Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
ii
iii
de
Ps-Graduao
em
Biologia
Banca Examinadora
iv
vi
Agradecimentos
A todos os meus familiares e amigos que, muitas vezes sem saber, foram
muito importantes durante o decorrer deste trabalho.
Ana Carolina dos Santos Valente, caadora oficial de artigos quase
impossveis de serem encontrados. Sua ajuda foi imensurvel em todos os sentidos.
No existe espao aqui e nem palavras com as quais eu possa expressar toda a
minha gratido.
A todos os amigos do Laboratrio de Fisiologia e Controle de Artrpodes
Vetores (LAFICAVE), principalmente pelo esprito de unio, o que torna nosso grupo
especial: Denise, Bento, Jutta, Patrcia, Ademir, Gustavo, Mrcia, Isabela, Luana,
Camila, Priscila, Diogo, Aline, Nathlia, Eliane, Tnia, Gilberto, Diego, dula, Ciroulo
e Bianca. Valeu Mosquiteiros!! Trabalhar com vocs demais!!
A todos os amigos da turma da Biologia Parasitria 2005: Marcelo, Flvio,
Alexandre, Vigoder, Marcos, Gabriel, Cntia, Jaline, Cris, Tamara, Tatiane, Simone,
Raquel Canto, Karen, Camila, Lidiane e Carol pelos inmeros churrascos, amigos
ocultos, festas, quadras etc etc etc. Fiz grandes amigos durante esse perodo que,
com certeza, vo estar presentes em toda a minha vida!!!
A Luana e Camila, amigas desde a graduao.
vii
Ao Gustavo Lazzaro Rezende, que alm de ser meu amigo uma pessoa
bastante prestativa. Suas sugestes e nossas conversas foram importantes no
decorrer deste trabalho.
querida "Mafaldinha" que possui uma capacidade gigantesca de ajudar
as pessoas! Muito obrigado por me ouvir, fazer companhia e me ajudar durante os
bioensaios na sala de resistncia.
Ao Diogo Bellinato, que me socorreu muitas vezes com sua ajuda durante
a realizao dos experimentos.
A Luana Rosa e Jaqueline Chaves pela reviso do portugus da
dissertao. Foi muito bom conhecer vocs duas, e sem dvida alguma, j fazem
parte da minha vida!
Dr Denise Valle, pela pacincia e orientao deste trabalho. Consideroa uma grande pessoa e excelente orientadora. O mais importante que, acima de
tudo, minha amiga!
Ao Dr Jos Bento Pereira Lima, pela orientao, conselhos e sugestes
durante estes dois anos de pesquisa.
Ao Dr Alexandre Peixoto, pelo uso de equipamento e orientao durante
os ensaios de atividade no Laboratrio de Biologia Molecular de Insetos.
Tamara Nunes de Lima Camara, pela amizade e orientao durante os
ensaios de atividade no Laboratrio de Biologia Molecular de Insetos.
Ao Instituto de Biologia do Exrcito (IBEx), onde todos os experimentos
foram realizados.
Ao Instituto Oswaldo Cruz (IOC) pelo apoio financeiro e tcnico para
realizao deste trabalho.
viii
ix
ndice
Resumo .................................................................................................................................... 1
Abstract.................................................................................................................................... 2
1) Introduo........................................................................................................................... 3
1.1)
Os mosquitos ......................................................................................................... 3
1.1.1)
Aedes aegypti................................................................................................. 4
1.1.2)
Aedes albopictus........................................................................................... 5
1.1.3)
1.2)
1.2.1)
1.2.1.1)
Organoclorados ..................................................................................... 9
1.2.1.2)
Organofosforados ............................................................................... 10
1.2.1.3)
Carbamatos........................................................................................... 11
1.2.1.4)
Piretrides ............................................................................................. 12
1.2.2)
1.3)
Inseticidas qumicos..................................................................................... 9
Resistncia a inseticidas........................................................................... 12
1.3.1)
Controle biolgico....................................................................................... 16
1.3.2)
1.3.2.1)
1.3.2.2)
1.3.2.3)
2.2)
Objetivos especficos......................................................................................... 26
3) Resultados........................................................................................................................ 27
3.1) artigo 1: Effect of a chitin synthesis inhibitor on Aedes aegypti populations
susceptible and resistant to the organophosphate temephos.................................... 27
3.2) artigo 2: Exposure of Aedes aegypti larvae to a chitin synthesis inhibitor sublethal dose: effects on the viability and reproduction of adults................................... 39
x
xi
Resumo
Aedes aegypti, Aedes albopictus e Culex quinquefasciatus so trs espcies de
mosquito difundidas principalmente nas regies tropicais e subtropicais do globo. No
Brasil, esto relacionadas com a transmisso de doenas como a dengue e a
filariose linftica e h grande preocupao com sua potencial participao na
transmisso de outras arboviroses, como o vrus do oeste do Nilo e o da febre
amarela. Atualmente, a principal forma de combate a vetores de doenas realizada
atravs do uso de inseticidas qumicos, cujo stio de ao o sistema nervoso
central do inseto. Como conseqncia do uso macio destes produtos, a freqncia
de indivduos resistentes em populaes de vrias espcies de insetos vetores tem
aumentado. Os reguladores do desenvolvimento de insetos aparecem como uma
nova alternativa de controle de mosquitos vetores. Neste grupo, encontram-se os
inibidores da sntese de quitina (CSI), substncias que prejudicam o processo de
muda, acarretando deficincias principalmente na cutcula dos insetos. No presente
trabalho, verificou-se que o CSI triflumuron foi eficaz contra estas trs espcies de
culicdeos vetores, em concentraes na ordem de g/L. Alm disso, foi investigado o
efeito da aplicao, em larvas, de dose parcialmente letal de triflumuron sobre vrios
aspectos da biologia de Ae. aegypti. De modo geral, a longevidade, aceitao do
repasto sangneo, volume de sangue ingerido, reproduo e postura so afetados
negativamente nos adultos sobreviventes ao tratamento na fase imatura. Triflumuron
foi eficaz contra diversas populaes de campo de Ae. aegypti, com diferentes nveis
de susceptibilidade ao organofosforado temephos e ao piretride deltametrina: no
houve emergncia de adultos viveis quando as larvas foram expostas IE99 de
triflumuron para a cepa-referncia Rockefeller. Embora no tenha sido detectada
resistncia cruzada entre aqueles inseticidas qumicos e o CSI, a mortalidade de
populaes resistentes ao temephos, mas no deltametrina, ocorreu mais
tardiamente. Estes resultados indicam que triflumuron pode ser considerado como
uma alternativa promissora para o controle de culicdeos no Brasil e apontam para a
necessidade de monitoramento constante do status de susceptibilidade das
populaes do vetor.
Abstract
Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are three mosquito
species spread allover the world, mainly in the tropical and subtropical regions. In
Brazil these are implicated in the transmission of diseases such as dengue and
lymphatic filariasis, and there is a great concern related to their potential in
transmitting several other arboviruses, like those responsible for West Nile and
yellow fever. Presently, the major tools against disease vectors are chemical
insecticides, whose target site is the insect central nervous system. A consequence
of the massive use of these products is the frequence increasing of resistant
individuals in populations of several species of medical importance. The Insect
Growth Regulators represent a novel alternative of mosquito vectors control. The
chitin synthesis inhibitors (CSI), members of this group, interfere with the molting
process, resulting in a series of defects, mainly in the insect cuticle. In the present
work we verified that the CSI triflumuron was effective against three species of
culicidae vectors, in concentrations in the range of g/L. The consequences of
exposing Ae. aegypti larvae to partial lethal doses of triflumuron over several aspects
of their biology were also investigated. In general longevity, blood feeding
acceptance, size of blood meal, reproduction and egg laying are negatively affected
in triflumuron surviving adults. Triflumuron was effective against several Ae. aegypti
field populations, exhibiting different levels of resistance to the organophosphate
temephos and to the pyrethroid deltamethrin: no emergence of viable adults was
detected after exposure of larvae to the EI99 triflumuron Rockefeller (a reference
strain) dose. Although no cross resistance among those chemical insecticides and
the CSI has been detected, in general, mortality of temephos (but not deltamethrin)
resistant populations was observed later. These results indicate triflumuron can be
envisaged as a promising alternative to the control of culicidae in Brazil and point to
the need of the permanent monitoring of the insecticide susceptibility status of vector
populations.
