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Bionomics of the black fly Simulium guianense (Diptera: Simuliidae) in northeast Brazil.

Simulium guianense Wise (Diptera: Simuliidae) is widely distributed in Brazil (Adler & Crosskey 2014), but it is more frequent in the northern portion of the country where it is the main vector of the nematode Onchocerca volvulus (Leuckart) (Spirurida: Onchocercidae) in the Amazon region. Despite the medical importance of this species (Shelley et al. 1997), its biology and ecology are poorly known. Two studies on this species were done in the Brazilian state of Amazonas, one providing water physical-chemical characteristics of the rivers where larvae and pupae were collected (Gomes & Py-Daniel 2002) and the other identifying the microalgae in the larval stomach contents (Gomes et al. 2002). Villamizar et al. (2011) provided the first information on the habitat of S. guianense immatures in the onchocerciasis area of Venezuela. Several studies on anthropophilic activity of this species have been done in Brazil (e.g., Shelley et al. 1997) and Venezuela (e.g., Grillet et al. 2001).

Because S. guianense has wide geographical distribution and some populations are zoophilic whereas others are anthropophilic, it is suspected that the species is a complex of 2 or more sibling species (i.e., morphologically similar but reproductively isolated species) (Shelley et al. 1997). A cytotaxonomic study conducted with 4 populations of S. guianense in Brazil indicated the presence of 4 cytotypes, according to geographical region (Charalambous et al. 1996).

The number of instars is not fixed in Simuliidae, in contrast, for example, to Chironomidae and Culicidae, which are 2 families of aquatic Diptera in which there are 4 instars. In Simuliidae, the number of instars may vary depending on the species; also, larval development can be related to environmental conditions such as food availability and the temperature of streams (Crosskey 1990). Water temperature can affect the population of black flies, reducing the development period, the number of instars, and the size of the larva at maturity (Crosskey 1990). However, some studies conducted in Brazil have indicated no change in either the number of instars or the larval size, considering either the season (Alencar et al. 2001) or the geographical distribution (Alvan-Aguilar & Hamada 2003).

Oviposition behavior by female black flies can be classified into 4 types, depending on how the eggs are placed and the timing (continued or interrupted) in which they are laid (Crosskey 1990). In Brazil, the dabbing technique (with eggs placed singly or in groups with interrupted timing) has been reported for 2 species of Simulium (Etecmnaspis) (Gorayeb 1981; Hamada 1998). One species of Simulium (Inaequalium) was observed laying eggs in the terrestrial environment on riparian vegetation in a splash zone of a water fall in southern Brazil (Moreira & Sato 1996). Improved knowledge of the distribution of this species is important for understanding the potential for onchocerciasis dispersion and to serve as a basis for other studies, such as phylogeographic and ecological analyses. The present study aimed to expand the geographical records of this species in Brazil and to provide biological information on the immature stages and on the oviposition behavior observed in Piaui State. Information on the biology and distribution of this species may assist in understanding its population dynamics and help in detecting the presence of potential sibling species.

The numbers of instars reported for black fly species of South America are: 6 (e.g., Coscaron-Arias & Bramardi 1996), 7 (e.g., Alencar et al. 2001; Alvan-Aguillar & Hamada 2003), and 8 (e.g., Coscaron-Arias & Bramardi 1996). Thus, we tested the hypothesis that the number of instars of S. guianense could be 6, 7 or 8.

Materials and Methods

The sampled area (Table 1) is located in a transition zone between deciduous Caatinga/Cerrado and Carnauba/Caatinga riparian forest, where the annual precipitation is between 800 and 1,600 mm (Giulietti et al. 2004). Sampling was done in the northern portion of Piaui State and in the western portion of Ceara State (both in northeastern Brazil), from 27 May to 8 Jun 2011 (at the beginning of the dry season). The width of rivers sampled was estimated visually. Temperature, pH, and electrical conductivity were measured using portable equipment (waterproof pH meter, conductivity meter, and thermometer; Oakton Instruments, Vernon Hills, Illinois, USA). The geographical position was obtained with a GPS (Garmin Map 76Cx; Garmin, Olathe, Kansas, USA).

