Susceptibility of Simuliidae (Diptera) larvae to a new Bti-based solid formulation in Cascavel, BrazilABSTRACT
Due to the hematophagous habits of females, some species of black fly (Diptera: Simuliidae) cause economic losses and health damages to humans and livestock. Several bioinsecticides based on the bacterium Bacillus thuringiensis var. israelensis (Bti) have been widely used to control the populations of these insects. This study evaluates the susceptibility of black fly larvae to a new Bti solid effervescent formulation, comparing it with a commercial standard formulation, Vectobac® liquid formulation, under laboratory conditions. Also, the diversity of Simuliidae, and aspects of the water quality, were analyzed from streams in the municipality of Cascavel, state of Paraná, Brazil. Larvae were collected from streams in the urban and rural areas of Cascavel. In laboratory, larvae were exposed of the Bti formulations. Eighteen hours after application of the formulations, larval mortality was evaluated. The control group did not exceed 20% mortality, for concentrations of 50 and 60 mg/L, the mean mortality rates were 50.6% and 64.2%, respectively, and neither differed significantly from the Vectobac®. Samples from streams showed external fecal contamination during the eight weeks of monitoring, and water quality parameters that could interfere with the efficacy of Bti application at the site. The following species of black flies were collected and identified in the two watersheds sampled: Simulium pertinax, S. subpallidum, S. nigrimanum, S. rubrithorax, S. perflavum, and S. inaequale. The bioecological aspects of the breeding site were investigated and are presented here. This is the first report of black fly species for the municipality of Cascavel. The potential of the solid effervescent formulation was proven under laboratory conditions and the bioecological evaluations demonstrated the relevance of environmental diagnosis and monitoring in the optimization of control protocols for Simuliidae.
KEYWORDS:
Black flies; biological control; entomopathogenic bacteria; entomological monitoring; tropical streams
INTRODUCTION
Black flies (Diptera: Simuliidae) are considered cosmopolitan (Duknić et al. 2019) and the immature stages have several morphological and physiological adaptations that allow them to develop in lotic ecosystems (Adler and Mccredie 2019). The larvae are key organisms in those ecosystems, due to their ability to process dissolved organic matter, making it more readily available in the trophic chain (Malmqvist et al. 2004). On the downside, adults bite and that can trigger irritation and allergic conditions in humans, leading to decreased well-being and economic losses to tourism and farming (Sá and Maia-Herzog 2003). In addition, some species are vectors of several human and animal diseases (Crosskey 1990). In Brazil, these insects transmit onchocerciasis and mansonellosis to humans (Shelley et al. 2010).
High densities of black fly populations often result from environmental problems, such as loss of biodiversity, reduction in the number of natural predators, and high levels of organic matter present in anthropized and polluted streams. This excess of organic load in lotic environments may be a predisposing factor for the reduction in black fly diversity, which can also increase densities of singular species, for example the anthropophilic Simulium pertinax Kollar, 1832 (Strieder et al. 2006).
Faced with the problem of using chemical insecticides to control black fly populations, integrated management protocols containing Bacillus thuringiensis var. israelensis Berliner, 1911 (Bti) are currently used. This bacterium, which can be found naturally in the soil of different regions of the world, has a parasporal body (crystal) composed of proteins that, when ingested by the black fly larvae, has the potential to destroy midgut epithelial cells, leading to sepsis and death (Bravo et al. 2007). The Bti-based bioinsecticides available in Brazil are imported and demand high investment from the consumer. Therefore, the development of national formulas, with a low acquisition cost when compared to imported products, would optimize their use in integrated management programs for black fly control (Angelo et al. 2010).
Efficient pest control also requires knowledge of the environmental conditions of the treated site. The water needs to be treated in intervals, since black fly larvae upstream of the treatment area will recolonize treated sections downstream (Docile et al. 2015). Indiscriminate treatments can lead to local extinction of black fly populations (Cheke et al. 2008), while not following recommendations may lead to product waste (Rivers-Moore et al. 2008).