1) Introduo
1.1)
Os mosquitos
Os mosquitos pertencem famlia Culicidae, grupo que se enquadra
dentro da ordem Diptera, que contm por volta de 151.000 espcies descritas
(Brusca e Brusca, 2003). Esta ordem, por sua vez, inclui os insetos portadores de
duas asas, como as moscas e os mosquitos (Ruppert e Barnes, 1996). Os
mosquitos so encontrados basicamente em todo o mundo, com exceo dos
lugares com clima muito hostil, que permanecem congelados o ano todo. A maior
parte das espcies de mosquitos vive nos trpicos e sub-trpicos, onde o clima
moderadamente quente e mido favorvel ao rpido desenvolvimento e
sobrevivncia dos adultos (Clements, 1992).
Atualmente, os mosquitos so classificados em trs subfamlias:
Toxorhynchitinae, Anophelinae e Culicinae. Entretanto, apenas as duas ltimas
apresentam importncia epidemiolgica, visto que os Toxorhynchitinae no realizam
hematofagia (Forattini, 2002). O ciclo de vida dos mosquitos holometablico, ou
seja, os insetos sofrem metamorfose completa. As fases imaturas compreendem os
estgios de ovo, larva e pupa, que alm de serem anatomicamente diferentes dos
adultos, vivem em habitats aquticos (Ruppert e Barnes, 1996).
As fmeas dos mosquitos, dependendo da espcie, so capazes de
colocar entre 50 a 500 ovos em um nico evento de oviposio. Em geral, os ovos
so depositados diretamente sobre a gua ou em locais midos que provavelmente
sero inundados em um futuro prximo (Clements, 1992). O desenvolvimento
embrionrio se inicia quase imediatamente aps os ovos serem depositados, dando
origem a uma larva entre um a sete dias, dependendo da espcie e da temperatura
ambiente. Na maioria das espcies, a larva eclode assim que formada. Entretanto,
ovos dos mosquitos da Tribo Aedini so capazes de resistir dessecao, de modo
que uma larva completamente formada no interior do ovo pode resistir por meses na
ausncia de gua, vindo a eclodir quando chuvas inundarem o stio de oviposio.
Muitos culicdeos so vetores de doenas, principalmente devido
necessidade de ingesto de sangue pelas fmeas, condio necessria para a
produo dos ovos na grande maioria das espcies. Dentre as principais doenas
transmitidas por culicdeos, pode-se destacar a malria, uma srie de arboviroses
(como a dengue e a febre amarela) e as filarioses.
3
1.2)
Controle de vetores
Alguns mtodos de controle de insetos datam de muitos sculos atrs,
como o uso de preparaes feitas atravs de produtos naturais (WHO, 1984). Antes
da 2 Guerra Mundial, as substncias qumicas usadas no combate aos insetos
eram produtos inorgnicos, como por exemplo, os compostos arsnicos. Apenas
algumas substncias orgnicas de origem vegetal eram usadas para o controle de
pragas naquele perodo, como a nicotina, piretrina e rotenona (WHO, 1997).
A dcada de 1940 representou o incio da era moderna dos pesticidas
orgnicos, conhecida tambm como a poca da revoluo dos pesticidas, quando
o DDT (diclorodifeniltricloroetano) foi usado pela primeira vez como inseticida
(DAmato et al., 2002). Essa substncia havia sido sintetizada originalmente por
Zeidler em 1874, mas suas propriedades de inseticida s foram descobertas por
Paul Mller em 1939, o que rendeu a este pesquisador o prmio Nobel de medicina
em 1948 (DAmato et al., 2002). A partir de ento, o DDT passou a ser o principal
inseticida utilizado at a dcada de 1970, quando seu uso foi proibido em diversos
pases, principalmente devido aos problemas causados ao meio ambiente (DAmato
et al., 2002).
De modo geral, o controle de mosquitos pode ser voltado tanto para as
larvas quanto para os adultos. Os mtodos mais utilizados no controle de larvas so:
1) fsico, 2) qumico e 3) biolgico (Neves et al., 2001). O controle fsico consiste na
eliminao dos criadouros das larvas visando interrupo do ciclo dos mosquitos.
O controle qumico baseia-se na aplicao de produtos capazes de eliminar de
modo eficaz os espcimes. Finalmente, o controle biolgico consiste na utilizao de
organismos que, de modo natural, controlam as populaes de mosquitos.
Atualmente, tem sido dada bastante nfase ao controle integrado de
vetores (WHO, 2006). Essa modalidade consiste na utilizao de dois ou mais
mtodos de controle de forma simultnea ou seqencial (Neves et al., 2001; WHO,
2006). De modo geral, inclui a vigilncia entomolgica, reduo dos criadouros,
controles qumico e biolgico, manejo da resistncia a inseticidas, assim como
campanhas de educao voltadas para a populao (Rose, 2001).
O controle de vetores tem importncia fundamental no combate a uma
srie de doenas, como a malria (Rey, 2001), filariose linftica (Ottesen &
Ramachandran, 1995) e dengue (Gubler, 2002). Em muitas ocasies representa a
nica alternativa de diminuio da transmisso, principalmente nos casos de
8
em
quatro
grandes
classes:
organoclorados,
organofosforados,
1.2.1.1)
Organoclorados
Os organoclorados so inseticidas que contm carbono, hidrognio e cloro
em
mamferos.
Aparentemente,
DDT,
seus
anlogos
os
1.2.1.2)
Organofosforados
Os osganofosforados (OPs) so assim chamados por possurem fsforo
10
1.2.1.3)
Carbamatos
Os
carbamatos
so
inseticidas
derivados
do
cido
carbmico,
11
1.2.1.4)
Piretrides
Os piretrides compreendem a mais antiga classe de inseticida de origem
12
resistncia
praticamente
todos
os
grupos
de
inseticidas,
incluindo os
onde havia sido aplicado o DDT, ou saam rapidamente delas (Roberts e Andre,
1994).
b) penetrao reduzida: a reduo da penetrao cuticular outro
mecanismo fisiolgico que pode permitir a sobrevivncia do inseto quando exposto a
determinado inseticida. Neste caso, como a taxa de penetrao do inseticida atravs
da cutcula reduzida, menor quantidade do xenobitico alcana o organismos e os
mecanismos de detoxificao internos presentes no inseto tm maior probabilidade
de metabolizar o inseticida (IRAC-BR, 2006). Apesar da penetrao reduzida
raramente ocorrer, ela responsvel por nveis dramticos de resistncia a diversas
classes de inseticidas. No existem relatos dos processos fisiolgicos ou dos
mecanismos moleculares envolvidos neste tipo de resistncia (Raymond et al.,
1989).
c) metabolizao dos inseticidas: a metabolizao dos inseticidas est
relacionada com o aumento da eficincia dos mecanismos enzimticos de
detoxificao naturais do inseto (resistncia metablica). Este aumento de eficincia
promove a inativao e eliminao do inseticida, impedindo que o mesmo alcance o
sistema nervoso, seu stio final de ao (Brogdon e McAllister, 1998; Hemingway e
Ranson, 2000). Trs principais grupos de enzimas, quando alteradas, so
responsveis pela resistncia metablica: Esterases, Oxidases de funo mista (ou
Monoxigenases P450), e Glutationa-S-Transferases (GSTs).
