Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes.

Mosquitoes are responsible for transmitting many pathogens to humans and Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are important vectors in the world. The microbiota plays an important role in developmental studies that involve impacts on the biological cycle of mosquitoes and vector control strategies. In this study, the aim was to understand the environment plays in the microbiota culturable diversity of Aedes aegytpi, Aedes albopictus and Culex quinquefasciatus. Midgut of studied mosquitoes (laboratory-reared and wild) were dissected and analyzed by MALDI-TOF MS to identify the microbiota. Most of the bacteria identified in the microbiota of mosquitoes from the laboratory and field belong to the phylum Proteobacteria. We reported on the microbial diversity among the mosquito species studied where Cx. quinquefasciatus and Ae. albopictus show greater bacterial similarity. The genus Rahnella was present in all mosquito species studied, both in those from the laboratory and those from the wild. Bacillus, Ewingella, Microccocus, Klebsiella and Pantoea are genera was predominant among the mosquitoes studied. The difference of microbiota diversity between mosquitoes laboratory-reared and wild shows that the environment plays an important role in the acquisition of bacteria, mainly in Ae. aegypti and Cx. quinquefasciatus.

Long-term exposure of Aedes aegypti to Bacillus thuringiensis svar. israelensis did not involve altered susceptibility to this microbial larvicide or to other control agents.

BACKGROUND: Bacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti. Its mode of action based on four protoxins disfavors resistance; however, control in endemic areas that display high mosquito infestation throughout the year requires continuous larvicide applications, which imposes a strong selection pressure. Therefore, this study aimed to investigate the effects of an intensive Bti exposure on an Ae. aegypti strain (RecBti), regarding its susceptibility to Bti and two of its protoxins tested individually, to other control agents temephos and diflubenzuron, and its profile of detoxifying enzymes. METHODS: The RecBti strain was established using a large egg sample (10,000) from Recife city (Brazil) and more than 290,000 larvae were subjected to Bti throughout 30 generations. Larvae susceptibility to larvicides and the activity of detoxifying enzymes were determined by bioassays and catalytic assays, respectively. The Rockefeller strain was the reference used for these evaluations. RESULTS: Bti exposure yielded an average of 74% mortality at each generation. Larvae assessed in seven time points throughout the 30 generations were susceptible to Bti crystal (resistance ratio RR ≤ 2.8) and to its individual toxins Cry11Aa and Cry4Ba (RR ≤ 4.1). Early signs of altered susceptibility to Cry11Aa were detected in the last evaluations, suggesting that this toxin was a marker of the selection pressure imposed. RecBti larvae were also susceptible (RR ≤ 1.6) to the other control agents, temephos and diflubenzuron. The activity of the detoxifying enzymes α- and ß-esterases, glutathione-S-transferases and mixed-function oxidases was classified as unaltered in larvae from two generations (F19 and F25), except for a ß-esterases increase in F25. CONCLUSIONS: Prolonged exposure of Ae. aegypti larvae to Bti did not evolve into resistance to the crystal, and no cross-resistance with temephos and diflubenzuron were recorded, which supports their sustainable use with Bti for integrated control practices. The unaltered activity of most detoxifying enzymes suggests that they might not play a major role in the metabolism of Bti toxins, therefore resistance by this mechanism is unlikely to occur. This study also highlights the need to establish suitable criteria to classify the status of larval susceptibility/resistance.

Insecticide susceptibility of Aedes albopictus and Ae. aegypti from Brazil and the Swiss-Italian border region.

