338 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 A B S T R A C T Aedes aegypti has overcome all kinds of mosquito control attempts over the last century. Strategies for population control resorts to the use of synthetic insecticides, which can lead to problems like human intoxication and environmental contamination. The effects of Bacillus thuringiensis var. israelensis (Bti), Ilex paraguariensis (yerba mate), and Ilex theezans (caúna herb) extracts against A. aegypti larvae were evaluated. The bioassays were conducted under controlled laboratory conditions of temperature (27 ± 3°C) and photoperiod (12 h). Hydroalcoholic extract of the leaves of I. theezans displayed better residual effect compared to the aqueous extract of I. paraguariensis fruits. The strongest residual effect of I. theezans was probably due to the presence of certain chemicals in its leaves, such as coumarins, hemolytic saponins, and cyanogenic glucosides, which were absent in I. paraguariensis. The results herein contributed to the prospection of natural insecticides and opened the possibility for subsequent studies on the use of plant extracts in field situations in a short-time scale. Keywords: dengue; vector control; inseticide; entomology; mate herb. R E S U M O Aedes aegypti superou todos os tipos de tentativas de controle do mosquito pelo homem no último século. Estratégias para controle populacional recorrem ao uso de inseticidas sintéticos, que podem levar a problemas como intoxicação humana e contaminação ambiental. Foram avaliados os efeitos de Bacillus thuringiensis var. israelensis (Bti), extratos de Ilex paraguariensis (erva-mate) e Ilex theezans (erva-caúna) contra a mortalidade de larvas de A. aegypti. Os bioensaios foram conduzidos sob condições laboratoriais controladas de temperatura (27 ± 3°C) e fotoperíodo (12 h). O extrato hidroalcoólico de folhas de I. theezans apresentou melhor efeito residual quando comparado ao extrato aquoso de frutos de I. paraguariensis. O efeito residual mais forte de I. theezans provavelmente ocorreu devido à presença de substâncias químicas em suas folhas, tais como cumarinas, saponinas hemolíticas e glicosídeos cianogênicos, ausentes em I. paraguariensis. Nossos resultados contribuíram para a prospecção de inseticidas naturais e abriram a possibilidade de estudos subsequentes sobre o uso de extratos vegetais em situações de campo em um curto espaço de tempo. Palavras-chave: dengue; controle vetorial; inseticida; entomologia; erva-mate. How long is long enough? Decreasing effects in Aedes aegypti larval mortality by plant extracts over time Quanto tempo é tempo suficiente? Redução dos efeitos na mortalidade larval de Aedes aegypti por extratos de plantas ao longo do tempo Gilberto Dinis Cozzer1 , Renan de Souza Rezende1 , Junir Antônio Lutinski1 , Walter Antônio Roman Júnior1 , Maria Assunta Busato1 , Daniel Albeny Simões2 1Universidade Comunitária da Região de Chapecó – Chapecó (SC), Brazil. 2BioVectors Soluções em Controle de Vetores – Chapecó (SC), Brazil. Correspondence address: Daniel Albeny Simões – Rua Marechal Borman, 317D – Centro – CEP: 89801-050 – Chapecó (SC), Brazil – E-mail: danielalbeny@gmail.com. Conflicts of interest: the authors declare there are no conflicts of interest. Funding source: Universidade Comunitária da Região de Chapecó. Received on: 05/25/2020. Accepted on: 10/27/2020. https://doi.org/10.5327/Z21769478806 Revista Brasileira de Ciências Ambientais Brazilian Journal of Environmental Sciences This is an open access article distributed under the terms of the Creative Commons license. Revista Brasileira de Ciências Ambientais Brazilian Journal of Environmental Sciences ISSN 2176-9478 Volume 56, Number 2, June 2021 http://orcid.org/0000-0003-4825-6032 http://orcid.org/0000-0002-4129-0863 http://orcid.org/0000-0003-0149-5415 http://orcid.org/0000-0001-8363-8795 http://orcid.org/0000-0003-0043-7037 http://orcid.org/0000-0002-8001-030X mailto:danielalbeny@gmail.com https://doi.org/10.5327/Z21769478806 http://www.rbciamb.com.br http://abes-dn.org.br/ How long is long enough? Decreasing effects in Aedes aegypti larval mortality by plant extracts over time 339 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 Introduction Over the last century, the mosquito Aedes aegypti (Linnaeus, 1762) has overcome all mosquito control attempts. A. aegypti fe- males are well known by their capacity of naturally and/or under laboratory conditions replicate and transmit over 100 kinds of vi- ruses (Weaver and Reisen, 2010). As an example, the viruses of