A superproduo de Esterases considerada uma resposta presso de
seleo por organofosforados ou carbamatos. Este fenmeno tem sido verificado em
uma srie de artrpodes, tais como mosquitos, carrapatos e baratas (Hemingway et
al., 2004). As Esterases amplificadas podem rapidamente seqestrar o inseticida,
impedindo-o de chegar ao stio-alvo (Karunaratne et al., 1993). Adicionalmente,
mudanas qualitativas na enzimas podem ser responsveis pela rpida hidrlise e
detoxificao de inseticidas (Hemingway et al., 2004).
As Oxidases de funo mista (MFO), comumente citadas como
Monoxigenases P450, constituem uma grande famlia de enzimas encontradas
desde bactrias at mamferos. Estas molculas esto envolvidas no metabolismo
de compostos endgenos e exgenos (Ren, 1999). Existem relatos que
demonstram que intensa atividade de Monoxigenases P450 est relacionada com
resistncia a inseticidas em mosquitos (Berg et al., 1998). Experimentos de
presso de seleo com Ae. aegypti tm sugerido que as P450 representam a
principal classe enzimtica envolvida na resistncia metablica a piretrides (Kumar
14
et al., 2002). Alm disto, como as P450 podem estar envolvidas com o metabolismo
de virtualmente todas as classes de inseticidas, levando ativao de xenobiticos
ou, mais freqentemente, sua detoxificao (Berg et al., 1998; Hemingway e
Ranson, 2000), esta famlia de enzimas a mais importante no fenmeno da
resistncia cruzada. Por exemplo, presso de uma populao de A. aegypti, em
laboratrio, com o OP temephos, por algumas geraes sucessivas, aumentou sua
resistncia ao piretride deltametrina em mais de 300 vezes. Ensaios com
sinergistas indicaram o envolvimento das P450 neste fenmeno (Rodriguez et al.,
2002).
Assim como as Monoxigenases P450, as Glutationa S-transferases
(GSTs) so amplamente difundidas entre os seres vivos e tambm tm como funo
primria a detoxificao de compostos endgenos e exgenos (Hemingway et al.,
2004). A elevada atividade das GSTs tambm est envolvida na resistncia a
inseticidas. Em geral, ocorre devido ao aumento quantitativo de um ou mais tipos de
GSTs, resultado de amplificao de genes, do aumento dos nveis transcricionais ou
de mudanas qualitativas nas enzimas (Ranson e Hemingway, 2005).
d) modificao do stio-alvo do inseticida: pequenas modificaes,
como uma mutao pontual no stio-alvo dos inseticidas, podem diminuir ou anular a
afinidade entre o inseticida e seu receptor no organismo do inseto (Beaty e
Marquardt, 1996). A Acetilcolinesterase, por exemplo, quando alterada, est
relacionada com resistncia a organofosforados e carbamatos (Ayad e Georghiou,
1979; Penilla et al., 1998). Mutaes pontuais nos receptores de GABA esto
relacionadas resistncia a ciclodienos, tais como dieldrin (Hemingway et al., 2004).
Da mesma maneira, alteraes no canal de sdio dos insetos podem ser
responsveis pela resistncia aos piretrides (Soderlund e Kniple, 2003).
1.3)
Alternativas de controle
Com o intuito de contornar o problema da resistncia a inseticidas, vrios
16
17
modo que sua viabilidade extremamente reduzida (Vasuki, 1992; Vasuki e Rajavel,
1992; Vasuki, 1999).
Os IGRs tm sido utilizados no controle de mosquitos vetores de doenas
em Sade Pblica (WHO, 2006). Em geral, so seguros para peixes, pssaros,
mamferos e para a maioria dos organismos aquticos no-alvo (Mian e Mulla, 1982;
WHO, 2006). Alm disso, apresentam toxicidade extremamente baixa para os seres
humanos.
Com base no modo de ao, os IGRs so classificados em trs categorias
principais: (1) hormnios juvenis e seus anlogos, tambm conhecidos como
juvenides ou mmicos, (2) agonistas da ecdisona (hormnio envolvido na muda) e
(3) inibidores da sntese de quitina (CSI, do ingls chitin synthesis inhibitor) (Staal,
1975; Marx, 1977; Graf, 1993; Boudjelida et al., 2005).
1.3.2.1)
(Tunaz e Uygun, 2004); hoje outros JHAs, como piriproxifen e fenoxicarb, esto
disponveis (Mulla et al., 1985; Schaefer et al., 1987; Vythilingam, 2005; Seng;
2006).
Os JHAs interferem com importantes mecanismos bioqumicos, tais como
a secreo e o transporte dos hormnios juvenis naturais do stio de secreo para o
stio de ao, ou para os locais de degradao e excreo (Retnakaran et al., 1985).
Seus efeitos biolgicos so muito complexos e variam de acordo com o tipo de
anlogo utilizado. Por exemplo, o JHA hidroprene pode estimular a sntese de JH
endgeno quando presente em baixas doses, mas pode ter efeito contrrio quando
administrado em grandes quantidades (Tobe e Stay, 1979).
Apesar de o efeito dos JHAs ocorrer principalmente nos estgios imaturos
(Axtell et al., 1979; Ali et al., 1995; Braga et al., 2005), existem alguns relatos que
evidenciam que adultos sobreviventes de exposio a pequenas doses possuem
alteraes fisiolgicas que comprometem sua sobrevivncia e reproduo (Arias e
Mulla, 1975; Sawby et al., 1992; Ritchie et al., 1997). Ritchie et al. (1997) verificaram
que baixas doses de methoprene diminuram a capacidade de Aedes vigilax (Skuse,
1889) de realizar o repasto sangneo. De modo geral, os adultos sobreviventes se
soltam da exvia com dificuldade (Kramer et al., 1993), apresentam longevidade
reduzida (Sawby et al., 1992), problemas morfolgicos (Arias e Mulla, 1975) e
reprodutivos (Sithiprasasna et al., 1996).
Em um dos poucos relatos sobre o efeito de JHA administrado diretamente
aos adultos, Divakar e Rao (1975) verificaram que cerca de 80% de ovos de
Anopheles stephensi alimentados com soluo de methoprene eram pequenos,
frgeis e esbranquiados. Os autores sugeriram que methoprene provavelmente age
nas clulas foliculares do ovrio, interferindo com a formao normal do crion e o
desenvolvimento do ocito. Apesar destas evidncias, Braga et al. (2005), no
constataram problemas em ovos de Ae. aegypti resultantes de tratamento com
methoprene.
Trabalhos que tratam do desenvolvimento de resistncia a JHAs em
campo tambm so escassos. Dame et al. (1998) verificaram que uma linhagem de
Aedes taeniorhynchus (Wiedemann, 1821) da Flrida era 14,9 vezes mais resistente
ao methoprene do que uma linhagem susceptvel de laboratrio. Em alguns casos, a
resistncia prvia a outros inseticidas pode estar relacionada com algum nvel de
resistncia ou tolerncia aos JHAs (Ortega et al., 1991).
