The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions.

BACKGROUND: In the tropics, the utilization of insecticides is still an important strategy for controlling Aedes aegypti, the principle vector of dengue, chikungunya and Zika viruses. However, increasing insecticide resistance in Ae. aegypti populations might hinder insecticide efficacy on a long-term basis. It will be important to understand the dynamics and evolution of insecticide resistance by assessing its frequency and the mechanisms by which it occurs. METHODOLOGY/PRINCIPAL FINDINGS: The insecticide resistance status of four Brazilian Ae. aegypti populations was monitored. Quantitative bioassays with the major insecticides employed in the country was performed: the adulticide deltamethrin (a pyrethroid-PY) and the larvicides, temephos (an organophosphate) and diflubenzuron (a chitin synthesis inhibitor). Temephos resistance was detected in all populations although exhibiting a slight decrease over time probably due to the interruption of field use. All vector populations were susceptible to diflubenzuron, recently introduced in the country to control Ae. aegypti. Resistance against deltamethrin was extremely high in three populations. Molecular assays investigated substitutions in the voltage gated sodium channel (NaV), the PY target site, at positions 1011, 1016 and 1534. Elevated frequencies of substitutions Val1016Ile and Phe1534Cys related to high PY resistance levels were identified. Biochemical assays detected alterations in the activities of two detoxifying enzyme classes related to metabolic resistance, glutathion-S-transferases and esterases. The results obtained were evaluated in the context of both recent insecticide use and the records of dengue incidence in each locality. CONCLUSIONS/SIGNIFICANCE: The four Ae. aegypti populations evaluated were resistant to the neurotoxic insecticides, temephos and deltamethrin. However, they were still susceptible to diflubenzuron. A probable correlation between adult insect resistance to PY and the domestic application of insecticides is discussed, pointing to the need for awareness measures regarding the correct utilization by citizens. This work aims to contribute to the efficient and rational management of Ae. aegypti control of both larvae and adults.

Resistance Status to the Insecticides Temephos, Deltamethrin, and Diflubenzuron in Brazilian Aedes aegypti Populations.

Insecticides are still largely applied in public health to control disease vectors. In Brazil, organophosphates (OP) and pyrethroids (PY) are used against Aedes aegypti for years. Since 2009 Insect Growth Regulators (IGR) are also employed in the control of larvae. We quantified resistance to temephos (OP), deltamethrin (PY), and diflubenzuron (IGR) of A. aegypti samples from 12 municipalities distributed throughout the country, collected between 2010 and 2012. High levels of resistance to neurotoxic insecticides were detected in almost all populations: RR95 to temephos varied between 4.0 and 27.1; the lowest RR95 to deltamethrin was 13.1, and values higher than 70.0 were found. In contrast, all samples were susceptible to diflubenzuron (RR95 < 2.3). Biochemical tests performed with larvae and adults discarded the participation of acetylcholinesterase, the OP target, and confirmed involvement of the detoxifying enzymes esterases, mixed function oxidases, and glutathione-S-transferases. The results obtained were discussed taking into account the public chemical control component and the increase in the domestic use of insecticides during dengue epidemic seasons in the evaluated municipalities.

Discrepancies between Aedes aegypti identification in the field and in the laboratory after collection with a sticky trap.

Currently, sticky traps are regularly employed to assist in the surveillance of Aedes aegypti infestation. We tested two alternative procedures for specimen identification performed by local health agents: directly in the field, as recommended by certain manufacturers, or after transportation to the laboratory. A total of 384 sticky traps (MosquiTRAP) were monitored monthly during one year in four geographically representative Brazilian municipalities. When the same samples were inspected in the field and in the laboratory, large differences were noted in the total number of mosquitoes recorded and in the number of specimens identified as Ae. aegypti by both procedures. Although field identification has the potential to speed vector surveillance, these results point to uncertainties in the evaluated protocol.

