Abstract

Objective:

to evaluate, under simulated field conditions, the efficacy of pyriproxyfen (juvenile hormone), novaluron (chitin inhibitor) and spinosad (biolarvicide) in controlling Aedes aegypti.

Methods:

periodic exposition of Ae. aegypti larvae collected in Itabuna, BA, Brazil, to recipients treated with larvicides and comparison of residual effect of treatment with the Rockefeller strain.

Results:

the inhibitory effect on adult emergence after 60 days was spinosad 89.5%, novaluron 96.5% and pyriproxifen 75.4% for Itabuna larvae, with no statistical difference (p=0.412) between treatments; spinosad and novaluron had a higher percentage of mortality in the larval stage, 98.8% and 97.9% respectively; pyriproxyfen showed higher mortality (95.1%) in the pupal stage.

Conclusion:

the three larvicides demonstrated similar control; however, pyriprofyxen might give a false impression of breeding ground positivity as it acts at the pupal stage, compromising the indicators of infestation that are strategic parameters for control actions.

Keywords:
Aedes; Arbovirus Infections; Control; Larvicides

Introduction

In several countries worldwide, the so-called arboviruses have been a cause for great concern with regard to Public Health. In Brazil, apart from the four dengue virus (DENV) serotypes in circulation, the population is at risk of infection by the chikungunya virus (CHIKV), the Zika virus (ZIKV) and the yellow fever virus (YFV), whereby the latter virus is capable of affecting unimmunized individuals.¹1. Donalisio MR, Freitas ARR, Von Zuben APB. Arboviroses emergentes no Brasil: desafios para a clínica e implicações para a saúde pública. Rev Saúde Pública [Internet]. 2017 mar [citado 2018 dez 4];51:30. Disponível em: Disponível em: http://www.scielo.br/pdf/rsp/v51/pt_0034-8910-rsp-S1518-87872017051006889.pdf . doi: 10.1590/S1518-8787.2017051006889
http://www.scielo.br/pdf/rsp/v51/pt_0034...

According to the Ministry of Health’s epidemiological bulletin, in 2017 a total of 249,056 suspected dengue cases were recorded in Brazil. The Northeast region had the highest number of probable cases - 86.110 -, in relation to the national total.²2. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Monitoramento dos casos de dengue, febre de chikungunya e febre pelo vírus Zika até a Semana Epidemiológica 50. Bol Epidemiológico [Internet]. 2017 [citado 2018 dez 4];48(45). Disponível em: Disponível em: http://portalarquivos2.saude.gov.br/images/pdf/2018/janeiro/10/2017-046-Publicacao.pdf
http://portalarquivos2.saude.gov.br/imag... Out of all municipalities in the state of Bahia, 277 (66.4%) reported 9,736 suspected dengue cases, 515 of which were registered by the municipality of Itabuna.³3. Secretaria de Saúde (Bahia). Diretoria de Vigilância Epidemiológica do Estado da Bahia. Situação epidemiológica das arboviroses na Bahia: 2015-2016. Bol Epidemiológico [Internet]. 2016 jan [citado 2018 dez 4];12. Disponível em: Disponível em: http://www.blogdogusmao.com.br/v1/wp-content/uploads/2016/02/Boletim-epidemiológico-nº-12-dengue_chikungunya_zika.pdf
http://www.blogdogusmao.com.br/v1/wp-con...