19
1.3.2.2)
Agonistas da ecdisona
1.3.2.3)
22
do novaluron (Su et al., 2006) e do triflumuron (Sulaiman et al., 1994; Smith e Wall,
1998; Rehini e Soltani, 1999).
Alm de interferir com a sntese de quitina, os CSIs provocam uma srie
de efeitos secundrios que prejudicam o desenvolvimento dos insetos. Parween
(1998) verificou que larvas do coleptero Tribolium castaneum (Herbst, 1797),
quando tratadas com triflumuron, se alimentavam significativamente menos quando
comparadas com as no tratadas, apresentando desenvolvimento reduzido, alm de
um comprimento corporal menor. Vasuki (1992) verificou que a longevidade de
adultos de Cx. quinquefasciatus, An. stephensi e Ae. aegypti sobreviventes ao
tratamento com hexaflumuron era drasticamente reduzida. Em um trabalho posterior,
Vasuki (1999) tambm observou que estes trs culicdeos vetores ingeriam menos
sangue do que os indivduos no tratados, e, consequentemente, colocavam uma
quantidade menor de ovos. Em outros estudos, nos quais fmeas foram alimentadas
diretamente com CSIs, tambm se observou a diminuio da oviposio, alm de
uma menor viabilidade dos ovos (Miura et al., 1976; Wilson e Cryan, 1997; Senzde-Cabezn et al., 2006). Vasuki e Rajavel (1992) verificaram que hexaflumuron
provocou anormalidades visveis nos imaturos e adultos de Cx. quinquefasciatus,
Ae. aegypti e An. stephensi, muitas das quais relacionadas com a mortalidade
precoce dos adultos. Os adultos sobreviventes apresentavam asas e tarsos
defeituosos que dificultavam sua locomoo. Amir e Peveling (2004) tambm
observaram menor atividade de vo e comprometimento da prole em colnias de
abelhas, quando expostas ao triflumuron.
O inseticida Starycide (Bayer), cujo composto ativo o triflumuron, tem
sido usado no controle de diversos artrpodes. Como os CSIs tm alvo diferenciado
dos inseticidas clssicos, pode-se considerar seu uso como uma possvel alternativa
ao combate de populaes de culicdeos vetores, incluindo aquelas resistentes aos
inseticidas comumente utilizados.
Espera-se, portanto, que triflumuron provoque uma srie de problemas
que resultem na morte das larvas ou na emergncia de adultos fisiologicamente
debilitados. Neste contexto, no presente trabalho larvas de Ae. aegypti, Ae.
albopictus e Cx. quinquefasciatus foram expostas a vrias concentraes de
triflumuron, um inibidor da sntese de quitina. Alm disto, investigou-se o efeito de
doses subletais de triflumuron, aplicadas a larvas de Ae. aegypti, sobre os adultos
sobreviventes. Finalmente, investigamos a eficincia deste CSI sobre populaes de
campo de Ae. aegypti provenientes de diferentes localidades do Brasil, com distintos
23
24
utilizados
no
controle
de
culicdeos,
as
formas
de
resistncia
25
2) Objetivos
2.1)
Objetivo geral
2.2)
Objetivos especficos
26
3) Resultados
3.1) artigo 1: Effect of a chitin synthesis inhibitor on Aedes aegypti populations
susceptible and resistant to the organophosphate temephos
Autores: Ademir Jesus Martins, Thiago Affonso Belinato, Jos Bento Pereira
Lima, Denise Valle
27
Ademir Jesus Martins, Thiago Affonso Belinato, Jos Bento Pereira Lima, Denise
Valle
Laboratrio de Fisiologia e Controle de Artrpodes Vetores Departamento de
Entomologia Instituto Oswaldo Cruz - FIOCRUZ
Abstract: In Brazil, control of the dengue vector is impaired due to resistance of field
Aedes aegypti populations to organophosphates. Insect Growth Regulators are a
promising alternative, since their mechanisms of action are distinct from conventional
insecticides. We verified that triflumuron, a chitin synthesis inhibitor, arrests
development and induces mortality of the insecticide-susceptible strain Rockefeller in
a dose-dependent way (ED50 and ED90 of 0.8 and 1.8 g/L, respectively). A direct
relationship between triflumuron concentration and the precocity of its effects was
noted. The efficacy of triflumuron against two Ae. aegypti field populations was
equivalent, irrespective of their organophosphate resistance status: TemS and TemR
resistance ratios (RR90) against temephos was 4.5 and 13.8, while RR90 against
triflumuron was 1.0 and 1.3, respectively. These results point chitin synthesis
inhibitors as potential alternatives in the control of Ae. aegypti.
1 INTRODUCTION
In the last two decades the number of dengue and dengue hemorrhagic fever cases
increased in Brazil.
urbanization in the country2,3. Both situations are correlated to the presence and the
28
29
2.3 Bioassays
In order to synchronize larvae age, eggs were allowed to hatch for one hour. Groups
of 1,000 larvae were reared in 33 X 24 X 8 cm plastic containers with 1L of
dechlorinated water and small amount cat food (Friskies, Purina, Camaqu/RS),
added only once. Larvae were kept at 26C into a BOD incubator (Electrolab) during
three days, when the beginning of L3 instar was reached.
Four replicas of transparent plastic cups filled with 150 mL dechlorinated water
(control) or triflumuron solutions of varying concentrations (0.5 to 5.0 g/L) and a
small amount of food, added on the first day, were prepared. Each replica contained
20 L3 larvae and each assay was performed four times.
30
Dead and alive larvae, pupae and adults were scored daily until death or adult
emergence of all individuals. Dead specimens and alive adults were removed from
the test cups daily. Adults that survived less than 24 hours were considered dead.
31
A direct relationship between triflumuron concentration and the precocity of its effects
was noted: the proportion of dead larvae increased in high triflumuron
concentrations, while the opposite was observed with the rate of adults (Figure 2).
The same has been previously observed for other IGR compounds.19-21
Table 1 and Figure 3 show results obtained with the exposure of mosquitoes from
two Ae. aegypti field populations to temephos or triflumuron. According to the criteria
proposed by Mazzari and Georghiou (1995),18 one population, TemR, is highly
resistant and the other, TemS, susceptible to temephos (Table 1). Triflumuron
induced a dose-dependent adult emergence inhibition effect in both populations.
However, comparison with results obtained for the insecticide susceptible strain
Rockefeller revealed no triflumuron resistance, even in the TemR population. In
opposition to temephos induced mortality, no significant difference was noted for
triflumuron EI lines shown in Figure 3 (F.05 [2,16]; P>0,05).
Chitin synthesis inhibitors are a good potential alternative against Ae. aegypti
populations resistant to conventional insecticides. We showed that two Brazilian field
populations, one of which resistant to the OP temephos, are as susceptible to the
BPU triflumuron as one insecticide-susceptible laboratory strain. Considering
triflumuron acts on chitin synthesis, this compound is not expected to be harmful to
vertebrates. Although the use of another CSI in potable water has already been
approved by WHOPES,22 when triflumuron is taken into account, toxicological assays
are still needed. We are presently evaluating the effects of sublethal triflumuron
concentrations on the development and reproduction of resulting Ae. aegypti adults.
ACKNOWLEDGEMENTS
This work was supported by the Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq), Fundao de Amparo Pesquisa do Estado do Rio de Janeiro
32
(Faperj),
Fundao
Oswaldo
Cruz
(FIOCRUZ)
and
Coordenao
de
REFERENCES
1. Barbosa-da-Silva JJr, Siqueira JBJr, Coelho GE, Vilarinhos PT and Pimenta
FGJr, Dengue in Brazil: current situation and control activities. Epidemiol Bull
23:3-6 (2002).
