Culex quinquefasciatus resistance to the Binary protoxin from Lysinibacillus sphaericus biolarvicides can rely on mutations in the cqm1 gene, which encodes its Cqm1 receptor. Bacillus thuringiensis svar. israelensis (Bti) biolarvicides can be used to manage L. sphaericus resistance since their protoxins act through distinct receptors from Cqm1. This study aimed to evaluate the effect of a L. sphaericus/Bti combined biolarvicide treatment on the frequency of three cqm1 resistance alleles in Cx. quinquefasciatus larvae from one subarea (SA2) compared to another (SA1) where only L. sphaericus was kept. Larvae collected before and during a two-year trial were subjected to bioassays and PCR assays. Susceptibility to L. sphaericus showed oscillations in both areas, but no significant alterations of the lethal concentrations were detected. The pre-trial frequency of the resistance alleles was 0.057, while during the trial, the mean frequency was significantly lower in SA2 (0.068) than in SA1 (0.088). All three alleles investigated were detected in the study subareas and the cqm1REC-D19 was the most frequent allele found, regardless the treatment employed. This study showed that the use of the L. sphaericus/Bti biolarvicide was associated with a lower frequency of the resistance alleles and can be a useful tool to prevent the onset of L. sphaericus resistance.

BACKGROUND:Aedes albopictus, the Asian tiger mosquito, originates from the tropical and subtropical regions of Southeast Asia. Over the recent decades it has been passively spread across the globe, primarily through the used tyre trade and passive transportation along major traffic routes. A. albopictus is a proven vector for many arboviruses, most notably chikungunya and dengue, with recent outbreaks also in continental Europe. In southern Switzerland, in the Canton of Ticino A. albopictus was spotted for the first time in 2003. Since then the local authorities have implemented a control programme based on larval source reduction. Despite these efforts, mosquito densities have increased over the last decade, casting doubts on the effectiveness of such larval control programmes. METHODOLOGY/PRINCIPAL FINDINGS:The Italian communities just across the Swiss-Italian border lack a control programme. This motivated us to compare the intervention and the non-intervention areas side by side in an attempt to find evidence for, or against, the effectiveness of larval A. albopictus control. Using ovitraps and a randomised sampling scheme, we examined the seasonal and spatial abundance of A. albopictus in sylvatic and urban environments across the Swiss-Italian border in 2012 and 2013. In the urban environments of the non-intervention area, egg densities were 2.26 times higher as compared to the intervention area. In the sylvatic environments, as compared to the urban environments, egg densities were 36% in the intervention area and 18% in the non-intervention area. CONCLUSIONS/SIGNIFICANCE:Though alternative explanations are also valid, the results support the hypothesis that the Ticino intervention programme does have an impact. At the same time the data also suggest that current larval interventions fall short in gaining full control over the mosquito, calling for the evaluation of additional, or alternative, approaches. Ideally, these should also consider inclusion of the neighbouring Italian communities in the surveillance and control efforts.

Aedes aegypti (L.) has become an efficient vector of important arboviruses due to its anthropophilic and domiciliary behaviors. Since the 1980s, dengue affects thousands of people every year in Brazil; in Fernando de Noronha (FN), a touristic archipelago, dengue cases have occurred since 2001. Once Ae. aegypti populations are well established in the inhabited areas of FN, the threat of dengue or another arbovirus epidemic is continuously imminent. This study aimed to monitor the DENV serotypes in mosquito samples collected in FN, where at least one resident was clinically diagnosed as dengue patient. Entomological surveillance was conducted in 2011 and 2012. Mosquitoes were sorted by sex and location and were stored in pools. DENV detection was performed using polymerase chain reaction with reverse transcription (RT-PCR) and the Platelia Dengue NS1 Ag. RNA integrity was checked by RT-PCR using rpL8 primers, and the minimum infection rate (MIR) was calculated. In total, 339 pools were analyzed, and only one was positive (DENV-1) by Multiplex RT-PCR (MIR = 1.53). When considering only pools with RNA integrity, the MIR was 2.92. Using the Platelia kit, the MIR was 9.18 (considering all the pools) and 17.54 (only 140 pools with RNA integrity). Our results showed the importance of a constant entomological surveillance in that area, the need to improve storage and transportation protocols, and an endogenous control in the RT-PCR to avoid false-negative results. Finally, our study indicated that the NS1-Ag detection was the most sensitive method and should be used routinely for DENV surveillance in mosquitoes if the serotype identification is not required.

