, Vossbrinck CR, Didier ES, Maddox JV, Shadduck JA. Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. J Eukaryot Microbiol 1995; 42:564-570.
  • 32
    Vossbrinck CR, Andreadis TG, Vavra J, Becnel JJ. Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae). J Eukaryot Microbiol 2004; 51:88-95.
  • 33
    Sokolova YY, Dolgikh VV, Morzhina EV, Nassonova ES, Issi IV, Terry RS, et al. Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus. Deg J Invertebr Pathol 2003; 84:159-172.
  • 34
    Muller A, Trammer T, Chioralia G, Seitz HM, Diehl V, Franzen C. Ribosomal RNA of Nosema algerae and phylogenetic relationship to other microsporidia. Parasitol Res 2000; 86:18-23.
  • 35
    Vossbrinck CR, Andreadis TG, Vavra J, Becnel JJ. Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae). J Eukaryot Microbiol 2004; 51:88-95.
  • 36
    Nilsen F, Endresen C, Hordvik I. Molecular phylogeny of microsporidians with particular reference to species that infect the muscles of fish. J Eukaryot Microbiol 1998; 45:535-543.
  • 37
    Malone LA, Broadwell AH, Lindridge ET, McIvor CA, Ninham J. Ribosomal RNA genes of two microsporidia, Nosema apis and Vavraia oncoperae are very variable. J Invertebr Pathol 1994; 64:151-152.
  • 38
    Docker MF, Kent ML, Hervio DL, Khattra JS, Weiss LM, Cali A, et al. Ribosomal DNA sequence of Nucleospora salmonis Hedrick, Groff and Baxa, 1991 (Microsporea: Enterocytozoonidae): implications for Phylogeny and Nomenclature. J Eukaryot Microbiol 1997; 44:55-60.
  • 39
    Silva AJ, Schwartz DA, Visvesvara GS, Moura H, Slemenda SB, Pieniazek NJ. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA. J Clin Microbiol 1996; 34:986-987.
  • 40
    Zhu X, Wittner M, Tanowitz HB, Cali A, Weiss LM. Nucleotide sequence of the small ribosomal RNA of Encephalitozoon Cuniculi. Nucleic Acids Res 1993; 21:1315.
  • 41
    Visvesvara GS, Silva AJ, Croppo GP, Pieniazek NJ, Leitch GJ, Ferguson D, et al. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. J Clin Microbiol 1995; 33:930-936.
  • 42
    Visvesvara GS, Leitch GJ, Silva AJ, Croppo GP, Moura H, Wallace S, et al. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J Clin Microbiol 1994; 32:2760-2768.
  • 43
    Gatehouse HS, Malone LA. The ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of a large tandem repeat unit size. J Invertebr Pathol 1998; 71:97-105.
  • 44
    Baker MD, Vossbrinck CR, Becnel JJ, Maddox JV. Phylogenetic position of Amblyospora Hazard & Oldacre (Microspora: Amblyosporidae) based on small subunit rRNA data and its implication for the evolution of the microsporidia. J Eukaryot Microbiol 1997; 44:220-225.
  • 45
    Nilse F, Che WJ. rDNA phylogeny of Intrapredatorus barri (Microsporida: Amblyosporidae) parasitic to Culex fuscanus Wiedemann (Diptera: Culicidae) Parasitology 2001; 122:617-623.
  • 46
    Andreadis TG, Vossbrinck CR. Life cycle, ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae). J Eukaryot Microbiol 2002; 49:350-364.

Data availability

Data citations

Online Archive of Instituto Brasileiro de Geografia e Estatística [Internet][cited 2013 May]. Available at: http://cod.ibge.gov.br/2335A.

Publication Dates

  • Publication in this collection
    Sep-Oct 2014

History

  • Received
    18 July 2014
  • Accepted
    9 Oct 2014
Sociedade Brasileira de Medicina Tropical - SBMT Caixa Postal 118, 38001-970 Uberaba MG Brazil, Tel.: +55 34 3318-5255 / +55 34 3318-5636/ +55 34 3318-5287, http://rsbmt.org.br/ - Uberaba - MG - Brazil
E-mail: rsbmt@uftm.edu.br