2. Mondet PB, Travassos da Rosa APA and Vasconcelos PFC. Les risques
Dpidmisation urbaine de la fivre jaune au Brsil par les vecteurs de la dengue
Aedes aegypti et Aedes albopictus. Bull Soc Path Exot 89:107-114 (1996).
3. Massad E, Coutinho FA, Burattin MN and Lopez LF. The risk of yellow fever in a
dengue-infested area. Trans R Soc Trop Med Hyg 95:370-4 (2001).
4. Franco O. Histria da Febre Amarela no Brasil. Rio de Janeiro: Superintendncia
de Campanhas de Sade Pblica, Ministrio da Sade Brasil (1976).
5. Teixeira MG, Barreto ML and Guerra Z. Epidemiologia e Medidas de Preveno
do Dengue. Informe Epidemiolgico do SUS 8:5-33 (1999).
6. Honrio NA, Cabelo PH, Codeo CT and Loureno-de-Oliveira R. Preliminary
data on the performance of Aedes aegypti and Aedes albopictus immatures
developing in water-filled tires in Rio de Janeiro. Mem Inst Oswaldo Cruz 101:
225-228 (2006).
7. SVS. Secretaria de Vigilncia em Sade Dados e indicadores selecionados.
Coordenao: Departamento de Anlise de Situao de Sade (2003).
8. Macoris MLG, Andrighetti MTM, Takaku L, Glasser CM, Garbeloto VC and Cirino
VCB. Alteration in susceptibility response of Aedes aegypti to organophosphates
in cities in the state of S. Paulo, Brazil. Rev Sade Pb 33:521-522 (1999).
33
9. Lima JBP, Pereira da Cunha M, Silva JRC, Galardo AKR, Soares SS, Braga IA,
Ramos RP and Valle D. Aedes aegypti resistance to organophosphates in several
localities in Rio de Janeiro and Esprito Santo, Brazil. Am J Trop Med Hyg 68:329333 (2003).
10. Braga IA, Lima JBP, Cunha SP, Soares SS, Melo RCGM and Valle D. Aedes
aegypti resistance to temephos during 2001 in several municipalities in Rio de
Janeiro, Sergipe and Alagoas States, Brazil. Mem Inst Oswaldo Cruz 99:199-203
(2004).
11. Funasa. Reunio Tcnica para discutir status de resistncia de Aedes aegypti e
definir estratgias a serem implantadas para monitoramento da resistncia no
Brasil. Braslia: Fundao Nacional de Sade, Ministrio da Sade (1999).
12. Lima JBP, Melo NV and Valle D. Residual effect of two Bacillus thuringiensis var.
israelensis products assayed against Aedes aegypti (Diptera: Culicidae) in
laboratory and outdoors at Rio de Janeiro, Brazil. Rev Inst Med Trop SP 47:125130 (2005).
13. Lima JBP, Melo NV and Valle D. Persistence of Vectobac WDG and Metoprag S2G against Aedes aegypti larvae using a semi-field bioassay in Rio de Janeiro,
Brazil. Rev Inst Med Trop SP 47:7-12 (2005).
14. Graf JF. The role of insect growth regulators in arthropod control. Parasitol Today
9:471-474 (1993).
15. Tunaz, H. and Uygun, N. Insect growth regulators for insect pest control. Turk J
Agric For 28:377-387 (2004).
16. Mondal KAMSH and Parween S. Insect growth regulators and their potential in
the management of stored-product insect pests. Int Pest Man Rev 5:255-295
(2000).
34
17. Raymond
M.
Presentation
dune
programme
danalyse
logprobit
pour
MB
and
Georghiou
GP.
Characterization
of
resistance
to
35
Legends to figures:
Figure 1. Effect of the chitin synthesis inhibitor triflumuron on the mortality and adult
emergence inhibition (EI) rates of Aedes aegypti, Rockefeller strain. The non-linear
regression curves shown were obtained after 14 days of exposure of L3 larvae.
Figure 3. Linear regression curves of Aedes aegypti (A) mortality after 24 hours
exposure to temephos or (B) adult emergence inhibition (EI) after 14 days in contact
with triflumuron.
36
Figure 1
100
80
60
40
EI
mortality
20
0
0
g/L triflumuron
Figure 2:
% Mortality
100
80
60
larvae
pupae
adult
40
20
0
0.0 0.5 0.6 0.7 0.8 0.9 1.0 2.0 5.0
g/ L triflumuron
Figure 3
B
100
100
80
80
60
60
% EI
% mortality
40
40
Rock
20
20
TemS
0
-4
-3
-2
log [temephos] (
g/L)
-1
TemR
-1
log [triflumuron] (
g/L)
37
Table 1. Resistance ratios to temephos and to triflumuron of Ae. aegypti F1 larvae from field populations. Comparison with results
obtained for the insecticide susceptible reference strain Rockefeller.
temephos
triflumuron
ED50
RR50
ED90
RR90
slope
ED50
RR50
ED90
RR90
slope
Rock
0.00139
0.00198
8.32 0.39
0.864
1.804
4.01 0.34
TemS
0.00581
4.2
0.00893
4.5
6.84 0.27
1.142
1.3
1.882
1.0
5.91 0.51
TemR
0.01522
10.9
0.02733
13.8
5.04 0.25
1.588
1.8
2.304
1.3
7.92 1.31
38
3.2) artigo 2: Exposure of Aedes aegypti larvae to a chitin synthesis inhibitor sublethal dose: effects on the viability and reproduction of adults
Autores: Thiago Affonso Belinato, Ademir Jesus Martins, Jos Bento Pereira
Lima, Tamara Nunes de Lima-Camara, Alexandre Afrnio Peixoto, Denise Valle
39
Thiago Affonso Belinato, Ademir Jesus Martins, Jos Bento Pereira Lima, Tamara
Nunes de Lima-Camara, Alexandre Afrnio Peixoto, Denise Valle
40
Introduction
The mosquito Aedes aegypti (L), widely distributed in the tropical and subtropical
regions, is highly adapted to the urban environment. It is frequently found inside or
near the houses, and it plays an important role in the transmission of arboviroses,
such as dengue and urban yellow fever (Consoli & Loureno-de-Oliveira, 1994;
Gubler, 2002).
Nowadays Ae. aegypti control is mainly performed with chemical larvicides that target
the insect central nervous system. However, the long historic of insecticide use has
led to the development of resistant populations all over the world. In this sense, the
study of novel tools to control Ae. aegypti and other insects of medical importance is
a major area of interest (Zaim & Guillet, 2002).
An alternative vectors control approach is the use of compounds with distinct
mechanisms of action and target sites, like the Insect Growth Regulators (IGR).
Those include the juvenile hormone analogs, the ecdysone agonists and the chitin
synthesis inhibitors (CSI) (Graf, 1993; Tunaz & Uygun, 2004).
Chitin synthesis inhibitors are benzoylphenylurea compounds discovered in the
decade of 1970 (Mian & Mula, 1982) that interfere with insect development,
disturbing the molt and resulting in deformations in the cuticle (Reynolds, 1987).
Additionally, adults deriving from CSI-exposed larvae can exhibit a series of
physiological constraints that ultimately lead to a diminished physical and
reproductive fitness (Mondal & Parween, 2000). These CSI effects vary according to
the species, developmental stage at the time of application, kind of compound and
administered dose (Wilson & Cryan, 1997; Mulla et al., 2003; Vasuki & Rajavel,
1992). Although several reports in the literature deal with CSI induced mortality or
adult emergence inhibition (Su et al., 2003; Rehini & Soltani, 1999; Batra et al.,
41
2005), CSI effects on the surviving adults and their implications in the vector fitness
are less exploited, especially when Culicidae are considered (Vasuki, 1992; Vasuki &
Rajavel, 1992; Vasuki, 1999).