Controlling the incidence of the arboviruses referred to above is based on the only vulnerable link in their transmission chain: vector mosquitoes. Among the vectors involved in transmission, the most relevant species is Aedes aegypti, which has a homometabolous life cycle with aquatic larvae and pupae and winged adults.⁴4. Tauil PL. O desafio do controle do Aedes aegypti e da assistência adequada ao dengue. Epidemiol Serv Saúde [Internet]. 2007 jul-set [citado 2018 dez 4];16(3):153-4. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000300001 . doi: 10.5123/S1679-49742007000300001
http://scielo.iec.gov.br/scielo.php?scri... In essence, Ae. aegypti control actions are aimed at the immature forms (larvae and pupae), with the use of larvicides aimed at reducing infestation rates.⁵5. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde [Internet]. 2007 out-dez [citado 2018 dez 4];16(4):295-302. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000400007 . doi: 10.5123/S1679-49742007000400007
http://scielo.iec.gov.br/scielo.php?scri... The choice of the types of insecticides used is determined by the identification of Ae. aegypti populations resistant to insecticide products.⁶6. Insecticide Resistance Action Committee. Prevention and management of insecticide resistance in vectors of public health importance [Internet]. 2nd ed. 2011 [cited 2018 Dec 4]. 72 p. Available in: Available in: http://www.irac-online.org/content/uploads/VM-layout-v2.6_LR.pdf
http://www.irac-online.org/content/uploa... It is therefore particularly important to monitor the susceptibility of Ae. aegypti to new larvicides, in order to obtain early diagnosis of resistant populations and to ensure the efficacy of these products in controlling vectors.

With effect from 1999, monitoring of Ae. aegypti susceptibility to different insecticide products began in Brazil.⁵5. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde [Internet]. 2007 out-dez [citado 2018 dez 4];16(4):295-302. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000400007 . doi: 10.5123/S1679-49742007000400007
http://scielo.iec.gov.br/scielo.php?scri... The tests were performed by the National Network for Monitoring Ae. aegypti Resistance to insecticides (MoReNa Network), responsible for assessing this vector’s resistance to the chemical products used to control it. The results of the Network’s work, as well as isolated studies, showed that with effect from 1998 temephos (organophosphate), a product that had been used for more than 30 years in Brazil, began to have low efficacy in combating Ae. aegypti in diverse Brazilian municipalities.⁵5. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde [Internet]. 2007 out-dez [citado 2018 dez 4];16(4):295-302. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000400007 . doi: 10.5123/S1679-49742007000400007
http://scielo.iec.gov.br/scielo.php?scri...

6. Insecticide Resistance Action Committee. Prevention and management of insecticide resistance in vectors of public health importance [Internet]. 2nd ed. 2011 [cited 2018 Dec 4]. 72 p. Available in: Available in: http://www.irac-online.org/content/uploads/VM-layout-v2.6_LR.pdf
http://www.irac-online.org/content/uploa... ^-77. Belinato DF, Viana-Medeiros PF, Araújo SC, Martins AJ, Lima JBP, Valle D. Resistance status to the insecticides temephos, deltamethrin, and diflubenzuron in Brazilian Aedes aegypti populations. BioMed Res Int [Internet]. 2016 [cited 2018 Dec 4];2016:1-12. Available in: Available in: https://www.hindawi.com/journals/bmri/2016/8603263/cta/ . doi: 10.1155/2016/8603263
https://www.hindawi.com/journals/bmri/20... These findings resulted in the Ministry of Health interrupting the use of organophosphates, given that purchasing insecticides to be used to control malaria, dengue, Zika and chikungunya is a federal government responsibility.⁸8. Brasil. Ministério da Saúde (MS). Portaria MS/GM nº 1.378, de 9 de julho de 2013. Regulamenta as responsabilidades e define diretrizes para execução e financiamento das ações de Vigilância em Saúde pela União, Estados, Distrito Federal e Municípios, relativos ao Sistema Nacional de Vigilância em Saúde e Sistema Nacional de Vigilância Sanitária [Internet]. Diário Oficial da União, Brasília (DF), 2013 jul 10 [citado 2018 dez 4]; Seção 1:48. Disponível em: Disponível em: http://bvsms.saude.gov.br/bvs/saudelegis/gm/2013/prt1378_09_07_2013.html
http://bvsms.saude.gov.br/bvs/saudelegis... Temephos was therefore replaced by so-called ‘alternative insecticides’, belonging above all to the biological insecticide and insect growth regulator groups.⁵5. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde [Internet]. 2007 out-dez [citado 2018 dez 4];16(4):295-302. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000400007 . doi: 10.5123/S1679-49742007000400007
http://scielo.iec.gov.br/scielo.php?scri... In Itabuna, for example, temphos was replaced by chitin synthesis inhibitors, starting with diflubenzuron from 2012 until the end of 2013. Novaluron (chitin synthesis inhibitor) was used throughout 2014, while from 2015 to date pyriproxyfen has been used,⁹9. Secretaria Municipal de Saúde (Itabuna). Programa municipal de controle de dengue. Equipe de educação em saúde. Relatório das atividades PMCD 2015. Itabuna: Secretaria Municipal de Saúde; 2015. this being a juvenile hormone analogue which acts on insect development thus inhibiting the emergence of adult insects.⁵5. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde [Internet]. 2007 out-dez [citado 2018 dez 4];16(4):295-302. Disponível em: Disponível em: http://scielo.iec.gov.br/scielo.php?script=sci_arttext&pid=S1679-49742007000400007 . doi: 10.5123/S1679-49742007000400007
http://scielo.iec.gov.br/scielo.php?scri...