Local production and obtaining national formulas, which are cheaper when compared to imported products, would optimize their use in integrated management programs in the control of black flies (Bhumiratana 1990, Ampofo 1995). The use of solid presentations for the control of black flies, presented in this work, is innovative, and aims to facilitate product application, especially in areas of difficult access. We envision the possibility of obtaining an effervescent product that can be applied directly to the stream by the very people who are affected by these flies (Becker 2000). Effervescent tablets based on Bti (Culinex®), have been used since 1992 with efficacy against Culex pipiens Linnaeus, 1758.
This study evaluates the susceptibility of the larvae of Simulium to an effervescent Bti formulation, under labo ratory conditions, and correlates it with the bioecological aspects of the breeding sites. In addition, we also describe the bioecological aspects of breeding sites in the streams of Cascavel, state of Paraná, Brazil and the species of Simulium found in the evaluated basins. Although there are records of Simulium spp. for the state of Paraná (Lozovei 2004), in the municipality of Cascavel there are no species’ records.
MATERIAL AND METHODS
Larvae and water river collection
Larvae collections were carried out with authorization #73993-1 from the Chico Mendes Institute for Biodiversity Conservation, Biodiversity Authorization and Information System - SISBIO, Ministry of the Environment, Brazil.
The insects were maintained in the laboratory in a controlled room (25 ± 2 °C), with permanent aeration maintained by two-outlet air compressors (Boyu®, SC-7500, 2 x 3 L/min, 127 V).
Bioinsecticide
To produce the bioinsecticide, the Bacillus thuringiensis var. israelensis (Bti) strain BR101 was used, from the Entomopathogenic Bacteria Bank of the Laboratory of Bacterial Genetics and Taxonomy, Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, Paraná.
Cultures of Bacillus thuringiensis strain BR101 were performed in a bioreactor containing 5 L of NYSM medium (Nutrient Yeast Extract Salt Medium) and maintained at 30 °C ± 2,300 rpm, pH 7.0, with constant air supply, for about 40 hours, until sporulation. Subsequently, the fermentation broth was centrifuged, the supernatant discarded and the final suspension was lyophilized.
Formulations
The effervescent solid dosage forms were prepared at the Laboratory of Chemical Ecology and Synthesis of Natural Products and Pharmaceutical Technological Laboratory of the Universidade Federal do Paraná, Curitiba, Paraná, Brazil. They were prepared by direct compression with a manual hydraulic press (Maxx press, Essence dental-SP), using a flat and circular punch, with a diameter of 11 mm. The compression force was standardized at 500 kgf for 20 seconds until the desired pressure was reached, and then maintained for another 10 seconds with 500 kgf.
Seven formulations were prepared (Table 1), keeping constant the presence of the effervescent mixture (citric acid, sodium bicarbonate, and sodium carbonate) as disintegrant, sucrose as soluble diluent, and powdered sodium lauryl sulfate as wetting/disintegrant. Corn starch was used as a diluent in varying concentrations to adjust the final mass of the tablet, established at 250 mg. The lyophilized solid containing the Bti protein crystals was used in increasing amounts (Table 1). The materials were weighed and hand mixed with low friction (plastic bag). After this process, compression was carried out, weighing the mass individually, using 250 mg of the corresponding mixture for each tablet produced.
Effervescent mix
The effervescent mixture was prepared at the time of final mixing (Prista et al. 1967); for every 10 parts of citric acid, 12 parts of sodium bicarbonate and 7.55 parts of sodium carbonate were used.
Standard formulation
The commercial product Vectobac® 12 AS (liquid formulation) with 1,200 UTI/mg (Lot 301-886-N930, expiration date: May, 2021), was used as a comparison standard for the test formulations. The suggested concentration for the product is from 0.5 to 25 ppm/L for one minute of exposure. However, in the tests, the amount of 0.2 mL/0.5 L (400 ppm) was stipulated as standard. This concentration was defined above that recommended by the manufacturer, considering the ease of application, and thus avoiding the need for serial dilutions. In addition, the product was also used with the objective of validating the applied methodology, by evaluating the response to the presence or absence of mortality due to Bti, with no intention of testing the potency of the commercial product.