Aedes aegypti is a domestic mosquito whose breeding sites, mostly water storage
recipients for human use, are subjected to frequent volume variations, resulting in
decrease of the insecticide residual effect. In this sense, detailed studies of CSI
effects of sublethal doses on the viability of resulting adults will contribute to evaluate
their impact in the field.
We confirmed the effectiveness of triflumuron against field Ae. aegypti populations,
including one strain resistant to the organophosphate temephos (Martins et al.,
2007). In the present study we evaluated the effects of exposure of Ae. aegypti
larvae to a partially lethal dose of the CSI triflumuron on the resulting adults. Several
parameters were investigated, such as the proportion of surviving males and
females, adult longevity and activity, blood feeding, mating and viability of the eggs.
Mosquitoes
Mosquitoes from the Rockefeller strain, an insecticide susceptible reference lineage,
were reared according to standard procedures (Braga et al., 2005) at 25 1 C and
80% r. h.
Bioassays
Starycide SC 0.48 (Bayer) was employed to prepare a stock solution of triflumuron
at 48 g/mL in DMSO (dimethyl sulfoxide) and aliquots were stored at -80C.
42
Male/female rate
Newly emerged adults surviving from triflumuron exposure were daily removed from
the cages and numbers of males and females from both experimental and control
cages were scored.
Longevity
Triplicate pools of 15 newly adult emerged couples derived from triflumuron-exposed
or control larvae were placed in small carton cages (8.5 cm diameter X 8.5 cm high),
and fed continuously with a 10% sucrose solution. Mortality was daily registered
during 16 days.
Activity
The locomotor activity pattern of triflumuron-surviving and control Ae. aegypti
females was evaluated with a locomotor activity monitor (TriKinetics) (Gentile et al.,
2006). Two-day old mosquitoes were individually placed in glass tubes with a cotton
plug soaked in 10% sucrose solution and the tubes placed in a Locomotor Activity
43
Blood feeding
Three-day old adult females were exposed to an anesthetized guinea pig during 30
minutes. The number of blood-fed females was counted in both triflumuron-exposed
and control groups.
In order to quantify the amount of ingested blood, groups of 10 experimental or
control mosquitoes were weighed before or after the blood meal in an analytic
balance (APX-200, Denver Instrument). The amount of ingested blood was
calculated by subtraction of these values.
Egglaying
Egglaying was induced in individual females three days after the blood meal,
according to the procedure adapted from Valencia et al. (1996), in inverted Petri
dishes with a wet filter paper at the bottom, at 26C. The number of egglaying
females, eggs per female and the viability of the eggs were investigated.
44
Results
Several bioassays were performed to accomplish the analysis here shown. From a
total of 12,000 experimental specimens exposed to triflumuron, 43.6% of adult
emergence was obtained. Delay in larvae development was not noted in the
experimental group, when compared to controls: in both cases adult emergence took
5-7 days after onset of the assays.
Male/female rate
In the control group equivalent rates of males (50.2 4.1%) and females were
obtained, while males accounted for 65.0 7.6% of triflumuron-surviving adults, a
highly significant difference between both sexes (ANOVA, p<0,005). This
represented a male/female proportion of 1.01 and 1.86 in control and experimental
groups, respectively.
45
Longevity
Figure 1 shows daily mortality of males and females from control and experimental
groups up to 16 days after adult emergence. In opposition to the control group, with
total mortality below 6% of both males and females during this period, triflumuron
survivors exhibited a reduced longevity. Moreover, females were more affected than
males: after 16 days the rate of surviving males (29.2%) was twice that of females
(13.8%).
Activity
Aedes aegypti adults showed diurnal habits, with peaks of activity in the beginning
and in the end of the photophase. Their pattern of activity was not altered by
triflumuron exposure. However, the intensity of activity was reduced, as exemplified
in Figure 2.
Blood feeding
The ability to ingest blood as well as the feeding rate were significantly altered in CSI
surviving females: while 90.4% (85/94) control females accepted the blood meal, this
rate decreased to 48.4% (77/159) in triflumuron exposed ones (20.05, 1; p<0.005).
Moreover, control and triflumuron surviving females ingested, respectively, 2.57
0.42 and 1.79 0.17 times their weigh in blood, also a significant difference (tS test;
p<0.05). In spite of this, the weigh of control (7.37 0.66 g) and triflumuron
surviving (7.71 1.02 g) females after adult emergence is equivalent (tS test;
p>0.05).
46
Egglaying
Table 1 summarizes oviposition data of control and triflumuron-exposed females.
Approximately 10% of experimental blood-fed females died up to 24 hours after
blood feeding; even higher mortality (35%) was observed after egglaying. The
number of eggs per female was also significantly different in both groups (t test;
p<0.001).
Among the triflumuron-surviving females that accomplished oviposition, 14.4%
presented eggs that broke when immersed in water (Figure 3). Spermathecae
dissection revealed these females had not been inseminated. On the other hand, no
significant difference was noted in the viability of eggs derived from triflumurontreated females that had effectively been inseminated (96.3 10.2%) compared to
control ones (97.5 7.5%).
Matings
In order to verify if triflumuron preferentially interferes with the reproductive ability of
one sex, individual matings with one control partner and one triflumuron survivor
were performed. Although 75 couples have been done for each condition, at the end
of three days one or both specimens of several couples had died (Table 2). When
surviving couples were taken into account, a reduction in the rate of inseminated
females was noted in all groups bearing at least one triflumuron-treated mate (Table
2). Chi-square analysis revealed difference between the non treated control and all
the experimental groups containing triflumuron survivors (p<0.05). On the other hand,
no significant differences among these later groups were found (p>0.05).
47
Discussion
Our data indicate that exposure of Ae. aegypti larvae to sublethal doses of the CSI
triflumuron results in mosquito adults affected in several aspects of their viability and
reproductive ability.
Disequilibrium in the proportion between males and females derived from treatment
with the triflumuron EI50 was observed. The smaller proportion of females was
attributed to their slower development kinetics when compared to males (Clements,
1992). As a consequence females would be exposed to the CSI for a longer period of
time in the larval stage. Exposure of the Coleoptera Tribolium confusum to
triflumuron also resulted in a greater proportion of males (3:1) (El-Sayed et al., 1984).
Adult longevity was highly reduced in triflumuron treated specimens. Females were
more severely affected than males, probably due to their longer period of contact with
the CSI during the larval stage, as mentioned above. Tenebrio molitor exposed to the
CSI diflubenzuron also exhibit reduced longevity (Soltani et al., 1983). Although a
drastic reduction in the longevity of Culex quinquefasciatus, Anopheles stephensi
and Ae. aegypti adults derived from treatment with the EI50 of the CSI hexaflumuron
has been reported (Vasuki, 1992), no differences in longevity between both sexes
were noted in these cases.
Triflumuron also reduced Ae. aegypti activity. Many triflumuron surviving adults had
structural abnormalities, such as deformed wings or easily broken legs, which could
account for this reduced activity. There are reports of structural defects produced by
treatment with CSI in several insect orders (Demark & Bennet, 1989; Wilson &
Cryan, 1997; Da-Silva et al., 2004). Vasuki & Rajavel (1992) observed that treatment
of the mosquitoes Cx. quinquefasciatus, Ae. aegypti and An. stephensi with a CSI
resulted in several visible abnormalities that could hamper locomotion and that were
48
probably related to the reduced longevity of adults. Accordingly, Amir & Peveling
(2004) verified that triflumuron-exposed Apis mellifera specimens exhibited a lower
flight activity.