Oviposition behavior was observed in one stretch of the Jacarei River, located in Piracuruca Municipality, Piaui State, while we were sampling for black flies. Two females were collected for species identification. Larvae used to estimate the number of instars came from the same river, which was the only place where oviposition was observed, thereby allowing the collection of 1st instars. Predation of larvae and pupae was observed in the Longa River, Batalha Municipality, Piaui, during one afternoon.

Larvae and pupae were collected directly from the substrate by using tweezers. The larvae and pupae were fixed in Carnoy's solution (acetic acid and absolute alcohol in a proportion of 3:1) and in absolute ethanol, respectively. Both fixatives were changed at least 4 times. Pupae containing pharate adults were kept alive in plastic containers with moist filter paper until adult emergence; exuviae and their respective adults were fixed in 80% ethanol for species identification in the laboratory. Vouchers of the analyzed specimens were deposited in the Invertebrate Collection of the Instituto Nacional de Pesquisas da Amazonia, Manaus, Amazonas, Brazil.

To estimate the number of instars, the following measurements were taken: total body length (TL), lateral length of the head capsule (LLH, a line drawn from the base of the labral fan stalk to the posterior limit of the head, passing through the ocelli), and the width of the frontoclypeal apotome (WFA) (as in Alencar et al. 2001). The measurements were taken under a stereomicroscope (Leica M165C model; Leica, Wetzlar, Germany) with a camera attached (DFC420 model; Leica, Wetzlar, Germany), using a self-assembly program (LAS version 3.7). A Pearson analysis was used to evaluate possible correlations between the measured structures (TL, LLH, WFA) and to eliminate correlated metrics.

To test the hypothesis that the number of instars of S. guianense could be 6, 7, or 8, we considered the following steps: a) determination of the size of the 1st and last instars, which were easily recognized by their structural characteristics (Fig. 1); b) analysis of the distribution frequency of the intermediate measurements to estimate the intermediate stages, testing the hypothesis that S. guianense has 6 (4 intermediate), 7 (5 intermediate), or 8 (6 intermediate) instars; c) performing 3 ANOVAs with an a posteriori Tukey test to determine if the number of instars of S. guianense was 6, 7, or 8, using the measured structures of the 1st and last instars and the sizes of the intermediate instars, which were estimated by frequency distribution analysis; d) finally, we chose the analysis where no overlapping instars were observed.


Out of 29 sites sampled in the study area, 9 had S. guianense: 8 in Piaui and 1 in Ceara State (Fig. 2). In total, 264 larvae from 1 river (Jacarei) were measured (LLH, TL, WFA). We verified the relationship between WFA, TL, and LLH, and all 3 measurements were highly correlated (Fig. 3). We therefore only considered LLH values in determining the number of instars. Measurements of the intermediate stages are shown in Fig. 4. The ANOVA and Tukey tests indicated that S. guianense has 6 instars, because the analysis with 7 or 8 instars resulted in overlapping instars (Fig. 5).

Simulium guianense is reported here for the first time in the states of Piaui and Ceara, in the Parnaiba River hydrographic basin (Table 1). Immatures of S. guianense were collected in rivers with an average width of 55 m (SD = 31.9; n = 9), average water temperature of 28.4[degrees]C (SD = 1.28; n = 9), pH of 7.28 (SD = 2.31; n = 9), and electrical conductivity of 157.3 [micro]S/cm (SD = 76.1; n = 9) (Table 1).

Immatures were distributed in high density (approximately 30 larvae per [cm.sup.2]) on different substrates such as twigs, branches, macrophytes (including Podostemaceae), and rock. Despite the high density of S. guianense immatures, adults were not observed in anthropophilic activity during the fieldwork.


Predation of immatures by ants (Solenopsis sp.; Hymenoptera: Formicidae) was observed in the Longa River (Fig. 6). At this site, we observed that the river water level went down very fast (observation at the beginning of the dry season), approximately 5 cm in 12 h, leaving larvae and pupae, which were using the bedrock as a substrate, exposed to this predator (Fig. 7). The rapid decrease in the water level of this river made it possible to observe many larvae confined to puddles of water formed in depressions in the rock (Fig. 8).