Bioassays
The bioassays were carried out in laboratory, based on Araújo-Coutinho et al. (2005), with modifications. Larvae were placed in plastic containers with water collected directly from where they were collected. Active searches for larvae, to perform the bioassay for the evaluation of larvicides, were carried out in seven streams in the urban and rural areas of Cascavel.
Among all the sample points studied, the Santa Rosa stream was selected for the bioassays, as it has more blackfly larvae and was not being treated with bioinsecticides or other products to control them. This stream is in the Sede Alvorada district, a rural area 23 km from the municipality of Cascavel. It is 7 km long and is a tributary of the São Francisco Verdadeiro river (24°49’15.9”S; 53°37’51.2”W). Only larvae collected on the same day of the test were used, to minimize the effect of laboratory time on their lifespan.
In the laboratory, the larvae were evaluated for motili ty and viability. Larvae that had dark gill histoblasts were avoided, because it indicates that they are in their last stages. The specimens were transferred to plastic containers filled with 400 ml of water from the collection point, individually and uninterruptedly oxygenated with an aerator compressor. After an acclimatization period of three hours, the larvae that had not been set were discarded and replaced on-site, ensuring that only healthy larvae were evaluated (Lacey 1997). Because of the source of the water, no external nutritional compounds were applied. The water temperature during collection, transport, and the experiment was maintained at between 20 and 25 °C.
For the application of the solid formulations, the tablets were diluted in 100 mL of water from the river itself. The mixture was applied to the respective recipients and one minute was allowed for the larvae to ingest the Bti. Subsequently, the same procedures described for the formulated commercial liquid were adopted. For each of the test products and for the control, three cups were prepared, each considered a repetition. The mortality of 20% of the control group was accepted as a cut-off point, since the larvae are sensitive to the process of collection, handling, transport, and the laboratory environment (Lacey 1997).
Data analysis
All bioassays were conducted in a completely randomized design. Larvae mortality data were submitted to analysis of variance and the Shapiro-Wilk (p < 0.05) resi dual normality test. The homogeneity of variance was then evalua ted using the Bartlett test (p < 0.05) and the means were compared using the Tukey test (p < 0.05).
Taxonomic identification
A representative sample of the material collected for the bioassay was sent to the Laboratory of Simulids and Onchocerciasis of the Instituto Oswaldo Cruz (LSO/IOC), for taxonomic identification.
Characterization of the bioecological parameters of the selected collection point
To assess the water quality parameters at the point of the bioassay, data collections were carried out to analyze the physical-chemical and microbiological characteristics of the water, weekly from October 3, 2021 to April 27, 2021. These data were analyzed at the Fundação para o Desenvolvimento Científico e Tecnológico (FUNDETEC - Cascavel).
The physicochemical parameters analyzed were the potential of Hydrogen (pH), electrical conductivity, turbidity, total solids, nitrite, nitrate, and phosphate. The microbiologi cal analysis investigated the presence of total coliforms and Escherichia coli (Escherich, 1885). Water temperature, air temperature, and relative air humidity were observed at the time of water collection. To verify the integrity of the stretch, the Protocol for the Rapid Assessment of Habitat Diversity in stretches of hydrographic basins (PAR) by Callisto et al. (2002), was applied on site. This protocol aims to assess the conditions observed in the basin stretch, anthropic influence, habitat conditions, and level of conservation, assigning a score for each characteristic observed in the location. The sum of points represents whether the stretch is impacted (0 to 40 points), altered (41 to 60 points), or natural (above 60 points).
RESULTS
Bioassay
The concentration of 50 mg/L presented a mean mortality of 50.6%, which did not differ significantly from the commercial standard formulation, or from the concentration of 40 mg/L. When the concentration was raised to 60 mg/L, significant mortality was observed, with an average of 64.2%, not significantly differing from the standard formulation. The commercial standard formula presented a mean mortality of 75.4% after the evaluation time of 18 hours (p < 0.05) (Table 2). Concentrations of 4 mg/L, 5 mg/L, and 10 mg/L were not associated with significant mortality.
Water parameters and physical integrity of the collection site
Weekly oscillations were observed in the parameters evaluated, mainly in the amount of Total Solids, with a minimum value of 72.1 and maximum of 423.9 ppm (CV% = 70.8) and in the parameters of Nitrate, from 0.9 to 1.6 ppm (CV% = 16.8) (Table 3). The presence of total coliforms and Escherichia coli was observed in all collections performed.