Triflumuron treatment interfered with the blood feeding ability of surviving females:
both the number of blood-fed females and the amount of ingested blood were
reduced in triflumuron-treated individuals. The same was reported by Vasuki (1999)
with other Culicidae species after treatment with the CSI hexaflumuron. In opposition
to this author, we observed mortality after egglaying in a high proportion of females,
probably due to their greater weakness.
Since blood feeding is a requisite to the production of eggs in hematophagous
insects, it was expected that oviposition of triflumuron-surviving females was
reduced. This was effectively the case: triflumuron-treated females laid roughly 25%
less eggs than control ones, a rate compatible with the 30% reduction in the amount
of blood ingested by this group when compared to the control. Reduction in the
oviposition of CSI-treated females of different insect orders has already been
reported (Miura et al., 1976; Wilson & Cryan, 1997; Senz-de-Cabezn et al., 2006).
However, Arias and Mulla (1975) did not detect any significant difference in the
amount of eggs laid by Culex tarsalis females treated with diflubenzuron, another
CSI. The number of eggs laid by methoprene-surviving Ae. aegypti females was also
equivalent to the control group.
A significative proportion of the eggs laid by triflumuron surviving females cracked
when immersed in water, and the remaining ones shrink (figure 3). Observation of
spermathecae confirmed the corresponding females had not been inseminated. We
asked if reduction of copula rates was due to preferential interference of triflumuron
49
with males or females, or if both sexes were affected. Crosses between control and
triflumuron-surviving adults revealed CSI interferes with both sexes.
Our results indicate that CSIs are a viable alternative to the control of Ae. aegypti.
Beyond its effectiveness against larvae (Martins et al. 2007), surviving adults exhibit
a series of physiological constraints that reduce their longevity and reproductive
ability. Hence, CSI survivors are expected to have a reduced vectorial capacity,
being less competent in the transmission of pathogens. Furthermore, the current
need of environmentally safer technologies requires the development of insecticides
with more selective target sites and with a reduced risk level to non-target organisms.
It was already verified that some CSI compounds have little or no effect on
mammals, birds and aquatic invertebrates (Mian & Mulla, 1982). Ecotoxicity studies
with triflumuron are a requisite towards its utilization against insect vectors of medical
importance.
Acknowledgements
We thank Diego de Lacerda Rosa, Nathalia Giglio Fontoura and Diogo Fernandes
Bellinato for technical assistance. This work was supported by Fundao Oswaldo
Cruz FIOCRUZ, Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
CNPq and Fundao de Amparo Pesquisa do Estado do Rio de Janeiro
FAPERJ. We are grateful to BayerCropscience for the triflumuron sample.
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comparison with Drosophila. Cryobiology, 33, 142-148.
Vasuki, V. & Rajavel, R. (1992) Influence of short time exposure to an insect growth
regulator, hexaflumuron, on mortality and adult emergence of vector mosquitoes.
Memrias do Instituto Oswaldo Cruz, 87, 275-283.
Vasuki, V. (1992) Adult longevity of certain mosquito species after larval and pupal
exposure to sublethal concentration of an insect growth regulator hexaflumuron.
Southeast Asian Tropical Medicine Public Health, 23, 121-124.
Vasuki, V. (1999) Influence of IGR treatment on oviposition of three species of vector
mosquitoes at sublethal concentrations. Southeast Asian Tropical Medicine Public
Health, 30, 200-203.
Wilson, T.G. & Cryan, J.R. (1997) Lufenuron, a chitin-synthesis inhibitor, interrupts
development of Drosophila melanogaster. The Journal of Experimental Zoology, 278,
37-44.
53
54
Legends to figures
Figure 1: daily mortality of control (circles) and triflumuron-surviving (squares) Ae.
aegypti adults. Opened and closed symbols refer to females and males, respectively.
Figure 2: Aedes aegypti females activity rhythm during four days under a 12:12 light
/ dark (gray bars) regimen. Solid and dashed lines represent control and triflumuronsurviving females, respectively.
Figure 3: Eggs laid by Ae. aegypti triflumuron-surviving females. Broken eggs derive
from unfertilized specimens.
55
Figure 1
% mortality
100
80
60
40
20
0
0
10
12
14
16
Figure 2
meandaactivity
(W(Wx)
Mdia
atividade
X)
control
controle
4,0
triflumuron
triflumuron
3,0
2,0
1,0
0,0
Tempo (dias)
time (days)
56
Figure 3
57
Mortality after
Females
Mortality after
Mean
blood meal
without eggs
egglaying
eggs/females
Control
100
112.9 41.1
triflumuron
139
14
14
44
86.4 44.0
Inseminated females
n (%)
n (%) (***)
cont x cont
75 (100)
74 (99)a
cont x trif
45 (60)
15 (33)b
trif x cont
66 (88)
22 (33)b
trif x trif
44 (59)
8 (18)b
(*) cont: control; trif: adult surviving from triflumuron exposure, performed in the larval
stage.
(**) 75 individual couples were made for each experimental condition.
(***) values followed by the same letter do not differ significantly.
58
Autores: Thiago Affonso Belinato, Ademir Jesus Martins, Jos Bento Pereira
Lima, Denise Valle
59
60
Introduo
O controle de mosquitos vetores de doenas tem sido dificultado em
decorrncia principalmente do desenvolvimento de resistncia aos inseticidas
tradicionais comumente utilizados (Bracco et al. 1999, Lima et al. 2003, Braga et al.
2004, Liu et al. 2004), assim como de problemas ao meio ambiente relacionados ao
uso indiscriminado dessas substncias (Zaim & Guillet 2002). Os reguladores do
desenvolvimento dos insetos (IGRs), com mecanismo de ao distinto e mais
seletivos quando em comparao com os inseticidas convencionais e, em
conseqncia, mais seguros para a maioria dos organismos no-alvo, so
considerados uma alternativa promissora (Mian e Mulla 1982, Graf 1993). Embora a
ao de muitos IGRs tenha sido testada contra uma variedade de insetos, um
nmero limitado destes compostos foi avaliado em culicdeos vetores (Estrada &
Mulla 1986, Su et al. 2003, Seng et al. 2006).
Os inibidores da sntese de quitina (CSI, do ingls chitin synthesis
inhibitors) so IGRs que interferem com o processo de muda nos insetos. Os CSIs
pertencem famlia das benzo-fenil-urias (BPUs), grupo qumico que tem sido
extensivamente estudado desde sua descoberta na dcada de 1970 (Mian & Mula
1982). De modo geral, a ao dos CSIs resulta em mal-formaes da nova cutcula
em desenvolvimento prejudicando principalmente as larvas, que geralmente tornamse incapazes de sobreviver prxima muda (Reynolds 1987, Tunaz & Uygun 2004).
Existem ainda relatos de sua eficincia sobre ovos (Wilson & Cryan 1997) e adultos
(Belinato et al. 2007, Vasuki 1992a,b, 1999).
Aedes aegypti, Aedes albopictus e Culex quinquefasciatus so trs
espcies
de
culicdeos
amplamente
difundidos
pelo
globo,
distribudos
61
63
64
65
primeiro
usado
inibidor
no
da
controle
sntese
de
de
diversas
quitina
espcies
disponibilizado
de
insetos,
66
Fundao
Oswaldo
Cruz
FIOCRUZ,
Conselho
Nacional
de
do
Estado
do
Rio
de
Janeiro
FAPERJ.