Despite larvicide replacement, dengue incidence rates have remained high in the municipality of Itabuna.³3. Secretaria de Saúde (Bahia). Diretoria de Vigilância Epidemiológica do Estado da Bahia. Situação epidemiológica das arboviroses na Bahia: 2015-2016. Bol Epidemiológico [Internet]. 2016 jan [citado 2018 dez 4];12. Disponível em: Disponível em: http://www.blogdogusmao.com.br/v1/wp-content/uploads/2016/02/Boletim-epidemiológico-nº-12-dengue_chikungunya_zika.pdf
http://www.blogdogusmao.com.br/v1/wp-con... Local surveillance teams have reported, for instance, the occurrence of live larvae following treatment using pyriproxyfen.⁹9. Secretaria Municipal de Saúde (Itabuna). Programa municipal de controle de dengue. Equipe de educação em saúde. Relatório das atividades PMCD 2015. Itabuna: Secretaria Municipal de Saúde; 2015. In view of this situation, the Bahia State Epidemiological Surveillance Directorate (DIVEP/BA), together with the Prof. Gonçalo Moniz Central Public Health Laboratory (Lacen/BA), identified the need to evaluate the effectiveness of the larvicides used in the National Dengue Control Program routine.

The objective of this study was to evaluate, in simulated field conditions, the efficacy of pyriproxyfen (juvenile hormone), novaluron (chitin inhibitor) and spinosad (biolarvicide) in controlling Ae. aegypti.

Methods

This is an experimental study using simulated field conditions. In order to evaluate the action of the larvicides, we used Ae. aegypti specimens from egg samples collected in the urban area of the municipality of Itabuna in the state of Bahia (BA). We used as controls eggs from susceptible strains (Rockefeller) of Ae. aegypti provided by the Center for Disease Control and Prevention in Puerto Rico, United States. The tests were carried out between August and October 2015 at the Lacen facilities in Salvador/BA.

Itabuna has 61,555 households and a resident population of 199,643 people distributed over 57 neighborhoods.¹⁰10. Instituto Brasileiro de Geografia e Estatística. Censo demográfico 2010. Informações completas [Internet]. Rio de Janeiro: Instituto Brasileiro de Geografia E Estatística; 2018 [citado 2018 dez 4]. Disponível em: Disponível em: http://www.cidades.ibge.gov.br/xtras/perfil.php?lang=&codmun=291480&search=bahia|itabuna|infograficos:-informacoes-completas
http://www.cidades.ibge.gov.br/xtras/per... The study area was divided geographically into 20 quadrants, whereby each quadrant covered two neighbourhoods on average and four properties were randomly selected in each quadrant. An ovitrap with three pallets was installed in each of the selected properties, using the methodology proposed by the MoReNa Network which is coordinated the by Health Ministry’s Health Surveillance Secretariat.¹¹11. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Rede nacional de monitoramento da resistência de Aedes aegypti a inseticidas (MoReNAa): metodologia de amostragem. Brasília: Ministério da Saúde; 2008. The traps were installed in July and the pallets were collected weekly for three months from July to September 2015.