BACKGROUND: Aedes aegypti and Ae. albopictus are two highly invasive mosquito species, both vectors of several viruses, including dengue, chikungunya and Zika. While Ae. aegypti is the primary vector in the tropics and sub-tropics, Ae. albopictus is increasingly under the public health watch as it has been implicated in arbovirus-transmission in more temperate regions, including continental Europe. Vector control using insecticides is the pillar of most control programmes; hence development of insecticide resistance is of great concern. As part of a Brazilian-Swiss Joint Research Programme we set out to assess whether there are any signs of existing or incipient insecticide resistance primarily against the larvicide Bacillus thuringiensis svar. israelensis (Bti), but also against currently applied and potentially alternative insecticides in our areas, Recife (Brazil) and the Swiss-Italian border region. METHODS: Following World Health Organization guidelines, dose-response curves for a range of insecticides were established for both colonized and field caught Ae. aegypti and Ae. albopictus. The larvicides included Bti, two of its toxins, Cry11Aa and Cry4Ba, Lysinibacillus sphaericus, Vectomax CG®, a formulated combination of Bti and L. sphaericus, and diflubenzuron. In addition to the larvicides, the Swiss-Italian Ae. albopictus populations were also tested against five adulticides (bendiocarb, dichlorodiphenyltrichloroethane, malathion, permethrin and λ-cyhalothrin). RESULTS: Showing a similar dose-response, all mosquito populations were fully susceptible to the larvicides tested and, in particular, to Bti which is currently used both in Brazil and Switzerland. In addition, there were no signs of incipient resistance against Bti as larvae were equally susceptible to the individual toxins, Cry11Aa and Cry4Ba. The field-caught Swiss-Italian populations were susceptible to the adulticides tested but DDT mortality rates showed signs of reduced susceptibility. CONCLUSIONS: The insecticides currently used for mosquito control in Switzerland and Brazil are still effective against the target populations. The present study provides an important reference as relatively few insecticide susceptibility surveys have been carried out with Ae. albopictus.

Characterization of the spatial and temporal dynamics of the dengue vector population established in urban areas of Fernando de Noronha, a Brazilian oceanic island.

Aedes aegypti has played a major role in the dramatic expansion of dengue worldwide. The failure of control programs in reducing the rhythm of global dengue expansion through vector control suggests the need for studies to support more appropriated control strategies. We report here the results of a longitudinal study on Ae. aegypti population dynamics through continuous egg sampling aiming to characterize the infestation of urban areas of a Brazilian oceanic island, Fernando de Noronha. The spatial and temporal distribution of the dengue vector population in urban areas of the island was described using a monitoring system (SMCP-Aedes) based on a 103-trap network for Aedes egg sampling, using GIS and spatial statistics analysis tools. Mean egg densities were estimated over a 29-month period starting in 2011 and producing monthly maps of mosquito abundance. The system detected continuous Ae. aegypti oviposition in most traps. The high global positive ovitrap index (POI=83.7% of 2815 events) indicated the frequent presence of blood-fed-egg laying females at every sampling station. Egg density (eggs/ovitrap/month) reached peak values of 297.3 (0 - 2020) in May and 295 (0 - 2140) in August 2012. The presence of a stable Ae. aegypti population established throughout the inhabited areas of the island was demonstrated. A strong association between egg abundance and rainfall with a 2-month lag was observed, which combined with a first-order autocorrelation observed in the series of egg counts can provide an important forecasting tool. This first description of the characteristics of the island infestation by the dengue vector provides baseline information to analyze relationships between the spatial distribution of the vector and dengue cases, and to the development of integrated vector control strategies.

Effect of Moringa oleifera lectins on survival and enzyme activities of Aedes aegypti larvae susceptible and resistant to organophosphate.