In a stretch of the Jacarei River, females of S. guianense were observed in oviposition activity in the morning (8:00 am), using as substrate submerged and/or partially submerged vegetation (Figs. 9 and 10). Because observation was not done in the afternoon, we do not know if this activity also occurs during this period of the day. Females hovered about 30 cm over the water, flying down to deposit small groups of eggs (8-40 eggs) over the substrate without landing on it.




The number of instars is not known for most Brazilian black fly species, but most of the species for which this parameter has been analyzed have 7 instars (e.g., Alencar et al. 2001; Alvan-Aguiar & Hamada 2003). Simulium guianense in the Jacarei River in Piaui State had 6 instars, which is similar to some species in Argentina and in the USA (e.g., Ross & Merritt 1978; Coscaron-Arias & Bramardi 1996).

The association of S. guianense larvae and pupae with Podostemaceae has been reported in the literature (e.g., Gomes & Py-Daniel 2002). In our study, this association was also observed, but immatures were also found in high density on other substrates provided by riparian vegetation and other aquatic macrophytes. Of the 9 rivers in which S. guianense was collected in the study area, only 1 (the Longa River) had larvae and pupae in high density on bedrock. This is not a common substrate for this species and may indicate the scarcity of the other substrates, considering the observed high larval and pupal densities at this site. In a study in the state of Amazonas, Gomes & Py-Daniel (2002) attributed the use of submerged rocks and roots as substrates for S. guianense larvae and pupae to environmental changes due to the construction of a dam, which nearly caused the disappearance of Podostemaceae in their study area.








The water characteristics in the study area in the northeast region of Brazil were somewhat different from those in S. guianense habitats in Amazonian rivers (Gomes & Py-Daniel 2002). In the Amazon region, the water pH was acidic (5.4-6.7) and electrical conductivity was much lower (8.2-3.2 [micro]S/cm) than we observed in northeastern Brazil. However, water temperature was similar in both regions (27.8-30.0[degrees]C, in Amazonia) (Gomes & Py-Daniel 2002). These differences suggest either that these variables do not limit the populations of S. guianense or that this species is composed of sibling species with different habitat requirements. Molecular and cytotaxonomic studies of these populations will help to evaluate this problem.

During the sampling period, ants (Solenopsis sp.) preyed on larvae and pupae of S. guianense in the Longa River when the water level in the river fell rapidly, leaving them exposed. Ants in this genus have been observed preying on larvae and pupae of Simulium perflavum Roubaud in a similar situation in the Amazon region (Hamada 1998). Another consequence of the rapid loss of water by the system at the beginning of the dry season was the trapping of larvae in puddles in depressions in the bedrock. Simulium guianense may be subjected to a high mortality rate during this time of the year.

Simulium guianense females used the dabbing technique (Crosskey 1990), laying their eggs in groups without landing on the substrate, similar to the behavior of Simulium rorotaense Floch & Abonnence (Gorayeb 1981) and S. perflavum (Hamada 1998).

The present study extends the distribution of S. guianense to the states of Piaui and Ceara in the northeastern region of Brazil, in the Parnaiba River drainage. Previously, the only state in the northeastern region reported with this species was Maranhao, in the Tocantins River (a large tributary of the Amazonas River drainage) (Charalambous et al. 1996; Adler & Crosskey 2014).


We thank Ranyse B. Querino and Paulo V. Cruz for help with the field work, and RBQ for the photographs. This paper was written as part of the project "Diversidade criptica em Simulium (Thyrsopelma) (Diptera: Simuliidae) e relates entre suas especies" (CNPq, processo n. 475663/2012-8). CRSN received a fellowship from PCI/INPA/MCTI and NH is a CNPq research fellow. P. M. Fearnside reviewed the English.

References Cited

Adler PH, Crosskey RW. 2014. World blackflies (Diptera: Simuliidae): a comprehensive revision of the taxonomic and geographical inventory, http://www. (last accessed 15 Jan 2014).

Alencar YB, Hamada N, Magni-Darwich S. 2001. Morphometric comparison of Simulium perflavum larvae (Diptera: Simuliidae) in relation to season and gender in central Amazonia, Brazil. Memorias do Instituto Oswaldo Cruz 96: 785-789.