The pallets removed from the traps were packaged individually in plastic containers and left to dry. Ae. aegypti eggs on the pallets were counted by the Itabuna-based Southern Health Region Group using a stereoscope; positive pallets were sent fortnightly to Lacen/BA where they were stored until the tests began. The length of egg storage, from initial collection to the tests being performed varied from 30 to 60 days.

One week before each exposure (test) began, the selected pallets were submerged in a plastic glass (500mL) containing tap water. The hatched larvae were transferred to plastic trays, fed with fish food (Tetramin®), at a temperature of 25ºC, until they reached stage L3. At the end of this process, 600 larvae, from all 20 quadrants, were kept to comprise the group exposed to the products (Table 1) using the dosage and concentrations recommended by the manufacturers¹²12. World Health Organization. WHOPES-recommended compounds and formulations for control of mosquito larvae [Internet]. Geneva: World Health Organization; 2017 [updated 2017 Jul 28; cited 2013 Oct 25]. Available in: Available in: http://www.who.int/whopes/Mosquito_Larvicides_25_Oct_2013.pdf
http://www.who.int/whopes/Mosquito_Larvi... (Table 2). The total of 600 larvae was comprised of 30 larvae per quadrant, this being the cut-off point, given that the maximum number of hatched larvae in one of the quadrants was 30.

Out of the 600 larvae kept in each pre-exposure stage, 100 were separated randomly for the purpose of species identification. Out of the remaining 500 larvae, 360 were randomly selected for the tests, comprising 12 groups of 30 Ae. Aegypti larvae each. Each group was placed in a 30 liter plastic bucket, totaling nine buckets for the test group (three buckets for each larvicide) and three control buckets (one for each test group). The larvae selection and identification procedure was repeated every 15 days, with new L3 larvae for each exposure.

The containers were kept in a shaded environment, treated and identified with the names of the products and distributed in a covered shed outside of the Entomology Laboratory.

The buckets were filled with 24 liters of water taken from the local water supply main. The test larvicides were also added and the larvae were exposed directly. At the time of exposure, 0.5g of fish food (Tetramin®) was added to each of the containers to feed the larvae. In order to simulate field conditions, the water in each of the buckets was changed twice a week by removing and replacing 1/3 of the container volume. Water temperature and pH were measured once a week. The containers remained closed with nylon mesh to prevent the entry of insects and any residues during the entire experimental period.

Following exposure, the first reading was taken after 48 hours, whereby reading is understood to mean the counting of larvae and pupae, whether alive or dead. The dead larvae and pupae were removed and those with intact morphologic structures were identified. The resulting information was recorded in a specific bulletin.

Following the first reading, new readings were performed every 24 hours and live pupae were transferred to a plastic glass with a lid containing water from the container they were taken from. The glasses were identified with the code of the container the pupae came from, as well as the date of the reading and the amount of pupae numbered sequentially; following this the glasses were sent to the laboratory to check for the emergence of adults and identification of species and sex.

The experiments were conducted in pairs comprised of Itabuna samples and Rockefeller strains.

Once the adults had emerged in the untreated containers (control), observation of the treated containers was ceased immediately, and the quantity of live and inhibited/dead larvae and pupae in all the containers was counted.

The larvicide effect of the products was analyzed by calculating the inhibition of the emergence of adult insects, taking as a parameter the percentage emergence of the control group. We calculated the mean percentage of inhibition whereby the mean was based on all the values obtained for the replicates in containers in which the same larvicide was used.

The following formula was used to calculate inhibition of the emergence of adults:

Where:

% IE = percentage emergence inhibition

% EC = percentage emergence in the control containers

% ET = percentage emergence in the treated containers

The tests were repeated every 15 days using new L3 larvae. The fortnightly evaluations lasted for 60 days, simulating the interval between the household visits made by health workers, this being the period in which the lethal effect of larvicide is expected to be above 80%.¹³13. World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides [Internet]. Geneva: World Health Organization; 2005 [cited 2018 Dec 4]. 44 p. Available in: Available in: https://www.who.int/whopes/resources/who_cds_whopes_gcdpp_2005.13/en/
https://www.who.int/whopes/resources/who...

