Comparison of the efficacy of long-lasting insecticidal nets PermaNet® 2.0 and Olyset® against Anopheles albimanus under laboratory conditions

Insecticide-treated nets provide a reduction in human-vector contact through physical barrier, mortality and/or repellent effects that protect both users and non-users, thereby protecting the wider community from vector-borne diseases like malaria. Long-lasting insecticide-treated nets (LLINs) are the best alternative. This study evaluated the bioefficacy of LLINs PermaNet® 2.0 and Olyset® under laboratory conditions with Anopheles albimanus. The laboratory strain was evaluated for insecticide susceptibility with selected insecticides used for malarial control. Regeneration time and wash resistance were evaluated with the standard bioassay cone technique following WHO guidelines. Heat assistance was used for Olyset® nets; the nets were exposed to four different temperatures to speed the regeneration process. The regeneration study of PermaNet® 2.0 showed that efficacy was fully recovered by 24 h after one and three washes and wash resistance persisted for 15 washes. Regeneration of Olyset® nets was not observed for nets washed three times, even with the different temperature exposures for up to seven days. Thus, for Olyset® the wash resistance evaluation could not proceed. Differences in response between the two LLINs may be associated with differences in manufacturing procedures and species response to the evaluated LLINs. PermaNet® 2.0 showed higher and continuous efficacy against An. albimanus.

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.

Malaria transmission blocking immunity and sexual stage vaccines for interrupting malaria transmission in Latin America

Malaria is a vector-borne disease that is considered to be one of the most serious public health problems due to its high global mortality and morbidity rates. Although multiple strategies for controlling malaria have been used, many have had limited impact due to the appearance and rapid dissemination of mosquito resistance to insecticides, parasite resistance to multiple antimalarial drug, and the lack of sustainability. Individuals in endemic areas that have been permanently exposed to the parasite develop specific immune responses capable of diminishing parasite burden and the clinical manifestations of the disease, including blocking of parasite transmission to the mosquito vector. This is referred to as transmission blocking (TB) immunity (TBI) and is mediated by specific antibodies and other factors ingested during the blood meal that inhibit parasite development in the mosquito. These antibodies recognize proteins expressed on either gametocytes or parasite stages that develop in the mosquito midgut and are considered to be potential malaria vaccine candidates. Although these candidates, collectively called TB vaccines (TBV), would not directly stop malaria from infecting individuals, but would stop transmission from infected person to non-infected person. Here, we review the progress that has been achieved in TBI studies and the development of TBV and we highlight their potential usefulness in areas of low endemicity such as Latin America.

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.

The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.

Characterization of the immunological properties of " Plasmodium falciparum " and " Plasmodium berghei " circumsporozoite proteins : antibody responses and recognition by specific CD8+ T cells