Den- gue, Chikungunya, Zika, and, most recently, the Mayaro (Weaver and Reisen, 2010) virus can be listed in Brazil, which represent a real threat to public health (Lopes et  al., 2014). Therefore,  its medical importance requires the population control of this spe- cies to reduce virus transmission and, consequently, its epidemic status. Although several chemical and natural products have been extensively used on attempts to reduce the population of adults and larvae (Liu, 2015; Apaire-Marchais et al., 2016), adequate mosquito control is not even close to become true, especially due to the ge- netic resistance selectivity because of the incorrect use of natural products and chemicals (Sun et  al., 2019). As a consequence, the most effective disease prevention method still focus on targeting the mosquito population by eliminating mosquito breeding places (Brasil, 2002). A very promising field for reducing the mosquito population is to focus on mosquito control strategies that target immature aquatic stages, when the insect is more vulnerable (Brasil, 2001; Crivelenti et  al., 2010). For this purpose, the use of synthetic in- secticides is well known for its efficacy, causing mosquito larval mortality (Busato et  al., 2015; Govindarajan et  al., 2018). Howev- er, those chemicals might affect humans, resulting in intoxication and environmental contamination, and affecting biodiversity (Bu- sato et  al., 2015; Tahir et  al., 2015; Baskar et  al., 2018; Govindara- jan et al., 2018). Regarding the environment, the continuous use of synthetic insecticides may present undesirable effects, such as the long-term permanence in the environment, selection of resistant populations, and the appearance of new pests (Tahir et  al., 2015; Baskar et al., 2018; Govindarajan et al., 2018). As to human health, the presence of such synthetic chemicals on the environment can cause neurological damage and is associated with a wide range of symptoms, with significant deficits in the nervous system function (Araújo et al., 2007). Alternatively to the use of synthetic chemicals, biological con- trol plays an important role on mosquito management (Zara et al., 2016; Coelho et al., 2017). The use of bacteria spores as a mosquito larvicide has stood out among the several components that are part of mosquito-integrated management programs (Zara et  al., 2016). Over the last decade, the use of inactivated spores of the bacteria Bacillus thuringiensis var. israelensis (Bti), spread in the water of mosquito breeding places, has met the expected results, reaching mortality rates above 99% (Soares-da-Silva et  al., 2017; Nakazawa et  al., 2020). Additionally, during the last few years, plant-derived compounds have been extensively used as an alternative method for controlling mosquitoes, not only because this is a new insecticidal agent, but also because it has been described as being environmen- tally friendly (Gomes et  al., 2016; Guarda et  al., 2016; Knakiewicz et  al., 2016; Rosa et  al., 2016). The use of natural insecticides has some advantages over traditional synthetic products, because nat- ural products are potentially less toxic to the environment. Envi- ronmentally-friendly compounds are less concentrated, have fast- er degradation, and are specific to certain insect groups, resulting in less occupational exposure and less environmental pollution (Krinski et al., 2014). Ilex paraguariensis A. St.-Hil (Aquifoliaceae), known as mate, is an abundant plant, native of South America, 20 m tall, with a dense crown, and very branched (Souza, 2009). After processing, its leaves are traditionally used in a regional tea known as mate in Argenti- na, Brazil, Paraguay, and Uruguay (Souza, 2009). I. paraguariensis is commercially important due to the presence of caffeine and theobro- mine, both recognized as having a stimulant effect in the nervous and cardiocirculatory systems (Castaldelli et al., 2011). The described pharmacological activities for I. paraguariensis leaf extracts include antioxidant, hypolipidemic (Gao et  al., 2013; Messina et  al., 2015), and hypoglycemic effects (Conceição et  al., 2017). Besides that, Ilex theezans Mart. Ex Reissek (Aquifoliaceae), popularly known as caú- na-herb, is commonly found in Southern Brazil (Souza, 2009). It is well known due to the physiological characteristics of its leaves as an adulterant of I. paraguariensis (Athayde et al., 1999). It is an ever- green tree, early secondary or late secondary species (Souza, 2009), 20-m tall and 70-cm diameter, on average (Athayde et al., 1999). Both