The mortality percentages and emergence inhibition percentages in the control group served as parameters for validating the tests, according to the criterion adopted by the Abbott method, according to which mortality greater than 20.0% would indicate that the tests had been manipulated and consequently result in them being invalidated. Mortality rates between 5.0 and 19.9% in the control containers indicated correction of mortality found in the treated containers using Abbott’s formula.¹⁴14. Abbott WS. A method of computing the effectiveness of an insecticide. 1925. J Am Mosq Control Assoc [Internet]. 1987 Jun [cited 2018 Dec 4];3(2):302-3. Available in: Available in: https://www.ncbi.nlm.nih.gov/pubmed/3333059
https://www.ncbi.nlm.nih.gov/pubmed/3333...

Where:

% C = percentage mortality in the control containers

% E = percentage mortality in the treated containers (exposed)

In order to ascertain differences in effectiveness and persistence among the formulations tested on the Ae. aegypti populations (Itabuna and Rockefeller), in simulated conditions, we performed analysis of variance (ANOVA)¹⁵15. Kruskal WH. A nonparametric test for the several sample problem. Annals of Mathematical Statistics [Internet]. 1952 Dec [cited 2018 Dec 4];23(4):535-40. Available in: Available in: https://www.jstor.org/stable/2236578
https://www.jstor.org/stable/2236578... with the aid of the Prism statistical application (GraphPad Software, Inc., 1999). In order to apply the analyses, the percentage emergence inhibition values were converted into arcsine values.¹³13. World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides [Internet]. Geneva: World Health Organization; 2005 [cited 2018 Dec 4]. 44 p. Available in: Available in: https://www.who.int/whopes/resources/who_cds_whopes_gcdpp_2005.13/en/
https://www.who.int/whopes/resources/who... We also compared the response of the Itabuna population with that obtained for the Rockefeller population for each larvicide using the Kruskal Wallis non-parametric test with the aid of the GraphPad Prism 3.0 application, this being considered to be statistically significant when p<0.05 (95% significance level).¹⁶16. Graphpad Prism 1999. User’s guide version 3.0: the fast, organized way to analyze and graph scientifi c data. San Diego. Available in: http:www.graphpad.com
http:www.graphpad.com...

With the aim of estimating the number of days following treatment needed to obtain a level of control equal to or greater than 80% in each treatment, we used the Polo-PC statistical package,¹⁷17. Polo PC: probit or logit analysis [computer program]. Berkele: LeOra Software; 1987. based on the emergence inhibition data per exposure time.

Results

Throughout the study, 100.0% of the adult and immature forms analyzed randomly belonged to the Ae. Aegypti species, totaling 500 larvae and 295 adults identified.

During the tests, temperature and pH did not oscillate greatly, both in the case of the treated and non-treated containers. Temperature varied between 29.5 and 31.2°C; while pH varied between 7.1 and 7.6.

The test validation parameter showed that mortality in the untreated containers was less than 20.0% in all weeks of exposure. However, we needed to correct the mortality (Abbott’s formula) of the untreated larvae forms in relation to the first exposure (1^st day) in the Itabuna population, in which we found 16.7% mortality, and also in relation to the final exposure (60^th day) in the Rockefeller strain, in which we found 13.3% mortality (Table 3).

There was variation in Ae. aegypti larvae mortality in the untreated containers, from 0.0 to 13.3% in the Rockefeller strain, and from 0.0 to 16.7% in the Itabuna sample, during the exposures occurring between day 1 and day 60. With regard to pupae in untreated containers, mortality varied between 0.0 and 8.4% for the Rockefeller strain and between 0.0 and 1.6% for the Itabuna population. With regard to percentage emergence of adult insects in untreated containers, variation ranged from 78.3 to 97.8% for the Rockefeller strain and from 81.7 to 100.0% for the Itabuna population (Table 3).