Agradecemos
67
Referncias
Ali A, Nayar JK, Rui-de Xue 1995. Comparative toxicity of selected larvicides and
insect growth regulators to a Florida laboratory population of Aedes albopictus. J
Am Mosq Control Assoc 36: 72-76.
Batra CP, Mittal PK, Adak T, Ansari MA 2005. Efficacy of IGR compound Starycide
480 SC (Triflumuron) against mosquito larvae in clear and polluted water. J Vect
Borne Dis 42: 109-116.
Belinato TA, Martins AJ, Lima JBP, Lima-Camara TN, Peixoto AF, Valle, D 2007.
Exposure of Aedes aegypti larvae to a chitin synthesis inhibitor sub-lethal dose:
effects on the viability and reproduction of adults. In preparation.
Boudjelida H, Bouaziz A, Soin T, Smagghe G, Soltani N 2005. Effects of ecdysone
agonist halofenozide against Culex pipiens. Pestic Biochem Physiol 83: 115-123.
Bracco JE, Barata, JMS, Marinotti O 1999. Evaluation of insecticide resistance and
biochemical mechanisms in a population of Culex quinquefasciatus (Diptera:
Culicidae) from So Paulo, Brazil. Mem Inst Oswaldo Cruz 94: 115-120.
Braga IA, Lima JBP, Cunha SP, Soares SS, Melo RCGM, Valle D 2004. Aedes
aegypti resistance to temephos during 2001 in several municipalities in Rio de
Janeiro, Sergipe and Alagoas States, Brazil. Mem Inst Oswaldo Cruz 99: 199203.
Braga IA, Mello CB, Peixoto AA, Valle D 2005. Evaluation of methoprene effect on
Aedes aegypti (Diptera: Culicidae) development in laboratory conditions. Mem
Inst Oswaldo Cruz 100: 435-440.
Da-Cunha MP, Lima JBP, Brogdon WG, Moya GE, Valle D 2005. Monitoring of
resistance to the pyrethroid cypermetrin in Brazilian Aedes aegypti (Diptera:
Culicidae) populations collected between 2001 and 2003. Mem Inst Oswaldo
Cruz 100: 441-444.
Davidson G, Zahar AR 1973. The practical implications of resistance of malaria
vectors to insecticides. Bull WHO 49: 475-483.
Estrada JG, Mulla MS 1986. Evaluation of two new insect growth regulators against
mosquitoes in the laboratory. J Am Mosq Control Assoc 2: 57-60.
Graf JF 1993. The role of insect growth regulators in arthropod control. Parasitol
Today 9: 471-474.
Gubler DJ 2002. Epidemic dengue/ dengue hemorrhagic fever as a public health,
social and economic problem in the 21st century. Tr Microbiol 10: 100-103.
68
MB,
Georghiou
GP
1995.
Characterization
of
resistance
to
69
70
71
Tabela 1: Nvel de susceptibilidade das populaes de Ae. aegypti ao organofosforado temephos e ao piretride deltametrina.
Regio
NE
SE
CW
Estado
temephosb
deltametrina
RR95
% mort DD
triflumuron, % mort
IE99
DDd
SE
Aracaju
19,3
88,1
100,0
100,0
AL
Macei
10,3
62,9
100,0
100,0
Belo Horizontea
5,4
74,5
100,0
100,0
Montes Claros
13,6
62,7
100,0
100,0
Dourados (Norte)
4,3
87,9
100,0
100,0
Dourados-(Sul)
7,1
61,9
100,0
100,0
Cuiab
4,0
89,2
100,0
100,0
MG
MS
MT
localidade
Georghiou 1995)
c
A dose diagnstica (DD) de deltametrina usada foi de 5 g ug/garrrafa (metodologia de Da-Cunha et al. 2005). Neste ensaio,
A dose diagnstica de triflumuron empregada, equivalente ao dobro da IE99, foi de 7,9 g/L.
72
73
Figura 1
100
100
Mortalidade (%)
IE (%)
80
60
40
20
0
larva
pupa
adulto
80
60
40
20
0
doses (ug/L)
0.9
1.25
1.5
1.75
2.5
2.75
3.5
doses (ug/L)
74
4.5
Figura 2
b
100
80
80
Mortalidade (%)
100
IE
(%)
60
40
20
60
la r va
pupa
a d u lto
40
20
0
0
0
10
doses (ug/L)
15
20
75
Figura 3
Mortalidade (%)
100
a,b
a,b
a,b
a,b
c
larva
pupa
80
60
40
20
0
Rock
D. Sul
Mortalidade (%)
100
80
larva
pupa
60
40
20
0
Rock
D. Sul
76
4) Discusso
Aedes aegypti, Ae. albopictus e Cx. quinquefasciatus so trs espcies de
mosquitos amplamente difundidas pelo territrio brasileiro, cuja presena constitui
um fato extremamente preocupante, uma vez que podem participar da transmisso
de diversas arboviroses. Atualmente, existe uma grande preocupao com a
possvel entrada do Vrus do Oeste do Nilo (VNO) na Amrica do Sul, j que esse
vrus se difundiu rapidamente pela Amrica do Norte, provocando epidemias, como
a de Nova York em 1999 (Goddard et al., 2003). O VNO pertence famlia
Flaviviridae, gnero Flavivirus, no qual tambm esto classificados os vrus da
hepatite C (HCV), da febre amarela, da encefalite japonesa, da encefalite de So
Lus e da dengue. Algumas aves constituem o reservatrio natural, e o ciclo do vrus
mantido na natureza atravs da relao entre as mesmas e mosquitos do gnero
Culex sp (Campbell et al., 2002). Culex quinquefasciatus e Ae. albopictus podem
representar potenciais vetores no Brasil, uma vez que linhagens destas espcies
susceptveis ao vrus tm sido encontradas na Amrica do Norte (Moore e Mitchell,
1997; Hayes et al., 2005).
Preocupao adicional existe com Ae. albopictus, principalmente devido a
sua grande valncia ecolgica. Aedes albopictus pode ser encontrado tanto em
habitats silvestres quanto em locais suburbanos ou urbanos (Juliano e Lounibos,
2005), podendo ser responsvel pela urbanizao de arboviroses, como a febre
amarela, por exemplo. Epidemias de febre amarela no existem no Brasil desde a
dcada de 1950, apesar da existncia do ciclo silvestre (Mondet et al., 1996).
Mondet et al. (2001) relatam que o risco de reurbanizao da febre amarela no Brasil
grande, uma vez que Ae. aegypti est amplamente distribudo no pas e muitas
vezes co-existe com Ae. albopictus. De acordo com Massad et al. (2001), o risco de
novas epidemias de febre amarela no Brasil iminente. Loureno-de-Oliveira et al.
(2003) verificaram que populaes brasileiras de Ae. albopictus so passveis de
infeco com o vrus da febre amarela.
O culicdeo Ae. aegypti, por sua vez, alm de ser um potencial vetor de
febre amarela urbana, continua sendo o nico vetor confirmado na transmisso de
dengue no Brasil. Atualmente a dengue considerada uma das arboviroses mais
importantes, sendo endmica em mais de 100 pases, localizados em regies
tropicais e sub-tropicais, predominando em reas urbanas e semi-urbanas (WHO,
1997, 2002). No Brasil, tm ocorrido epidemias freqentes de dengue em grande
77
78
80
5) Concluses
81
6) Perspectivas
Nosso
Laboratrio
investiga
diversos
aspectos
da
fisiologia
do
82
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