the I. paraguariensis fruit extract and the I. theezans hydroacoholic leaf extracts have larvicidal effect against A. aegypti larvae (Busato et al., 2015; Knakiewicz et al., 2016). Some studies showed that Ilex spp. leaves and fruit extracts kill A. aegypti larvae within a 24-h observational time (Busato et  al., 2015; Knakiewicz et al., 2016). However, there are no studies in the current literature evaluating the effects of time on the bioinsecticide lethal ac- tivity. How long is long enough for the bioinsecticide to maintain its ability to kill? (Resende and Gama, 2006; Santos et  al., 2007; Guira- do and Bicudo, 2009). In this context, the lethal residual effect of B. thuringiensis var. israelensis, and leaf and fruit extracts of I. theezans, and I. paraguariensis, respectively, against A. aegypti larvae were evalu- ated. Time would positively affect A. aegypti larvae survival due to de- cay of the lethal compounds, as a hypothesis, but the mortality caused by I. theezans was higher when compared to I. paraguariensis, due to the difference in physical and chemical characteristics. Material and Methods Animal source The A. aegypti larvae used in this experiment were provided by Laboratório de Entomologia Ecológica (LABENT-Eco). A filter paper Cozzer, G.D. et al. 340 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 holding about 300 eggs was placed in a plastic tray (30 × 20 cm) hold- ing 1 L of tap dechlorinated water. After hatching, the larvae were dis- tributed among three plastic trays of equal size and fed with 2 g of fish food. The mosquito larvae were raised for about 4-5 days until reaching 3rd and 4th instars. Plant source and extract preparation Fruits and leaves were obtained from native trees located at the Marechal Bormann district (27°19’05’’S; 52°65’11’’W), Chapecó City (Santa Catarina State), in December 2016. Plant parts were dehydrated at room temperature (± 20°C), pulverized in a knife mill (Cielamb®, CE 430), and stored away from light and humidity. Plant extracts were prepared according to Busato et al. (2015) and Knakiewicz et al. (2016). Samples of 20 g of I. paraguariensis dehydrated fruits and I. th- eezans leaves were used. Both samples were extracted by turbolysis, using 200 mL of distilled and deionized water and a hydroalcoholic solution (90% ethanol; 200 mL) as solvent, respectively (ANVISA, 2019). The  extracts were filtered in Büchner funnel, concentrated by rotavapor under reduced pressure, lyophilized, weighed, identified, and stored in a freezer at -20°C. Hydroalcoholic and aqueous extracts were prepared using I. theezans and I. paraguariensis leaves and fruits, respectively. Leaves at a concentration of 1,000μg/mL were used, and fruits were diluted to 2,000 μg/mL. B. thuringiensis var. israelensis (Bti), strain WG®, was used in a concentration of 0.004 g/L, the lethal dose specified by the manufacturer. Experimental microcosms and design Plastic cups of 300 mL with 100 mL of dechlorinated water plus the treatment proposed were adopted. In each individual sample, 20 3rd and 4th instar A. aegypti larvae were added. Each container was covered with a mosquito net held by a rubber elastic band. Tests for the effects of Bti spores; I. theezans, and I. paraguariensis leaves and fruits, respec- tively, were conducted; clean aged water (control) on the A. aegypti lar- val mortality after seven days of exposure was also performed (Naka- zawa et al., 2020). Before running the mortality test, each experimental treatment aged from one to eight weeks. Each week was considered as one age block with each treatment replicated six times. With  this ex- perimental design, the independence of each set of treatments was as- sured. The aged treatments were used to test for larval survival in each experimental week. At the end of the 7th day, larval survival was record- ed, with both pupae and emerged adults being considered as survivals. The experiment was performed for eight weeks (56 days) and carried out between April and May 2017 at the LABENT-Eco mosquito colo- ny room, under controlled conditions of temperature and photoperiod (27 ± 3°C, 12h D:L). Statistics Since both negative (Bti) and positive (tap water) control surviv- al rates were 0.16% and 100%, respectively, the data were analyzed in both ways, with (complete model) and without (simple model) these two categories. In order to evaluate differences in the percentage of lar- val mortality (response variable) between simple (I. paraguariensis and I. theezans) and the complete models (only water, Bti spores, I.  