The three products we evaluated, Mosquiron Ready 5 ME® (novaluron), Sumilarv® (pyriproxyfen) and Natular DT® (spinosad), applied using the dosage recommended by the manufacturers in simulated field conditions, were efficacious in inhibiting the emergence of Ae. Aegypti adults. In the case of Sumilarv® (Itabuna population), we estimated that on the 57^th day (52.64 confidence interval [CI] 64), there was 80.0% inhibition of the emergence of adults; in the case of the other treatments, percentage inhibition of emergence varied between 89.5 and 100.0% over the 60-day period (Table 4). We did not find significant statistical difference in the action of the three larvicides (treatments) for the Rockefeller strain (p=0,451), nor in the action of these products on the Itabuna population (p=0,412); nor was there significant difference between the Itabuna Ae. aegypti population and the Rockefeller strain (p<0.05).

In the case of Natular DT® and Mosquiron®, mortality in both populations studied (Rockefeller and Itabuna) occurred predominantly in the larval stage. With regard to the Itabuna population, we found that Natular DT® and Mosquiron® had 98.8% and 97.9% mortality, respectively, in the larval stage, while for the Rockefeller strain, mortality in the larval stage was 99.7% for Natular DT® and 100.0% for Mosquiron®. In the case of Sumilarv®, mortality was greater in the pupal stage in both populations (Table 5).

Discussion

The three larvicides tested in this study were found to be efficacious in controlling Ae. aegypti. Nevertheless, evidence in relation to pyriproxyfen needs to be considered: this product acts, almost exclusively, during the insect’s pupal stage. This particularity should serve as a warning to the Ministry of Health, in the sense of it reconsidering the use of pyriproxyfen as a larvicide to control the species, given that actions to combat the vector take the insect’s larval stage to be the indicator of infestation. Moreover, the continued and indiscriminate use of insecticides in inadequate concentrations has given rise to the selection of populations of resistant vector insects, thus causing difficulties in controlling pathogen transmission.¹⁸18. Gambarra WPT, Martins WFS, Lucena Filho MLL, Albuquerque IMC, Apolinário OKS, Beserra EB. Spatial distribution and sterase activity in populations of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) resistant to temephos. Rev Soc Bra Med Trop [Internet]. 2013 mar-abr [citado 2018 dez 4];46(2):178-84. Disponível em: Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822013000200178&lng=en. doi: 10.1590/0037-8682-1727-2013
http://www.scielo.br/scielo.php?script=s... Several studies have demonstrated resistance of Ae. aegypti populations to insecticides, thus hindering arbovirus control.⁷7. Belinato DF, Viana-Medeiros PF, Araújo SC, Martins AJ, Lima JBP, Valle D. Resistance status to the insecticides temephos, deltamethrin, and diflubenzuron in Brazilian Aedes aegypti populations. BioMed Res Int [Internet]. 2016 [cited 2018 Dec 4];2016:1-12. Available in: Available in: https://www.hindawi.com/journals/bmri/2016/8603263/cta/ . doi: 10.1155/2016/8603263
https://www.hindawi.com/journals/bmri/20... ^,99. Secretaria Municipal de Saúde (Itabuna). Programa municipal de controle de dengue. Equipe de educação em saúde. Relatório das atividades PMCD 2015. Itabuna: Secretaria Municipal de Saúde; 2015.^,1010. Instituto Brasileiro de Geografia e Estatística. Censo demográfico 2010. Informações completas [Internet]. Rio de Janeiro: Instituto Brasileiro de Geografia E Estatística; 2018 [citado 2018 dez 4]. Disponível em: Disponível em: http://www.cidades.ibge.gov.br/xtras/perfil.php?lang=&codmun=291480&search=bahia|itabuna|infograficos:-informacoes-completas
http://www.cidades.ibge.gov.br/xtras/per...

