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

IntroductionMicrosporidia 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.MethodsLarvae 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.ResultsThe 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.ConclusionsSamples 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.

Effects of Polydispyrenia simulii (Microspora; Duboscqiidae) on Development of the Gonads of Simulium pertinax (Diptera; Simuliidae)

The effects of the infection caused by the microsporidium Polydispyrenia simulii in gonads of the simuliid Simulium pertinax were evaluated. This blackfly species is the main target of control programmes in southeastern Brazil. Infected and noninfected blackflies were reared from larval stage to adults using a laboratory rearing system. There was no significant difference between the oogenic development and dimensions of ovarian follicles of healthy females compared to those reared from infected larvae. Otherwise, there was a significant difference between the fecundity of healthy females and those reared from infected larvae. In males, there was an evident reduction of motility of spermatozoids of males reared from infected larvae. The results obtained suggest that the microsporidium P. simulii have an important effect on the natural population dynamics of S. pertinax.

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinanting B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.

Evaluation of the impact of Bacillus thuringiensis serovar israelensis and Temephos, used for the control of Simulium (Chirostilbia) pertinax Kollar, 1832 (Diptera, Simuliidae) on the associated entomofauna, Paraty, state of Rio de Janeiro, Brazil

The study was set up to evaluate the impact of two commercial larvicide formulations, Bacillus thuringiensis serovar israelensis base (Bti) at 15 ppm/1 min and temephos at 0.03 ppm of active ingredient, used to control Simulium pertinax populations, on associated non-target entomofauna occupying the same breeding sites. The experiments were carried out on the Pedra Branca and Muricana rivers, on the slopes of Serra do Mar massif, municipality of Paraty, state of Rio de Janeiro, Brazil. Bti was applied to the river Pedra Branca and temephosto theriver Muricana. On both rivers, treatment and control sections were labeled as such, each one with two observation posts: slow moving water and fast water regions respectively. Artificial substrata was used to evaluate the abundance of associated entomofauna. Attached immature stages of arthropods were removed from both of its surfaces fortnightly. Were collected, from the two rivers, 28 477 specimens of the entomofauna associated with S. pertinax. The families Hydropsychidae, Chironomidae, Bactidae, Simuliidae, Blephariceridae and Megapodagrionidae were represented. These was an impact of temephos on the entomofauna associated with S. pertinax only in Simuliidae and Chironomidae, and to Bti only in Simuliidae. However, the reduction in their numbers was not statistically significant.

The phylogeny of Simulium (Chirostilbia) (Diptera: Simuliidae) and perspectives on the systematics of the genus in the Neotropical Region

Recently, knowledge of Neotropical Simuliidae has been accumulating quickly. However, information about supra-specific relationships is scarce and diagnoses of Simulium subgenera are unsatisfactory. To investigate the relationships among Simulium (Chirostilbia) species and test the subgenus monophyly, we performed a cladistic analysis. The ingroup included all species of this subgenus and the outgroup included representatives of the 17 species groups of Neotropical Simulium and three Holarctic species. The study was based on a data matrix with 31 terminal taxa and 45 morphological characteristics of adult, pupa and larva. The phylogenetic analysis under equal weights resulted in eight most-parsimonious trees (length = 178, consistency index = 34, retention index = 67). The monophyly of the S. (Chirostilbia) was not supported in our analysis. The Simulium subpallidum species group was closer to Simulium (Psilopelmia) and Simulium (Ectemnaspis) than to the Simulium pertinax species group. Additionally, we describe the three-dimensional shape of the terminalia of male and female of Simulium (Chirostilbia) for the first time and provide comments about the taxonomic problems involving some species of the subgenus: Simulium acarayense, Simulium papaveroi, S. pertinax, Simulium serranum, Simulium striginotum and S. subpallidum.