SUMMARY Interest in developing intervention strategies against malaria by targeting the liver stage of the Plasmodium life cycle has been fueled by studies which show that sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites. Anti-malarial drugs and insecticides have been widely used to control the disease, but in the hope of developing a more cost-effective intervention strategy, vaccine development has taken centre stage in malaria research. There is currently no vaccine against malaria. Attenuated sporozoite-induced immunity is achieved by antibodies and T cells against malaria liver stage antigens, the most abundant being the circumsporozoite protein (CSP), and many vaccine formulations aim at mimicking this immunity. However, the mechanisms by which the antibody and T cell immune responses are generated after infection by sporozoites, or after immunization with different vaccine formulations are still not well understood. The first part of this work aimed at determining the ability of primary hepatocytes from BALB/c mice to process and present CSP-derived peptides after infection with P. berghei sporozoites. Both infected hepatocytes and those traversed by sporozoites during migration were found to be capable of processing and presenting the CSP to specific CD8+ T cells in vitro. The pathway of processing and presentation involved the proteasome, aspartic proteases and transport through a post-Endoplasmic Reticulum (ER) compartment. These results suggest that in vivo, infected hepatocytes contribute to the elicitation and expansion of a T cell response. In the second part, the antibody responses of CB6F1 mice to synthetic peptides corresponding to the N- and C-terminal domains of P. berghei and P. falciparum CS proteins were characterized. Mice were immunized with single peptides or a combination of N- and C-terminal peptides. The peptides were immunogenic in mice and the antisera generated could recognize the native CSP on the sporozoite surface. Antisera generated against the N-terminal peptides or against the combinations inhibited sporozoite invasion of hepatocytes in vitro. In vivo, more mice immunized with single P. berghei peptides were protected from infection upon a challenge with P. berghei sporozoites, than mice immunized with a combination of N- and C-terminal peptides. Furthermore, P. falciparum N-terminal peptides were recognized by serum samples from people living in malaria-endemic areas. Importantly, recognition of a peptide from the N-terminal fragment of the P. falciparum CSP by sera from children living in a malaria-endemic region was associated with protection from disease. These results underline the potential of using such peptides as malaria vaccine candidates. RESUME L'intérêt de développer des stratégies d'intervention contre la malaria ciblant le stade pré-erythrocytaire a été alimenté par des études qui montrent qu'il est possible d'obtenir une immunité par l'injection de sporozoites irradiés. Les médicaments et les insecticides anti-paludiques ont été largement utilisés pour contrôler la maladie, mais dans l'espoir de développer une stratégie d'intervention plus rentable, le développement de vaccins a été placé au centre des recherches actuelles contre la malaria. A l'heure actuelle, il n'existe aucun vaccin contre la malaria. L'immunité induite par les sporozoites irradiés est due à l'effet combiné d'anticorps et de cellules T qui agissent contre les antigènes du stade hépatique dont le plus abondant est la protéine circumsporozoite (CSP). Beaucoup de formulations de vaccin visent à imiter l'immunité induite par les sporozoites irradiés. Cependant, les mécanismes par lesquels les anticorps et les cellules T sont génerés après infection par les sporozoites ou après immunisation avec des formulations de vaccin ne sont pas bien compris. La première partie de ce travail a visé à déterminer la capacité de hépatocytes primaires provenant de souris BALB/c à "processer" et à présenter des peptides dérivés de la CSP, après infection par des sporozoites de Plasmodium berghei. Nous avons montré que in vitro, les hépatocytes infectés et ceux traversés par les sporozoites pendant leur migration étaient capables de "processer" et de présenter la CSP aux cellules T CD8+ spécifiques. La voie de présentation implique le protéasome, les protéases de type aspartique et le transport à travers un compartiment post-reticulum endoplasmique. Ces résultats suggèrent que in vivo, les hépatocytes infectés contribuent à l'induction et à l'expansion d'une réponse immunitaire spécifique aux cellules T. Dans la deuxième partie, nous avons caractérisé les réponses anticorps chez les souris de la souche CB6F1 face aux peptides N- et C-terminaux des protéines circumsporozoites de Plasmodium berghei et Plasmodium falciparum. Les souris ont été immunisées avec les peptides individuellement ou en combinaison. Les peptides utilisés étaient immunogéniques chez les souris, et les anticorps produits pouvaient reconnaître la protéine CSP native à la surface des sporozoites. In vitro, les sera contre les peptides N-teminaux et les combinaisons étaient capables d'inhiber l'invasion de hépatocytes par les sporozoites. In vivo, plus de souris immunisées avec les peptides individuels de la CSP de P. berghei étaient protégées contre la malaria que les souris immunisées avec une combinaison de peptides N- et C-terminaux. De plus, les peptides N-terminaux de la CSP de P. falciparum ont été reconnus par les sera de personnes vivant dans des régions endémiques pour la malaria. Il est intéressant de voir que la reconnaissance d'un peptide N-terminal de P. falciparum par des sera d'enfants habitant dans des régions endémiques était associé à la protection contre la maladie. Ces résultats soulignent le potentiel de ces peptides comme candidats-vaccin contre la malaria.

Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos

In Brazil, decades of dengue vector control using organophosphates and pyrethroids have led to dissemination of resistance. Although these insecticides have been employed for decades against Aedes aegypti in the country, knowledge of the impact of temephos resistance on vector viability is limited. We evaluated several fitness parameters in two Brazilian Ae. aegypti populations, both classified as deltamethrin resistant but with distinct resistant ratios (RR) for temephos. The insecticide-susceptible Rockefeller strain was used as an experimental control. The population presenting the higher temephos resistance level, Aparecida de Goiânia, state of Goiás (RR95 of 19.2), exhibited deficiency in the following four parameters: blood meal acceptance, amount of ingested blood, number of eggs and frequency of inseminated females. Mosquitoes from Boa Vista, state of Roraima, the population with lower temephos resistance level (RR95 of 7.4), presented impairment in only two parameters, blood meal acceptance and frequency of inseminated females. These results indicate that the overall fitness handicap was proportional to temephos resistance levels. However, it is unlikely that these disabilities can be attributed solely to temephos resistance, since both populations are also resistant to deltamethrin and harbour the kdr allele, which indicates resistance to pyrethroids. The effects of reduced fitness in resistant populations are discussed.

Susceptibility and resistance to deltamethrin of wild and domestic populations of Triatoma infestans (Reduviidae: Triatominae) in Bolivia: new discoveries

Bolivia is a high-endemic country for Chagas disease, for which the principal vector is Triatoma infestans (Triatominae). This is a mainly domestic species that is also found in the wild environment. Recently, an increasing number of studies have shown the importance of Triatominae resistance to insecticides, especially in Bolivia. Data regarding the susceptibility/resistance of wild and domestic populations of T. infestans to deltamethrin are presented. For the first time, domestic populations of the department of Santa Cruz were tested, showing low resistance. Although most of the wild populations were found to be susceptible to deltamethrin, three populations from three departments showed a mortality rate of less than 100%. This result is emphasised here.

