José Bento Pereira Lima

Aedes aegypti resistance to temephos during 2001 in several municipalities in the states of Rio de Janeiro, Sergipe, and Alagoas, Brazil

For more than 30 years temephos, an organophosphate insecticide, has been the sole larvicide used in Brazil in the control of Aedes aegypti. Organophosphates were also used for adult control, being replaced by pyrethroids since 1999. In this same year the Brazilian Health Foundation started the coordination of the Ae. aegypti Insecticide Resistance Monitoring Program. In the context of this program, our group was responsible for the detection of temephos resistance in a total of 12 municipalities in the states of Rio de Janeiro (RJ), Alagoas (AL), and Sergipe (SE) during 2001. In each municipality, a pool of mosquitoes collected from different districts was used, with the exception of Rio de Janeiro city, where eight districts have been separately evaluated. Exposure of larvae to the diagnostic dose of temephos revealed resistance in all localities examined, with mortality levels ranging from 4% (Pilares district, Rio de Janeiro, RJ) to 61.9% (Campos dos Goytacazes, RJ). Quantification of mortality showed resistance ratios from 6.1 (Aracaju, SE) to 16.8 (São Gonçalo, RJ and Penha district, Rio de Janeiro, RJ). The national dengue control program is presently using these data to subside insecticide resistance management.

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.

Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Undesirable consequences of insecticide resistance following Aedes aegypti control activities due to a dengue outbreak.

Background During a dengue outbreak with co-circulation of DENV-1 and -2 in the city of Boa Vista, one patient was diagnosed with DENV-4, a serotype supposed absent from Brazil for almost 30 years. The re-emergence of DENV-4 triggered the intensification of mechanical and chemical Aedes aegypti control activities in order to reduce vector density and avoid DENV-4 dissemination throughout the country. Methods/Principal Findings Vector control activities consisted of (a) source reduction, (b) application of diflubenzuron against larvae and (c) vehicle-mounted space spraying of 2% deltamethrin to eliminate adults. Control activity efficacy was monitored by comparing the infestation levels and the number of eggs collected in ovitraps before and after interventions, performed in 22 Boa Vista districts, covering an area of ∼80% of the city and encompassing 56,837 dwellings. A total of 94,325 containers were eliminated or treated with diflubenzuron. The most frequently positive containers were small miscellaneous receptacles, which corresponded to 59% of all positive breeding sites. Insecticide resistance to deltamethrin was assessed before, during and after interventions by dose-response bioassays adopting WHO-based protocols. The intense use of the pyrethroid increased fourfold the resistance ratio of the local Ae. aegypti population only six months after the beginning of vector control. Curiously, this trend was also observed in the districts in which no deltamethrin was applied by the public health services. On the other hand, changes in the resistance ratio to the organophosphate temephos seemed less influenced by insecticide in Boa Vista. Conclusions Despite the intense effort, mosquito infestation levels were only slightly reduced. Besides, the median number of eggs in ovitraps remained unaltered after control activity intensification. The great and rapid increase in pyrethroid resistance levels of natural Ae. aegypti populations is discussed in the context of both public and domestic intensification of chemical control due to a dengue outbreak.

Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations.

One of the major insect pyrethroid resistance mechanisms affects its target site, the voltage‐gated sodium channel (Nav). In Aedes aegypti, the Val1016Ile substitution of the AaNav gene is associated to resistance in several Latin American countries. Genotyping of susceptible and resistant mosquitoes from seven Brazilian localities detected the 1016Ile mutation in five populations with a higher frequency of this substitution in resistant specimens in all cases. Furthermore, analysis of nine additional field populations revealed that five also presented the 1016Ile mutation. Our data suggest a recent dissemination and involvement of this substitution with pyrethroid resistance in Brazil.

Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: allozymes and mitochondrial DNA restriction fragment length polymorphisms.

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM.