The indiscriminate use of synthetic insecticides to control Aedes aegypti has led to emergence of resistant populations. Moringa oleifera seeds contain the lectins WSMoL and cMoL. WSMoL has larvicidal activity on fourth-stage of A. aegypti organophosphate-susceptible larvae (Rockefeller L4). This study reports on the effects of cMoL on the survival of Rockefeller L4 as well as of WSMoL and cMoL on L4 from an organophosphate-resistant population (Rec-R). The effects of lectins on digestive (amylase, trypsin, and protease) and detoxifying (superoxide dismutase (SOD), α- and ß-esterases) enzymes from larvae were also determined. cMoL (0.1-0.8 mg/ml) did not kill Rockefeller L4 as well as WSMoL and cMoL (0.1-0.8 mg/ml) were not larvicidal for Rec-R L4. WSMoL stimulated protease, trypsin-like, and α-amylase from Rockefeller L4 while cMoL inhibited these enzymes. WSMoL had no effect on trypsin-like activity from Rec-R L4 but inhibited protease and α-amylase. Among digestive enzymes of Rec-R L4, cMoL inhibited only trypsin-like activity. cMoL inhibited SOD activities from Rockefeller and Rec-R L4 in a higher level than WSMoL while ß-esterase from Rockefeller L4 was more inhibited by WSMoL. The lectins promoted low stimulation or inhibition of α-esterase activities from both populations. In conclusion, Rockefeller and Rec-R larvae were distinctly affected by M. oleifera lectins, and larvicidal mechanism of WSMoL on Rockefeller L4 may involve deregulation of digestive enzymes. cMoL interfered mainly on SOD activity and thus it can be investigated as a synergistic agent for controlling populations whose resistance is linked to an increased detoxifying process mediated by this enzyme.

Susceptibility status of Culex quinquefasciatus (Diptera: Culicidae) populations to the chemical insecticide temephos in Pernambuco, Brazil.

BACKGROUND: Culex quinquefasciatus is the vector of many agents of human diseases, including Wuchereria bancrofti, the parasite that causes bancroftian filariasis, an endemic disease in Pernambuco State, Brazil. Although temephos is not currently used to control C. quinquefasciatus, the species might be under a selection process from incidental exposure to this compound. This study aimed to evaluate the susceptibility status of C. quinquefasciatus to temephos, using bioassays, and to investigate its putative resistance mechanisms through biochemical assays and screening of the G119S mutation in the acetylcholinesterase gene, which is associated with organophosphate resistance, carried out by PCR and sequencing. RESULTS: The results showed that only mosquitoes from Santa Cruz do Capibaribe (SC) had an alteration in their susceptibility status (RR = 7.2-fold), while the other populations were all susceptible to the insecticide. Biochemical assays showed increased activity for all esterases in SC, as well as evidence of acetylcholinesterase insensitivity. The G119S mutation was detected in this population with a frequency of 0.11, but it was not found in the remaining populations. CONCLUSION: These data show that mechanisms of temephos resistance have been selected in natural C. quinquefasciatus populations from Pernambuco, which could undermine future control actions.

The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for management.

BACKGROUND: Aedes aegypti is the vector of dengue virus, and its control is essential to prevent disease transmission. Among the agents available to control this species, biolarvicides based on Bacillus thuringiensis serovar israelensis (Bti) are an effective alternative to replace the organophosphate temephos for controlling populations that display resistance to this insecticide. The major goal of this study was to determine the baseline susceptibility of Brazilian Ae. aegypti populations to Bti, taking into account their background in terms of larvicide exposure, status of temephos resistance and the level of activity of detoxifying enzymes involved in metabolic resistance to insecticides. METHODS: Population samples were established under insectarium conditions. Larval susceptibility to temephos and Bti was evaluated through bioassays and lethal concentrations of these compounds were determined. Biochemical assays were performed to determine the specific activity of five detoxifying enzymes in these samples. RESULTS: Fourteen populations were characterized and, except for one case, all displayed resistance to temephos. Most populations were classified as highly resistant. The populations also showed increased activity of one or more detoxifying enzymes (glutathione-S-transferases, esterases and mixed function oxidases), regardless of their temephos resistance status. All populations analyzed were susceptible to Bti, and the lethal concentrations were similar to those detected in two laboratory susceptible colonies. The response to Bti showed little variation. A maximum resistance ratio of 2.1 was observed in two untreated populations, while in two Bti-treated populations, the maximum resistance ratio was 1.9. No positive correlation was found between temephos resistance, increased activity of detoxifying enzymes, and susceptibility to Bti. CONCLUSIONS: Data from this study show that all populations were susceptible to Bti, including twelve untreated and two treated populations that had been exposed to this agent for more than ten years. The temephos resistance and increased activity of detoxifying enzymes observed in thirteen populations was not correlated with changes in susceptibility to Bti. Our data show a lack of cross-resistance between these two compounds; thus, Bti can be used in an integrated control program to fight Ae. aegypti and counteract the temephos resistance that was found among all populations analyzed.