Levantamento das espécies de Simulium (Diptera, Simuliidae), Riacho dos Padres, município de Almirante Tamandaré, Paraná, Brasil

De janeiro a agosto de 2002, quinzenalmente, foi levada a efeito a coleta de simulídeos imaturos em substratos naturais e antrópicos no Riacho dos Padres. O trabalho visou proceder ao inventário das espécies e verificar as freqüências relativas. A área de propriedade particular localizada na Região Metropolitana de Curitiba constitui uma microempresa de agronegócio, com criação de peixes, coelhos, ovinos, bovinos, aves domésticas e, ainda, área de lazer com eqüinos para cavalgada de visitantes. A presença constante de animais hospedeiros e a ração fornecida aos peixes garantiram farta alimentação aos simulídeos, o que favoreceu a explosão desta praga. Em todos os substratos foram coletadas 24.021 pupas e um total de 197.812 larvas. A partir de pupas, foram identificadas com as seguintes freqüências: Simulium inaequale (55,24%), Simulium perflavum (16,81%), Simulium pertinax (13,93%), Simulium orbitale (8,03%), Simulium subnigrum (4,92%), Simulium distinctum (1,03%) e Simulium incrustatum (0,04%). Verificou-se também a quantidade de larvas que colonizaram todos os substratos.

Controle integrado de espécies de Simulium (Diptera, Simuliidae) por Bacillus thuringiensis e manejos mecânicos no riacho e nos vertedouros de tanques de piscicultura, Almirante Tamandaré, Paraná, Brasil

No Riacho dos Padres na Chácara Evíssima, numa microempresa de agronegócio com criação de peixes e animais domésticos, área de lazer e salão de eventos, localizada na Região Metropolitana de Curitiba, os simulídeos em grande proliferação causaram sério impacto com as picadas a tal ponto que dificultaram o convívio habitual. De janeiro a agosto de 2002, coletou-se 24.021 pupas por meio das quais foram identificadas as espécies: Simulium inaequale, Simulium perflavum, Simulium pertinax com 85,98% e Simulium orbitale, Simulium subnigrum, Simulium distinctum e Simulium incrustatum com somente 14,02% de freqüência. O combate a estas populações iniciado em 28 de fevereiro terminou em 08 de agosto de 2002. O controle integrado consistiu na aplicação de biopesticida Bacillus thuringiensis var. israelensis (Bti) e em manejos mecânicos não só removendo os imaturos (ovos, larvas e pupas) por meio de escovação de vertedouros, mas também pela retirada de substratos naturais e antrópicos do leito. A densidade larvária média inicial, antes da aplicação de Bti no leito do riacho (75 m) e no último vertedouro, verificada em substratos antrópicos fitilhos (área 13.500 cm²), foi de 6,7 e 11,5 larvas/cm², respectivamente. Nestes habitats, o controle integrado com Bti de 1,77 a 2,09 mg/litro do ingrediente ativo, suplementado com manejos mecânicos, monitorado em substratos fitilhos (área 20.250 cm²), resultou em redução larvar de 72,61% a 99,97% e de 74,91% a 99,45%, respectivamente, nas vazões de 0,39 a 0,45 e de 0,38 a 0,43 m³/min. No primeiro vertedouro (área 5.110 cm²), porém, só com manejos mecânicos, atingiu-se a redução larvária de 53,81% a 99,59%. O controle efetuado reduziu as picadas hematófagas ao nível esperado, surtindo o efeito positivo almejado.