Discrepancies between Aedes aegypti identification in the field and in the laboratory after collection with a sticky trap

Currently, sticky traps are regularly employed to assist in the surveillance of Aedes aegypti infestation. We tested two alternative procedures for specimen identification performed by local health agents: directly in the field, as recommended by certain manufacturers, or after transportation to the laboratory. A total of 384 sticky traps (MosquiTRAP) were monitored monthly during one year in four geographically representative Brazilian municipalities. When the same samples were inspected in the field and in the laboratory, large differences were noted in the total number of mosquitoes recorded and in the number of specimens identified as Ae. aegypti by both procedures. Although field identification has the potential to speed vector surveillance, these results point to uncertainties in the evaluated protocol.

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.

Cocirculation of two dengue virus serotypes in individual and pooled samples of Aedes aegypti and Aedes albopictus larvae.

INTRODUCTION: To detect dengue virus, eggs of Aedes sp were collected in the city of Belo Horizonte, Brazil, in 2007. METHODS: Egg samples were subsequently hatched and the larvae were tested for the presence of dengue virus RNA by RT-PCR. RESULTS: Among the Aedes aegypti larvae samples, 163 (37.4%) out of 435 were positive, including 32 (10.9%) of 293 individual larvae samples concomitantly positive for two serotypes. CONCLUSIONS: Virological surveillance detecting coinfected vectors in the field could represent an important strategy for understanding the numerous factors involved in the transmission and clinical presentation of dengue.

Cocirculation of two dengue virus serotypes in individual and pooled samples of Aedes aegypti and Aedes albopictus larvae / Cocirculação de dois sorotipos do vírus dengue em amostras individuais e pools de larvas de Aedes aegypti e Aedes albopictus

INTRODUCTION: To detect dengue virus, eggs of Aedes sp were collected in the city of Belo Horizonte, Brazil, in 2007. METHODS: Egg samples were subsequently hatched and the larvae were tested for the presence of dengue virus RNA by RT-PCR. RESULTS: Among the Aedes aegypti larvae samples, 163 (37.4 percent) out of 435 were positive, including 32 (10.9 percent) of 293 individual larvae samples concomitantly positive for two serotypes. CONCLUSIONS: Virological surveillance detecting coinfected vectors in the field could represent an important strategy for understanding the numerous factors involved in the transmission and clinical presentation of dengue.

INTRODUÇÃO: Para a detecção do vírus da dengue, ovos de Aedes sp foram coletados em Belo Horizonte, Brasil, em 2007. MÉTODOS: Amostras de ovos eclodiram e suas larvas foram testadas para a presença de RNA do vírus dengue por RT-PCR. RESULTADOS: Das amostras de larvas de Aedes aegypti, 163 (37,4 por cento) de 435 foram positivas, incluindo 32 (10,9 por cento) das 293 amostras individuais que foram concomitantemente positivas para dois sorotipos. CONCLUSÕES: A vigilância virológica de vetores no campo poderia representar uma estratégia importante para a compreensão dos diversos fatores envolvidos na transmissão e apresentação clínica da dengue.

Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil.

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.

Identification and molecular cloning of insecticidal toxins from the venom of the brown spider Loxosceles intermedia.

The venom of Loxosceles intermedia was investigated for the presence of insecticidal toxins active against Spodoptera frugiperda (Lepdoptera: Noctuidade), an insect that has caused great reductions in corn production in Brazil. A combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (Carboxymethyl Cellulose, CM 52) resulted in four major fractions that were submitted to biological assay. Fraction 4 was further purified by a reverse phase HPLC (C18 Column) resulting in peptides active against Spodoptera frugiperda. Three new potential insecticidal toxins named LiTx x 1, LiTx x 2 and LiTx x 3 were identified. The partial amino terminal sequences of these peptides were obtained and used to clone the corresponding cDNAs with the help of degenerate oligonucleotides. The amino acid sequence deduced from the cDNA of LiTx x 1, LiTx x 2 and LiTx x 3 revealed mature proteins of approximately 7.4, 7.9 and 5.6 kDa.

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3' region with 218 nucleotides. LiD1 was expressed as a protein fused with beta-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.