Evaluation of a sticky trap (AedesTraP), made from disposable plastic bottles, as a monitoring tool for Aedes aegypti populations.

BACKGROUND: Dengue virus, which is transmitted by Aedes aegypti mosquitoes is the most important emerging viral disease, infecting more than 50 million people annually. Currently used sticky traps are useful tools for monitoring and control of A. aegypti, despite differences in efficiency, labor requirements and cost. In the present work, a field assay was carried out to evaluate the performance of a sticky trap (AedesTrap), produced using disposable material, in capturing gravid Aedes spp. females. Additionally, conditions necessary for the improved performance of the device, such as number of traps per site and location (indoors or outdoors) were evaluated. METHODS: During a one year period, traps were placed in a dengue endemic area in 28 day cycles. The trap, named AedesTrap, consisted of a disposable plastic soda bottle coated inside with colophony resin, which served as a sticky substrate. Disposable bottles were donated by restaurants, and traps were made by laboratory staff, reducing the cost of the sticky trap (less than U$3). Mosquito capture in indoor and outdoor areas was compared by placing the traps in laundry room, kitchen or bedroom (indoors) and front or back yard (outdoors). The relationship between the number of AedesTraps and quantity of captured mosquitoes was investigated by utilizing one or three traps/site. RESULTS: During a 28 day cycle, a single AedesTrap was capable of capturing up to 15 A. aegypti in a house, with a mean capture of 0.5 to 2.63 females per premise. The AedesTrap collected three times more outdoors versus indoors. Similarly, the capability of detecting Aedes spp. infestation, and of capturing females, was three times higher when using three AedesTraps per house, compared with one trap per house. CONCLUSIONS: AedesTrap was shown to be capable of capturing A. aegypti and other culicidae, providing information on the adult mosquito population, and allowing the identification of areas critically infested by mosquitoes. Low requirements for skilled labor together with easy maintenance and low cost are additional advantages of using this sticky trap.

Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools.

Bacillus sphaericus binary toxin action on Culex quinquefasciatus larvae relies on the binding to Cqm1alpha-glucosidases, which act as midgut receptors. Resistance of two laboratory-selected colonies is associated with the allele cqm1(REC) that prevents Cqm1 expression as membrane-bound molecules. This study evaluated stability of resistance after the interruption of selection pressure and introduction of susceptible individuals in these colonies. Bioassays showed that frequency of resistant larvae did not decrease throughout 11 generations, under these conditions, and it was associated to a similar frequency of larvae lacking the Cqm1alpha-glucosidase receptor, detected by in gel enzymatic assays. Direct screening of the cqm1(REC) allele, by specific PCR, showed that its frequency remained stable throughout 11 generations. Parental resistant colony did not display biological costs regarding fecundity, fertility and pupal weight and data from susceptibility assays, enzymatic assays and PCR screening showed that cqm1(REC) was not disfavored in competition with the susceptible allele and persisted in the progenies, in the lack of selection pressure. Characterization of molecular basis of resistance is essential for developing diagnostic tools and data have relevant implication for the establishment of strategies for resistance management.

Cytopathological effects of Bacillus sphaericus Cry48Aa/Cry49Aa toxin on binary toxin-susceptible and -resistant Culex quinquefasciatus larvae.

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.

Detection of an allele conferring resistance to Bacillus sphaericus binary toxin in Culex quinquefasciatus populations by molecular screening.

The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound alpha-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1(REC)) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1(REC) allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1(REC) allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through alpha-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1(REC) allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.