In the investigated stretch of the Santa Rosa stream, the Protocol for the Rapid Assessment of Habitat Diversity in stretches of hydrographic basins (Callisto et al. 2002) indicated that the stretch can be characterized as a natural environment.
Taxonomic identification
Altogether, 22 exuviae, 55 pupae, ten adults, 42 mature larvae, and 299 immature larvae were collected and examined. Six species belonging to the genus Simulium Latreille, 1802 were identified and distributed in five subgenera: Simulium (Chirostilbia) Enderlein, 1921 (two species), S. (Hemicnetha) Enderlein, 1934 (one species), S. (Psaroniocompsa) Enderlein, 1934 (one species) S. (Psilopelmia) Enderlein, 1934 (one species), and S. (Trichodagmia) Enderlein, 1934 (one species) (Table 4).
At points 1 and 2, only two species were found, whereas at Point 3 the six species identified in this study were present, this being consequently the point of greatest diversity (Table 4). However, this may have been due to the greater number of samples collected at this location.
DISCUSSION
Larvicidal activity of bioinsecticides
The exposure time of one minute was associated with high concentrations of Bti. However, total larval mortality was not observed in any of the treatments. This likely means that one minute did not provide long enough exposure. Histological alterations in the midgut epithelium were observed in S. pertinax larvae after 1 hour of Bti exposure (6 mg/L), in addition to total larval mortality (Cavados et al. 2004). However, this exposure time is much longer than usual for field applications.
Water parameters and physical integrity of the collection site
Regarding water parameters, in an experiment conducted by Wilson et al. (2005), the best mortality results of a Bti-based product against Simulium damnosum Theobald, 1903 larvae were observed when the temperature increased. According to the authors, temperature influences metabolism and, consequently, greater ingestion of toxins by larvae. Atwood et al. (1992), in an experiment conducted with larvae of Cnephia pecuarum (Riley, 1887), observed a reduction in larval mortality in response to Bti when the water temperature decreased, recommending that the control be carried out before the temperature drops below 9 °C. This recommendation could be applied to the Santa Rosa stream, especially in the winter: Bti should only be applied if the water temperature is above 9 °C.
Wilson et al. (2005) found that the high conductivity of the water provided better larvicidal performance of the product, obtaining the best result in the conductivity of 323 mS (millisiemens). This number is much higher than that observed in the Santa Rosa stream, with an average conductivity of 16.2 to 19.1 µS/cm. According to the authors, most streams do not have such high conductivities under natural conditions, and this parameter should not be a decisive factor in the use of the product.
For the turbidity parameter, Wilson et al. (2005) observed better responses to Bti when the water turbidity was below 4.2 Nephelometric Turbidity Units (NTU), and recommended that, for the operational use of the product, turbid water should be avoided. Therefore, if a simulid control protocol was applied on the evaluated stream, it would be important to monitor the water quality and rainfall, comparing the information with the larvicidal efficiency of the product, since the water parameters oscillated due to rainfall and discharges of organic matter.
In the Santa Rosa stream, where S. pertinax was identified, the mean variation in nitrate was 0.9 to 1.6 ppm. This is similar to the results of a study carried out in an impacted basin in southern Brazil, where a correlation was found between the occurrence of S. pertinax and nitrate concentrations from 1.0 mg/L to 1.75 mg/L. These concentrations indicate greater organic pollution and environmental degra dation and are typical of poultry and swine production sites (Strieder. 2006).
The averages of the variables pH, turbidity, total solids, nitrite, and nitrate in the water of the Santa Rosa Stream were framed in CONAMA (2005) Resolution #357, of March 17, 2005, as values observed for Class 1 of freshwater quality standards (which is the best classification standard and can be used for human consumption after disinfection). However, the microbiological analysis indicated external fecal contamination during the eight weeks of monitoring, which are likely associated with the fact that cattle and other animals are raised in nearby properties. It is also believed that external contamination may be the source of organic matter for the maintenance of immature breeding sites in the stream.