para- guariensis and I. theezans), regarding week (1 to 8) and week-treatment interaction (explicative variables), we used factorial GLM, with bino- mial correct to quasi-binomial (link = logit, test = Chi-square) distri- butions (Crawley, 2007). All analyzed GLMs were corrected for cases of under- or overdispersion. Differences among the categorical variables were assessed with a contrast analysis (Crawley, 2007). In this analysis (orthogonal), the dependent variables (different treatment and weeks) were ordered in- creasingly and tested pairwise (with the closest values); sequentially, adding to the model values with no differences and testing with the next values in a stepwise model simplification (for more details see also Chapter 9 of Crawley, 2007). All analyses were performed using the R program (Venables et al., 2019). Results The A. aegypti larval mortality was not affected by the water age (positive control). In contrast, the Bti resulted in the death of all the larvae until the age of seven weeks, with only 6.6% of lar- vae alive on the age of eight weeks (negative control). In this way, due to these extreme results in controls (0% of mortality in the positive control and 100% of mortality in the negative control), mortality data were analyzed only between treatments (I. para- guariensis and I. theezans). Larval mortality was significantly different between treatments (I. paraguariensis and I. theezans), weeks (1 to 8), and interaction fac- tors (week:treatment) for both GLMs models (with and without pos- itive and negative controls; Table 1). The highest larval mortality was found in the Bti treatment (negative control), followed by I. theezans, I. paraguariensis, and positive control (Table 1; Figure 1A). In addition, the I.  theezans hydroalcoholic leaf extract, regardless of extract’s age, killed significantly more A. aegypti larvae than the aqueous I. para- guariensis fruit extract (Table 1; Figure 1B). A positive relationship between the survival of the larvae and the plant extract age was observed. In general, both I. paraguariensis and I. theezans killed less (mainly after seven weeks) mosquito larvae as the plant extracts aged (Figure 2). A higher significant larval mortality was found in week 1, followed by weeks 2 and 3, weeks 4 and 6, week 5, and weeks 7 and 8 (Figures 1B and 1C). Residual deviance (estimate of the variance of the tested variables) in GLM with positive and negative controls, showed that differences in all treatments (74%) was the main responsible for larval mortality, followed by extracts’ age (18%; Table 1). On the other hand, residual deviance in GLM, without positive and negative controls, showed that differences between all weeks (68%) was the main responsible for larval mortality, followed by treatments (6%; Table 1). How long is long enough? Decreasing effects in Aedes aegypti larval mortality by plant extracts over time 341 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 Discussion Transformation of the larvicide effect into food resource over time I. theezans and I. paraguariensis extracts are promising against mosquito larvae (if applied and monitored in the first weeks). The po- tential of using these plant extracts as larvicides for A. aegypti may be an advantage, since they are natural extracts and do not leave toxic waste in the environment. These extracts are an abundant and acces- sible alternative in Southern Brazil, where A. aegypti infestation and dengue cases have been observed in the last decade (Busato et  al., 2015). However, extracts’ age should be considered; the main objec- tive of the present study is not to discourage the use of such alter- native method, but to warn about the importance of extract aging before using it for mosquito-control purposes. Better results may also be obtained with the development of additional studies, evaluating the larvicidal activity of pure compounds isolated from these plants. Furthermore, better results might be obtained by evaluating if there is a supporting effect of more than one active principle with larvicidal action against A. aegypti. Plant extracts may degrade as time goes by, and these organic compounds with previous larvicidal activity may become food for A. aegypti larvae (explaining the residual deviance percentage in GLMs models). The transformation of larvicides into food probably took place, especially in those treatments with seven- and eight-weeks old plant extracts, which presented the highest survival rate. Therefore, the age of plant extract should be considered (Albeny-Simões et al., 2015). Aedes aegypti larval mortality between plant extracts Plant extract age plays an important role on mosquito larvae mortality (mainly with positive and negative controls). Despite the plant species, plant parts, and extraction method, mosquito larval mortality decreases with the aging of plant extracts. However, the ex- tracts tested were highly efficient in the first weeks of the experiment (high mortality). The higher larval mortality found in the I. theezans extracts can be partially explained by the use of solvents during the extraction process (Lee and Houghton, 2005). The hydroalcoholic ex- traction method used to obtain the I. theezans extracts removed low polarity chemical constituents from plant tissues, and these mole- cules have a higher ability to penetrate mosquitoes’ larvae cells and modify their metabolic activities. On the other hand, aqueous extraction, used for the I. paraguarien- sis fruits, preferentially removes high-polarity chemical compounds, which are not able to easily penetrate such cells (Lee and Houghton, 2005). Moreover, the susceptibility of A. aegypti larvae to I. theezans may be explained by the presence of secondary metabolites of the cou- marin class and absence of alkaloids when compared to I. paraguarien- sis (Valduga et al., 1997). Coumarins are part of the secondary metab- olism of several plants, being well known for presenting insecticidal activities, acting as a repellent of adult insects, preventing oviposition, impairing feeding and growth, promoting morphogenetic and hor- monal system alterations, sexual behavior changes, and adult steriliza- tion, among other effects (Dietrich et al., 2011). Furthermore, I. para- guariensis has a higher content of caffeoyl derivatives and flavonoids than I. theezans. Flavonoids are recognized for their potent larvicidal activity, which may partially explain the results obtained herein (Filip et al., 2001; Garcez et al., 2013). In raw plant extracts, the active constit- uents are usually found in small concentrations (Krinski et al., 2014). Larvae mortality and extract age of Aedes aegypti In both I. theezans and I. paraguariensis, the mortality of A. aegypti larvae exposed to one-week plant extracts was 100%. Table 1 – Generalized linear models (GLM), degrees of freedom (Df ), Residual Deviance (total and in), and p values, comparing the percentage of Aedes aegypti larvae mortality after exposure to treatments (water control, Bacillus thuringiensis israelensis — Bti, hydroalcoholic dried leaves extract of Ilex theezans, and aqueous Ilex paraguariensis fruits extract), time (8 weeks) and interaction among treatments and weeks, under laboratory conditions. GLM Df Resid. Dev. Resid. Dev. % Pr(>Chi) Analysis of contrast a. With positive and negative controls Treatments 3 132.9 74.1 < 0.001 Control < Ilex paraguariensis < Ilex theezans < Bti Weeks 7 33.4 18.6 < 0.001 Week 8 = 7 < 5 < 4 = 6 < 3 = 2 < 1 Treatment: weeks 21 3.0 1.7 < 0.001 Residual 160 10.0 5.6 b. Without positive and negative controls Treatments 1 3.1 6.6 < 0.001 Ilex paraguariensis < Ilex theezans Weeks 7 32.3 68.7 < 0.001 Week 8 = 7 < 5 < 4 = 6 < 3 = 2 < 1 Treatment:weeks 7 2.4 5.1 0.002   Residual 80 9.2 19.6 Cozzer, G.D. et al. 342 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 However, the plant extracts for both species reduced the mor- tality of mosquito larvae as the plant extracts aged. These results pointed out the need for carefully selecting the right age for an Ilex spp. plant extract before using it to control mosquito larvae. This is especially true since a product is considered efficient for pest population control when it reduces individuals above 80%, otherwise resistance genes are selected (Jagadeesan et al., 2016). In addition, besides the decaying of lethal chemical compounds which were toxic for mosquito larvae on the first week, as time goes by, the organic compounds present on the plant extracts may act as a useful food source for mosquito larvae. In this way, since organic compounds are well known for being an important component of sev- eral larval habitats, forming the basis of many food webs (Merritt et al., 1992; Moore et  al., 2004), microorganisms such as bacteria play an important role in the cycling and breaking of large organic molecules (Sinsabaugh and Linkins, 1990). Therefore, microorganisms may act making them more easily absorbed by aquatic organisms, such as mos- quito larvae, especially those belonging to the Culicidae family (Merritt et al., 1992). As a result, decomposing microbial