Alvan-Aguilar MA, Hamada N. 2003. Larval biometry of Simulium rubrithorax (Diptera: Simuliidae) and size comparison between populations in the states of Minas Gerais and Roraima, Brazil. Memorias do Instituto Oswaldo Cruz 98: 507-511.

Charalambous M, Shelley AJ, Herzog MM, Dias APAL. 1996. Four new cytotypes of the onchocerciasis vector blackfly Simulium guianens in Brazil. Medical and Veterinary Entomology 10: 111-120.

Coscaron-Arias CL, Bramardi SJ. 1996. Determinacion del numero de estadios de tres especies de Simuliidae (Diptera) del Parque Nacional Lanin (Neuquen, Argentina). Revista de la Sociedad Entomologica Argentina 55: 95-105.

Crosskey RW. 1990. The Natural History of Blackflies, p. 711. The Museum of Natural History Press, JS Wiley, London, United Kingdom.

Giulietti AM, Du Bocage Neta AL, Castro AAJF, Gamarra-Rojas CFL, Sampaio EVSB, Virginio JF, Queiroz LP, Figueiredo MA, Rodal MJN, Barbosa MRV, Harley RM. 2004. Diagnostico da vegetado nativa do bioma Caatinga, pp. 4896 In Silva JMC, Tabarelli M, Fonseca MT, Lins LV [eds.], Biodiversidade da Caatinga: areas e a$oes prioritarias para a conservado. Ministerio do Meio Ambiente, Brazil.

Gomes SAG, Py-Daniel V. 2002. Caracterizado fisicoquimica de dois criadouros de larvas de Thyrsopelma guianense (Wise, 1911) (Diptera, Culicomorpha, Simuliidae) da Amazonia brasileira. Entomologia y Vectores 9: 443-446.

Gomes SAG, Marques L, Py-Daniel V, Meira P. 2002. Caracterizado alimentar do ultimo estadio larval de Thyrsopelma guianense (Wise, 1911) (Diptera, Culicomorpha, Simuliidae) em duas cachoeiras da Amazonia brasileira. Entomologia y Vectores 9: 375-421.

Gorayeb IS. 1981. Comportamento de oviposicao e ciclo evolutivo de Simulium fulvinotum Cerq. e Mello, 1968 (Diptera, Nematocera). Acta Amazonica 11: 595-604.

Grillet ME, Basanez MG, Martinez SV, Villamizar N, Frontado H, Cortez J, Coronel P, Botto C. 2001. Human onchocerciasis in the Amazonian area of southern Venezuela: spatial and temporal variations in biting and parity rates of black fly (Diptera: Simuliidae) vectors. Journal of Medical Entomology 38: 520-530.

Hamada N. 1998. Bionomics of Simulium perflavum Roubaud (Diptera: Simuliidae) in Central Amazonia, Brazil. Revista Brasileira de Entomologia 41: 523-526.

Moreira GRP, Sato G. 1996. Blackfly oviposition on riparian vegetation of waterfalls in an Atlantic rain forest stream. Anais da Sociedade Entomologica do Brasil 25: 557-562.

Ross DH, Merritt RW. 1978. The larval instar and population dynamics of five species of black flies (Diptera: Simuliidae) and their responses to selected environmental factors. Canadian Journal of Zoology 56: 1633-1642.

Shelley AJ, Lowry CA, Maia-Herzog M, Luna-Dias APA, Moraes MAP. 1997. Biosystematic studies on the Simuliidae (Diptera) of the Amazonia onchocerciasis focus. Bulletin of the Natural History Museum, Entomology Series 66: 1-120.

Villamizar N, Cortez J, Alarcon ON, Escalona M, Botto C, Grillet ME. 2011. Primera descripcion del habitat acuatico de Simulium guianense s.l. (Diptera: Simuliidae) en el area endemica de oncocercosis, al sur de Venezuela. Boletin de Malariologia y Salud Ambiental 51: 97-101.