Although the Protocol for the Rapid Assessment of the Diversity of Habitats in stretches of hydrographic basins (PAR) presented 66 points, which is configured as a “natural” stretch, several alterations were diagnosed in the stream, including the presence of contamination by thermotolerant coliforms, erosion, and deforestation. These alterations, if they persist, may change the score of the section in the future, considering that it is already very close to being classified as an altered environment (60 points). Therefore, the association between loss of water and decreased quality of the water, and the presence of breeding sites, may indicate that the community needs to get involved and take responsibility for the health of their environment.
Considerations on Taxonomic Identification
Among the identified species, some stand out for their medical, veterinary, or socioeconomic importance. In particular, Simulium nigrimanum Macquart, 1838 is an anthropophilic species that has been considered a potential vector of onchocerciasis in Minaçu, Goiás (Shelley et al. 2010). Additionally, it has been associated with the development of the human autoimmune disease known as pemphigus foliaceus, which is characterized by extensive bullous lesions and cutaneous erosions (Eaton et al. 1998, Shelley et al. 2010).
The females of Simulium inaequale (Paterson & Shannon, 1927) are highly anthropophilic, however, they have also been recorded biting mules (Shelley et al. 2010). Simulium pertinax is the most common anthropophilic species in southern Brazil and has also been recorded biting horses in Brazil and cows in Bolivia (Shelley et al. 2010). This species can be found at high densities in anthropic streams, being the main target in several simulid control programs. The presence of this species at Point 3 may be associated with the variations in nitrate (Table 1), which are consistent with the results of Strieder et al. (2006), who found a significant association between nitrate concentration and the presence of S. pertinax.
Furthermore, females of Simulium perflavum (Roubaud, 1906), Simulium subpallidum Lutz, 1910, and Simulium rubrithorax Lutz, 1909 are zoophilic, the latter having been recorded feeding on horses and probably cattle (Coscarón and Coscarón-Arias 2007, Shelley et al. 2010). The feeding habit of S. rubrithorax is compatible with the place where it was collected, Point 3, since the presence of farm animals, such as cattle, was observed in nearby properties.
Although simulids are widely known for their nuisance potential, their larvae are key organisms in aquatic ecosystems. They are an important source of food for vertebrate and invertebrate organisms, and, due to their ability to process dissolved organic matter, they make it more readily available in the food chain (Malmqvist et al. 2004). In addition, numerous species that do not interfere with humans or their activities are also affected by control methods, reinforcing the need for previous knowledge of the biodiversity of species existing in target locations of control, in order to subsidize sustainable and efficient actions (Malmqvist et al. 2004).
This is the first record of Simuliidae for the municipality of Cascavel and considering that the collections were carried out in only three fragments of lotic environments, and only two of the three hydrographic basins, it is believed that the diversity of species in the urban and rural areas of the city may be greater.
Final remarks
The innovative use of a solid Bti compound for black fly control, presented in this work, aims to facilitate the process of product application, especially in areas of difficult access, envisioning the possibility of obtaining an effervescent product that can be applied directly to the water without the need for prior dilution. The data obtained indicate the larvicidal potential of this new effervescent formulation, with proven efficacy in the laboratory.
The present work also demonstrates that in the study area there is biodiversity of black fly fauna, of human and veterinary health importance, associated with urban and rural streams. The bioecological assessments reinforce the relevance of environmental diagnosis and monitoring in the optimization of Simuliidae control protocols.
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ADDITIONAL NOTES
- ZooBank register
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How to cite this article
Lis PC, Alves LFA, Zequi JAC, Cardoso PF, Vilas-Bôas GT, Marques FA, Schorr RR, Andreazza IF, Molina ÓS (2025) Susceptibility of Simuliidae (Diptera) larvae to a new Bti-based solid formulation in Cascavel, Brazil. Zoologia 42: e24020. https://doi.org/10.1590/S1984-4689.v42.e24020
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Published by
Sociedade Brasileira de Zoologia at Scientific Electronic Library Online - https://www.scielo.br/zool
Publication Dates
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Publication in this collection
21 Feb 2025 -
Date of issue
2025
History
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Received
07 Mar 2024 -
Accepted
06 Sept 2024