Distribuição e abundância de larvas de Simulium spp.em córregos do estado de São Paulo nos diferentes níveis de qualidade da água

The aim of this work was to determine the distribution and abundance the species of Simulium: in 5 streams of the north coastal region of the state of São Paulo. Simulium larval and water samples were collected every 2 weeks in summer and winter, 2001. The larval were collected on artificial substrates (triplicate) preserved in alcohol (70%) and after analyzed quantitative and qualitative. From the streams the values of temperature, dissolved oxygen, conductivity, pH and stream discharge were measured. The contents of total nitrogen and dissolved nitrogen, ammoniacal nitrogen, nitrate, nitrite, orthophosphate, dissolved phosphorus and total phosphorus were determined in the laboratory. Water quality has great influence in the distribution and abundance of the species. S. pertinax was more abundant in streams without or with low levels of pollution, S. inaequale in streams more polluted and S. incrustatum in intermediary levels of pollution. S. pertinax was the species more abundant with 57.5% of presence in the samples, the second more abundant was S. inaequale with 42.1% of presence and S. incrustatum was rare with 0.4% of presence. The larval abundance was lower in the polluted environment. © 2012 by Unisinos.

Methods for field detection of resistance to temephos in simuliids. Larval esterase level and topical application of the insecticide to adults

Two practical field methods for indirect detection of simuliid populations resistant to temephos are proposed. The first is based on high esterase activity in resistant larvae and involves adaptations of a filter paper test in which faintly stained spots indicate susceptible populations and strongly stained ones reveal populations resistant to temephos. The second is based on the resistance to the larvicide when adults are topically exposed, and involves the use of diagnostic doses obtained by the comparison between the LD50 for susceptible and resistant populations. The relevance of such methods is discussed in order to help resistance detection in Simulium pertinax Kollar control programmes.

Local distribution of blackfly (Diptera, Simuliidae) larvae in two adjacent streams: the role of water current velocity in the diversity of blackfly larvae

This study aimed to evaluate the influence of water velocity speed on the local distribution and taxocenosis structure of blackfly larvae. The larvae were collected from two adjacent streams located in the municipality of Angra dos Reis (RJ): Caputera River and one of its tributaries. Riffle litter patches were sampled randomly using a 30 x 30 cm quadrat. Four blackfly species were found: Simulium incrustatum s. l. Lutz, 1910; Simulium (Inaequalium) sp. ; Simulium pertinax s. l. Kollar, 1832 and Simulium subpallidum s. l. Lutz, 1909. Among these species, Simulium pertinax s. l. was clearly associated with higher water current speeds, while Simulium subpallidum s. l. showed association with lower water velocities, and Simulium (Inaequalium) sp. had a relatively constant distribution along the water current gradient.

Susceptibilidade de populações de Simulium (Chirostilbia) pertinax Kollar, 1832 (Culicomorpha, Simuliidae) ao temephos e a um formulado à base de Bacillus thuringiensis var. israelensis

O uso de pequenas calhas de madeira no leito de riachos, manualmente colonizadas com borrachudos, é proposto para avaliações da eficiência de larvicidas 3 ou 4 horas após as aplicações. A susceptibilidade larval também foi avaliada pelo critério de TL50. Em três ensaios no litoral do Estado de São Paulo e Rio de Janeiro, populações de Simulium (C.) pertinax Kollar, 1832 mostraram-se resistentes ao temephos, mesmo em altas concentrações. Vectobac 12 AS, à base de Bacillus thuringiensis var. israelensis mostrou-se mais potente contra larvas dos últimos estádios e eficiente para controle em concentrações a partir de 7.200 UIP/1 (10 min.). O TL50para 3.744 UIP/1 (10 min.) foi de 70,9 minutos.

Chromosomal comparisons among and within populations of Simulium (Chirostilbia) pertinax (Diptera, Simuliidae)

Chromosomal studies were carried on six larval populations of Simulium (Chirostilbia) pertinax from different locations in Brazil. Larvae were collected in the states of Paraná, Rio Grande do Sul, Rio de Janeiro and São Paulo. Polytene chromosome map comparisons within and among populations showed no differences in banding pattern, except for some limited polymorphism (secondary NOR and four band polymorphisms). There were no chromosomal variations associated with the resistance or susceptibility of the larvae to temephos. The chromosomal homosequentiality found among the six populations suggests that S. pertinax may be a monomorphic species.