communities present a relevant contribution to the diet of culicid larvae, being ingested with the organic remains over time (Merritt et  al., 1992; Cochran-Stafira; Von Ende, 1998; Kaufman et al., 1999; Eisenberg et al., 2000). The Bti of the present study resulted in 93.4% mortality in the eight-week solution. The residual effect described in the technical manual of the manufacturer is 30 days. Moreover, the values obtained herein were much higher than those described in the literature, with a lethality of 100% for 49 days and 6.6% of larval survival in up to 56  days. In this way, evaluating the effectiveness of the products that are already being used by state programs to control and combat A. ae- gypti could be performed. The use of the methodology without water renewal in the experiment resulted in a longer residual effect. Thus, not evaluating the effect of this renewal in reducing the residual effect of larvicidal substances (Pontes et  al., 2005). Other studies in the field should encourage this water renewal by constantly emptying and re- placing water, which would probably contribute to the reduction of the residual effect for all treatments tested. Conclusions The present study emphasized the need to implement alternative methods for vector control, since they represent a long-term risk. Fur- thermore, in the short term, it reported potential alternative pathways for mosquito-population control using natural products originated from the native flora. The plants presented a high larvicidal effect against A. aegypti, contributing to the maintenance of the quality of life and well-being of the population, since they are easily accessed by the local populations, reducing public expenditure with vector control and treat- ment of confirmed cases of dengue. Finally, time positively affected the survival of A. aegypti larvae due to the decay of lethal compounds from plant extracts, corroborating our first hypothesis. We also found that mortality by B. thuringiensis var. israelensis was constant throughout the experimental period and A. aegypti larvae survival was lower in the treatments with plant extracts than with B. thuringiensis var. israelensis. High mortality was observed in extracts of I. theezans compared to I. *Different letters (“a”, “b”, “c”, “d”, and “e”) indicate significant differen- ces. Boxes represent the quartiles, bold line represents the median, horizontal dashed line represents the mean, vertical dashed line repre- sents the upper and lower limits, and circles represent the outliers. Figure 1 – (A) Aedes aegypti larvae mortality among treatments, (B) sample weeks with positive and negative controls, and (C) sample weeks without positive and negative controls among them*. How long is long enough? Decreasing effects in Aedes aegypti larval mortality by plant extracts over time 343 RBCIAMB | v.56 | n.2 | Jun 2021 | 338-345 - ISSN 2176-9478 Contribution of authors: Cozzer, G.D.: Investigation, Data Curation, Writing – original draft. Rezende, R.S.: Software, Formal Analysis, Writing – review & editing. Lutinski, J.A.: Conceptualization, Methodology, Supervision, Writing – review & editing. Roman Júnior, W.A.: Methodology, Writing – review & editing. Busato, M.A.: Writing – review & editing. Simões, D.A.: Supervision, Formal Analysis, Funding Acquisition, Writing – review & editing. References Agência Nacional de Vigilância Sanitária (ANVISA). 2019. Farmacopeia Brasileira. 6. ed. Anvisa, Brasília, 874 pp. Albeny-Simões, D.; Murrell, E. G.; Vilela, E. F.; Juliano, S. A., 2015. A multifaceted trophic cascade in a detritus-based system. Ecosphere, v. 6, (3), 32. https://doi.org/10.1890/es14-00365.1. 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Efeito da erva mate ( Ilex paraguariensis a . st . -hill) no comportamento e fisiologia de ratos Wistar. Revista Brasileira de Biociências, v. 9, (4), 514-519. *Significant statistical difference between I. paraguariensis and I. theezans affecting larvae survival. Figure 2 – A. aegypti larvae survival’s mean as a function of plant extracts’ age. Extracts’ age are represented by weeks. The circles represent, aqueous I. paraguariensis fruits extract (open) and I. theezans hydroalcoholic leaves extract (closed). paraguariensis, corroborating our second hypothesis. The strongest re- sidual effect of I. theezans was probably due to the presence of chemi- cals on their leaves, such as coumarins, hemolytic saponins, and cyano- genic glucosides, which were absent in I. paraguariensis. 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