The representative sample of Ae. aegypti eggs collected in the municipality of Itabuna, as well as the regularity of temperature and pH parameters throughout the exposure tests, contributed to the analysis and to the consistency of the results. The test validation parameter showed that mortality in the untreated containers was less than 20% in all weeks of exposure, demonstrating that adequate conditions were ensured throughout the experiments. This is confirmed since according to the criterion proposed by Abbott’s method, larva mortality greater than 20% is a result that indicates that the test has been manipulated and, consequently, leads to its being invalidated. Mortality of between 5.0 and 19.9% in the control containers indicates the need for correction, according to the same method. In this study, the need for correction using Abbott’s method only arose twice in larvae in untreated containers, namely on the 1^st day for the Rockefeller strain and on the 60^th day for the Itabuna population.

The efficacy of the three alternative products - Mosquiron Ready 5 ME®, Sumilarv® and Natular DT® -, with no statistical difference between them (p=0.412) in controlling Ae. aegypti, confirms the results of other studies.¹⁹19. Braga IA, Mello CB, Peixoto AA, Valle D. Evaluation of methoprene effect on Aedes aegypti (Diptera: Culicidae) development in laboratory conditions. Mem Inst Oswaldo Cruz [Internet]. 2005 Jul [cited 2018 Dec 4];100(4):435-40. Available in: Available in: https://www.ncbi.nlm.nih.gov/pubmed/16113894
https://www.ncbi.nlm.nih.gov/pubmed/1611...

20. Fontoura NG, Bellinato DF, Valle D, Lima JBP. The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo Cruz [Internet]. 2012 May [cited 2018 Dec 4];107(3):387-95. Available in: Available in: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000300014. doi: 10.1590/S0074-02762012000300014
http://www.scielo.br/scielo.php?script=s...

21. Marina CF, Bond G, Muñoz J, Valle J, Chirino N, Williams T. Spinosad: a biorational mosquito larvicide for use in car tires in southern Mexico. Parasit Vectors [Internet]. 2012 May [cited 2018 Dec 4];5:95. Available in: Available in: https://www.ncbi.nlm.nih.gov/pubmed/22608138. doi: 10.1186/1756-3305-5-95
https://www.ncbi.nlm.nih.gov/pubmed/2260... ^-2222. Resende MC, Gama RA. Persistência e eficácia do regulador de crescimento pyriproxyfen em condições de laboratório para Aedes aegypti. Rev Soc Bras Med Trop [Internet]. 2006 jan-fev [citado 2018 dez 4];39(1):72-5. Disponível em: Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822006000100014. doi: 10.1590/S0037-86822006000100014
http://www.scielo.br/scielo.php?script=s... This finding is important: the three alternative larvicides tested are on the list of products which the Health Surveillance Secretariat/Ministry of Health can buy, as alternatives to other chemical insecticides used by vector-borne disease control programs which, given their toxicity, can affect non-target organisms as well as the environment.

The results we obtained show that the tested larvicides had an adequate residual effect. This information can be useful for additional field studies, since it is possible that the test conditions (shaded location, plastic container) may have contributed to product non-degradation and bioavailability.²³23. Garcia Neto LJ. Efeito residual do Diflubenzuron sobre larvas de Aedes aegypti em condições simuladas de campo, no laboratório [dissertação]. Fortaleza (CE): Universidade Estadual do Ceará; 2011.

The residual effect of pyriproxyfen (Sumilary®), novaluron (Mosquiron Ready 5 ME®) and spinosad (Natular DT®) declined after between 45 and 60 days. The residual effect of pyriproxyfen declined after 45 days, in keeping with findings of authors in the state of Minas Gerais.²²22. Resende MC, Gama RA. Persistência e eficácia do regulador de crescimento pyriproxyfen em condições de laboratório para Aedes aegypti. Rev Soc Bras Med Trop [Internet]. 2006 jan-fev [citado 2018 dez 4];39(1):72-5. Disponível em: Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822006000100014. doi: 10.1590/S0037-86822006000100014
http://www.scielo.br/scielo.php?script=s... Likewise, the residual effects obtained for Mosquiron Ready 5 ME® and Natular DT® are similar to the findings of other studies, according to which the efficacy of these larvicides lasted for 6 to 8 weeks.²⁰20. Fontoura NG, Bellinato DF, Valle D, Lima JBP. The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo Cruz [Internet]. 2012 May [cited 2018 Dec 4];107(3):387-95. Available in: Available in: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000300014. doi: 10.1590/S0074-02762012000300014
http://www.scielo.br/scielo.php?script=s... ^,2121. Marina CF, Bond G, Muñoz J, Valle J, Chirino N, Williams T. Spinosad: a biorational mosquito larvicide for use in car tires in southern Mexico. Parasit Vectors [Internet]. 2012 May [cited 2018 Dec 4];5:95. Available in: Available in: https://www.ncbi.nlm.nih.gov/pubmed/22608138. doi: 10.1186/1756-3305-5-95
https://www.ncbi.nlm.nih.gov/pubmed/2260...