Effectiveness of Methoprene, an Insect Growth Regulator, Against Temephos-Resistant Aedes aegypti Populations from Different Brazilian Localities, Under Laboratory Conditions

Abstract The susceptibility of Aedes aegypti (L.) larvae from several Brazilian populations to the juvenile hormone analog methoprene and the organophosphate insecticide temephos were investigated. Populations from Natal (northeastern region), Macapá (northern region), and Jardim América, Rio de Janeiro (southeastern region) are temephos-resistant (RR90 = 24.4, 13.3, and 15.8, respectively), whereas populations from Presidente Prudente (southeastern region) and Porto Velho (northern region) exhibit only an incipient temephos-altered susceptibility status (RR90 = 1.8 and 2.6, respectively). Biochemical assays revealed alterations of the enzymes implicated in metabolic resistance, glutathione S-transferase, mixed function oxidases and esterases, among these populations. Dose–response assays showed at most a low resistance to methoprene of all populations tested, irrespective of their temephos resistance level. However, sequential exposure of Macapá and Natal populations to temephos and methoprene indicated a potential cross-resistance when larvae are exposed to both insecticides. Nevertheless, susceptibility of the Brazilian Ae. aegypti populations to methoprene alone suggests this insect growth regulator could substitute for temephos in the control of the dengue vector in the country.

Ecology of Sand Flies (Diptera: Psychodidae: Phlebotominae) in the North of the State of Mato Grosso, Brazil

Peixoto de Azevedo is located in the north of State of Mato Grosso, where environmental alterations led to an outbreak of American cutaneous leishmaniasis in the 80s. The parasite from patients was characterized as Leishmania (V.) braziliensis. The aim of this study is to contribute to the sand fly ecology of Central-West Brazil. Captures were carried out monthly using CDC light traps. Twenty-six species of sand fly were characterized; among which Lutzomyia (Lutzomyia) spathotrichia, L. runoides and L. (Psychodopygus) llanosmartinsi were recorded in the State of Mato Grosso for the first time. L. (Nyssomyia) whitmani, L. (N.) antunesi, L. (L.) spathotrichia, L. (P.) c. carrerai, L. (P.) complexa, L. (P.) lainsoni and L. (N.) umbratilis constituted 92.4% of the local fauna, among which L. (N.) whitmani and L. (N.) antunesi, accounting for about 53% of the fauna at the stations of capture. On the vertical distribution of sand flies on the Beira-Rio Farm, L. (N.) whitmani and L. (N.) antunesi prevailed at ground level and in the canopy, respectively, whereas on the BR-080, L. (P.) llanosmartinsi was prevalent on the ground and L. (P.) c. carrerai, in the canopy. It is suggested that L. (N.) umbratilis is the local vector.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Chitin synthesis inhibitor effect on Aedes aegypti populations susceptible and resistant to organophosphate temephos.

BACKGROUND In Brazil, dengue vector control is hampered by the resistance of Aedes aegypti L. populations to organophosphates (OPs). Insect growth regulators (IGRs) are a promising alternative, as their mechanisms of action are different from those of conventional insecticides. The authors analysed the effect of the IGR triflumuron, a chitin synthesis inhibitor, on the Ae. aegypti insecticide-susceptible strain Rockefeller, as well as on field populations both susceptible (TemS) and resistant (TemR) to the OP temephos. RESULTS Triflumuron arrested development and inhibited adult emergence of the Rockefeller strain in a dose-dependent way (EI(50) and EI(90) of 0.8 and 1.8 microg L(-1) respectively). A direct relationship between triflumuron concentration and the precocity of its effects was evident. TemS and TemR temephos resistance ratios (RR(90)) were 4.5 and 13.8, triflumuron RR(90) being 1.0 and 1.3 respectively. CONCLUSION The IGR triflumuron exhibited a dose-dependent effect against the reference Ae. aegypti Rockefeller strain. It was also effective against two field populations, regardless of their OP resistance status. The present results are discussed in the context of utilization of chitin synthesis inhibitors as potential alternatives in the control of Ae. aegypti in Brazil.