Potential sources of Triatoma infestans reinfesting peridomiciles identified by morphological characterization in Los Llanos, La Rioja, Argentina

The presence of Triatoma infestans in habitats treated with insecticides constitutes a frequent problem in endemic areas. Basing our study on the hypothesis that descendants of a residual population should be more similar to the pre-treatment population than to any other, we compared the indications of two quantitative morphological approaches. This study seeks to find the origin of 247 T. infestans from three populations found in two chicken coops and a goat corral after treatment with insecticides. The results obtained by quantitative morphology suggest that the T. infestans found between three-34 months after the application of insecticides formed mixed populations with insects derived from residual foci and neighbouring habitats. Our analyses also showed the presence of a phenotype which does not resemble neither the pre-treatment phenotype nor the one from neighbouring populations, suggesting the presence of a particular post-treatment phenotype. The heads size showed some variations in males from different populations and remained unchanged in females, which reinforces the hypothesis of an intraspecific competition for food with priority for females. This article presents, for the first time, the combined analysis of geometric morphometry of heads and antennal phenotypes to identify the composition of reinfesting populations.

Toxicological and biochemical analysis of the susceptibility of sylvatic Triatoma infestans from the Andean Valley of Bolivia to organophosphate insecticide

To increase our knowledge of the natural susceptibility of Triatoma infestans to an organophosphate insecticide, we performed toxicological and biochemical studies on three sylvatic populations from Bolivia and two populations from domestic dwellings from Bolivia and Argentina. Fifty-per-cent lethal doses (LD50) were determined based on the topical application of fenitrothion on first instar nymphs and mortality was assessed at 24 h. Both type of populations exhibited LD50ratios significantly higher than 1 with a range of the values (1.42-2.47); the maximum value were found in a sylvatic (-S) population, Veinte de Octubre-S. Samples were biochemically analysed using a glutathione S-transferase activity assay. The highest significant activity was obtained for Veinte de Octubre-S and the lowest activity was obtained for the reference population (102.69 and 54.23 pmol per minute per mg of protein respectively). Two out of the three sylvatic populations (Veinte de Octubre-S and Kirus Mayu-S) exhibited significantly higher glutathione S-transferase activity than that of the reference population. Based on this analysis of the natural susceptibility of this organism to organophosphate insecticides, continental and focal surveys of organophosphate susceptibility should be conducted to evaluate the evolution and distribution of this phenomenon.

Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP).

Is imidacloprid an effective alternative for controlling pyrethroid-resistant populations of Triatoma infestans (Hemiptera: Reduviidae) in the Gran Chaco ecoregion?

The prevention of Chagas disease is based primarily on the chemical control of Triatoma infestans (Klug) using pyrethroid insecticides. However, high resistance levels, correlated with control failures, have been detected in Argentina and Bolivia. A previous study at our laboratory found that imidacloprid could serve as an alternative to pyrethroid insecticides. We studied the delayed toxicity of imidacloprid and the influence of the blood feeding condition of the insect on the toxicity of this insecticide; we also studied the effectiveness of various commercial imidacloprid formulations against a pyrethroid-resistant T. infestans population from the Gran Chaco ecoregion. Variations in the toxic effects of imidacloprid were not observed up to 72 h after exposure and were not found to depend on the blood feeding condition of susceptible and resistant individuals. Of the three different studied formulations of imidacloprid on glass and filter paper, only the spot-on formulation was effective. This formulation was applied to pigeons at doses of 1, 5, 20 and 40 mg/bird. The nymphs that fed on pigeons treated with 20 mg or 40 mg of the formulation showed a higher mortality rate than the control group one day and seven days post-treatment (p < 0.01). A spot-on formulation of imidacloprid was effective against pyrethroid-resistant T. infestans populations at the laboratory level.

Estimation of Aedes aegypti (Diptera: Culicidae) population size and adult male survival in an urban area in Panama

Traditional mosquito control strategies rely heavily on the use of chemical insecticides. However, concerns about the efficiency of traditional control methods, environmental impact and emerging pesticide resistance have highlighted the necessity for developing innovative tools for mosquito control. Some novel strategies, including release of insects carrying a dominant lethal gene (RIDL®), rely on the sustained release of modified male mosquitoes and therefore benefit from a thorough understanding of the biology of the male of the species. In this report we present the results of a mark-release-recapture study aimed at: (i) establishing the survival in the field of laboratory-reared, wild-type male Aedes aegypti and (b) estimating the size of the local adult Ae. aegypti population. The study took place in Panama, a country where recent increases in the incidence and severity of dengue cases have prompted health authorities to evaluate alternative strategies for vector control. Results suggest a life expectancy of 2.3 days for released male mosquitoes (confidence interval: 1.78-2.86). Overall, the male mosquito population was estimated at 58 males/ha (range 12-81 males/ha), which can be extrapolated to an average of 0.64 pupae/person for the study area. The practical implications of these results are discussed.