In the Rockefeller and Itabuna populations, pyriproxyfen acted above all in the pupal stage, with mortality of 97.9% and 95.1%, respectively, while larvae mortality was 2.1% and 4.9%. Pyriproxyfen prevents the emergence of adult Aedes, acting to inhibit the development of the characteristics of the adult insect (wings, maturation of the reproductive organs and external genitalia),²⁴24. Chena CD, Andy-Tana WA, Lokea SR, Lee HL, Yasmin AR, Sofian-Azirun M. Effectiveness of pyriproxyfen-controlled release block against larvae of Aedes (Stegomyia) aegypti in Kuala Lumpur, Malaysia. Dengue Bull [Internet]. 2008 [cited 2018 Dec 4];32:199-206. Available in: Available in: http://apps.who.int/iris/bitstream/handle/10665/170713/db2008v32p199.pdf?sequence=1&isAllowed=y
http://apps.who.int/iris/bitstream/handl... acting almost exclusively in the pupal stage. A similar result was found by Braga et al. (2005), in their laboratory study in which they found greater Ae. Aegypti pupae mortality.¹⁹19. Braga IA, Mello CB, Peixoto AA, Valle D. Evaluation of methoprene effect on Aedes aegypti (Diptera: Culicidae) development in laboratory conditions. Mem Inst Oswaldo Cruz [Internet]. 2005 Jul [cited 2018 Dec 4];100(4):435-40. Available in: Available in: https://www.ncbi.nlm.nih.gov/pubmed/16113894
https://www.ncbi.nlm.nih.gov/pubmed/1611...

The results obtained in our study, using field condition simulation, do have limitations. It would be valuable to go into these analyses in greater depth by means of a study carried out directly in the field. Biotic and abiotic factors can bring influence to bear on the development of the insect’s biological cycle: for instance, it is known that intra- and inter-specific larval competition, or atmospheric variations such as temperature, rainfall and light, can fluctuate in the field to such an extent that they may not be fully reproduced in simulation experiments.²⁵25. Oliveira S, Villela DAM, Dias FBS, Moreira LA, Maciel de Freitas R. How does competition among wild type mosquitoes influence the performance of Aedes aegypti and dissemination of Wolbachia pipientis? PLoS Negl Trop Dis [Internet]. 2017 Oct [cited 2018 Dec 4];11(10):e0005947. Available in: Available in: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0005947 . doi: 10.1371/journal.pntd.0005947
https://journals.plos.org/plosntds/artic... With the aim of minimizing these limitations, over the course of the study we changed the water to simulate rain and/or water supply systems; we also maintained the periodicity of light (day and night) and the ambient temperature.

Although it makes pupae incapable of turning into winged adults, pyriproxyfen can cause a false impression of positivity in treated containers, since as the larvae remain alive, they can suggest incorrect levels of Ae. Aegypti infestation, thus compromising the targeting of control actions: in Brazil, Ae. aegypti infestation rates are measured based on larvae levels.

This study concludes that the tested products are efficacious in controlling Ae. Aegypti. Notwithstanding, we recommend the preferential use of larvicides that act above all in the larval stage, with residual effects that accompany the duration of the cycle of visits made by health workers, that do not result in operational difficulties and have low environmental impact.

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