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

Introduction: Microsporidia constitute the most common black fl y 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 artifi cial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 x 5.0 ± 0.7μm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 x 3.5μm in sporophorous vesicles. Results: The molecular analysis confi rmed 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 identifi ed 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 fl y-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.

Black fl ies (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 production [1][2][3] .Black fl y control remains a major public health challenge.Microsporidia are unicellular, eukaryotic organisms that are obligate, intracellular parasites with public health relevance 4 .Several studies have suggested a new classifi cation for microsporidia as fungi, but Ebersberger 5 stated that phylogenetic analysis did not support fungal characterization for this group.
Microsporidia are the most common black fl y pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood 6,7 .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 vigor 8 .
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 METHODS fl y bites annoy visitors and have deleterious effects on the local economy.Monitoring and controlling black fl ies are essential to avoiding seasonal population outbreaks.

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 458,097km 2 and had a population at that time of 100,840 9 .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 removed 10 .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, fi xed 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 overnight 11 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 Sprague 12 .

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 gene 13 .
The amplifi cation 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 purifi ed with the Illustra GFX PCR DNA and Gel Band Purifi cation Kit (GE Healthcare Limited, Little Chalfont, Buckinghamshire, UK) and were quantifi ed 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 software 15 .

Phylogenetic analysis
The analyses were performed using the Seaview software 16 .A phylogenetic tree was constructed, with reference sequences [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] from Table 1 (supplementary fi le), using the maximum likelihood method with the general time reversible (GTR) model of nucleotide substitution and gamma distribution (G) (GTR + G) 17 .The model was selected by the Modeltest software, version 3.0.6 18, 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 signifi cant.

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 Polydispyrenia spp.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%.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 x 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 x 3.5µm (n = 12).
The PCR products targeting the 16S region and electrophoresis agarose gel analysis confi rmed 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

DISCUSSION
Herein, we reported microsporidia parasitizing S. pertinax larvae in the State of São Paulo, with a prevalence of 0.51%.Araújo-Coutinho 6 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 Crosskey 19 in other populations of black fl ies, 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 Janeiro 6 .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 x 5.0 ± 0.7μm in width.Araújo-Coutinho 6 reported spores of a similar size for a Polydispyrenia sp. from S. pertinax that was ovocylindrical and measured 7.0 ± 0.6 x 4.9 ± 0.8μm.However, Castello-Branco and Andrade 20 reported larger-sized spores measuring 8.3μm in length x 6.3μm in width for P. simulii from S. pertinax collected in State of São Paulo, Brazil.Sprague 12 stated that the spore dimensions were 4.5 to 5.5μm x 2.5 to 3.5μm for P. simulii with the hosts listed as S. pertinax and S. perfl avum from Brazil.
In this study, for Amblyospora spp.from Caraguatatuba, the spore measurement was 4.5μm in length x 3.5μm in width, similar to that found by Araújo-Coutinho 6 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 fl ies in North America and Europe 21 .According to Sprague 12 , the morphological similarity between species of microsporidia, particularly the spore measurements, makes identifi cation diffi cult, and other methods are needed for identifi cation.Such evidence indicates that spore dimension diversity is too variable; thus, molecular analysis could help in species identifi cation.
Because the Amblyospora group is divided into two clades, corresponding to the hosts (Culex or Aedes/Ochlerotatus) 28 , 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 Paleartic 29 , ; confi rming 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 origins 31 .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.
The authors declare that there is no confl ict of interest.

FIGURE 3 -
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 signifi cant.

Carvalho IMVG et al. -
Characterization of microsporidia in Simulium pertinax