Claudio Rabelo dos Santos-Neto (1) *, Neusa Hamada (1), and Sheyla R. M. Couceiro

(1) Instituto Nacional de Pesquisas da Amazonia--INPA, Coordenado de Biodiversidade--CBIO, Laboratorio de Citotaxonomia e Insetos Aquaticos--LACIA, Avenida Andre Araujo 2936, CEP 69067-375, Manaus, AM, Brazil

(2) Universidade Federal do Oeste do Para, Instituto de Ciencias e Tecnologia das Aguas, Laboratorio de Ecologia de Ictioplancton e Invertebrados Aquaticos, Anexo ao Campus Amazonia Boulevard. Av. Mendonga Furtado, 2946, Fatima, CEP 68040-470, Santarem, PA, Brazil

* Corresponding author; E-mail:

Caption: Fig. 1. Simulium guianense larvae. First instar, arrows indicate the egg-burster (A and B); the last instar is characterized by the presence of a completely formed gill histoblast (C).

Caption: Fig. 2. Map of sampling sites with Simulium guianense in the northeast region of Brazil.

Caption: Fig. 3. Relationship between width of the frontoclypeal apotome (WFA), total body length (TL), and lateral length of the head capsule (LLH) of Simulium guianense larvae collected in the Jacarei River, Piracuruca Municipality, Piaui, Brazil, in Jun 2011.

Fig. 4. Histogram with lateral length of the head capsule (LLH) values to establish 4 (A), 5 (B), and 6 (C) intermediate instars of Simulium guianense larvae collected in the Jacarei River, Piracuruca Municipality, Piaui, Brazil, in Jun 2011.

Caption: Fig. 5. Evaluation of the number of instars of Simulium collected in the Jacarei River in the municipality of Piracuruca, Piaui, Brazil in Jun 2011. Instars are distinguished by the mean LLH (lateral length of the head capsule). Panel A has larvae grouped into 6 instars, Panel B into 7 instars, and Panel C into 8 instars. Vertical bars denote 95% confidence intervals and equal lowercase letters indicate lack of statistical difference between instars. The last 2 instars are statistically indistinguishable in Panels B and C, meaning that 6 instars (Panel A) represents the correct number.

Caption: Fig. 6. Ant (Solenopsis sp.), indicated by arrow, preying on larvae of Simulium guianense in the Longa River, Batalha Municipality, Piaui, Brazil, in Jun 2011.

Caption: Fig. 7. Pupae of Simulium guianense, indicated by arrow, on bedrock in the Longa River, Batalha Municipality, Piaui, Brazil, in Jun 2011.

Caption: Fig. 8. Larvae of Simulium guianense confined to puddles formed by depressions in the bedrock in the Longa River, Batalha Municipality, Piaui, Brazil, in Jun 2011.

Caption: Fig. 9. Female of Simulium guianense together with egg masses on a macrophyte, in the Jacarei River, Piracuruca Municipality, Piaui, Brazil, in Jun 2011.

Caption: Fig. 10. Group of eggs of Simulium guianense on a leaf of an aquatic macrophyte in the Jacarei River, Piracuruca Municipality, Piaui, Brazil, in Jun 2011.
Table 1. Sites where Simulium guianense (Diptera: Simuliidae) were
collected in Piaui (PI) and Ceara (CE) States, Brazil, and some
habitat characteristics of the sampled site.

State   Municipality,    River                 Water
         river name,     width
             and          (m)

                                 Temperature    pH    Conductivity
                                 ([degrees]C)         ([micro]S/cm)

PI      Batalha, Longa    70         29.6       7.3        71

PI      Batalha, river     8         29.8       7.6        204
        without name,

PI      Piracuruca,       10         28.1       6.6        48
        Cabra Bom

PI      Sao Joao da       13         28.0       7.3        216

PI      Brasileira,       40         29.5       7.2        169

PI      Piracuruca,       15         28.4       7.1        209
        Jacarei River,

PI      Sao Miguel do     20         29.4       7.8        98
        Tapuio, Sao
        Nicolau River,

PI      Aroases,          23         26.5       7.3        101
        Sambito River,

CE      Vinosa do         18         26.1       7.3        213
        Ceara, Pirangi
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Author:dos Santos-Neto, Claudio Rabelo; Hamada, Neusa; Couceiro, Sheyla R.M.
Publication:Florida Entomologist
Article Type:Report
Date:Jun 1, 2015
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