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.

Lysinibacillus sphaericus (Ls) strains that produce insecticidal proteins show high activity against mosquito larvae. The most active of these is the binary (Bin) toxin that acts following ingestion and, after midgut processing and binding to specific receptors, provokes cytopathological effects and leads to larval death. Bin toxin displays specific action against some species of medical importance (e.g. Culex and Anopheles) and it is safe to non-target organisms. These features have led to the production of biolarvicides based on this bacterium and its effectiveness to control mosquito larvae has been widely related in the literature. The field utilisation of Ls has also shown that resistance could be selected among exposed populations and the mechanisms and genes involved in this process have been described. Management strategies can be successfully employed to avoid resistance and Ls can be used within integrated programmes as a selective and efficient agent to control mosquitoes.

Lysinibacillus sphaericus (Ls) strains that produce insecticidal proteins show high activity against mosquito larvae. The most active of these is the binary (Bin) toxin that acts following ingestion and, after midgut processing and binding to specific receptors, provokes cytopathological effects and leads to larval death. Bin toxin displays specific action against some species of medical importance (e.g. Culex and Anopheles) and it is safe to non-target organisms. These features have led to the production of biolarvicides based on this bacterium and its effectiveness to control mosquito larvae has been widely related in the literature. The field utilisation of Ls has also shown that resistance could be selected among exposed populations and the mechanisms and genes involved in this process have been described. Management strategies can be successfully employed to avoid resistance and Ls can be used within integrated programmes as a selective and efficient agent to control mosquitoes.

Aedes aegypti has developed evolution-driven adaptations for surviving in the domestic human habitat. Several trap models have been designed considering these strategies and tested for monitoring this efficient vector of Dengue. Here, we report a real-scale evaluation of a system for monitoring and controlling mosquito populations based on egg sampling coupled with geographic information systems technology. The SMCP-Aedes, a system based on open technology and open data standards, was set up from March/2008 to October/2011 as a pilot trial in two sites of Pernambuco Brazil: Ipojuca (10,000 residents) and Santa Cruz (83,000), in a joint effort of health authorities and staff, and a network of scientists providing scientific support. A widespread infestation by Aedes was found in both sites in 2008-2009, with 96.8%-100% trap positivity. Egg densities were markedly higher in SCC than in Ipojuca. A 90% decrease in egg density was recorded in SCC after two years of sustained control pressure imposed by suppression of >7,500,000 eggs and >3,200 adults, plus larval control by adding fishes to cisterns. In Ipojuca, 1.1 million mosquito eggs were suppressed and a 77% reduction in egg density was achieved. This study aimed at assessing the applicability of a system using GIS and spatial statistic analysis tools for quantitative assessment of mosquito populations. It also provided useful information on the requirements for reducing well-established mosquito populations. Results from two cities led us to conclude that the success in markedly reducing an Aedes population required the appropriate choice of control measures for sustained mass elimination guided by a user-friendly mosquito surveillance system. The system was able to support interventional decisions and to assess the program's success. Additionally, it created a stimulating environment for health staff and residents, which had a positive impact on their commitment to the dengue control program.

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.

Entomological indices based on larval surveillance methods are not sensitive enough to accurately monitor Aedes aegypti population densities in areas differing in levels of infestation. Here, we present and discuss results obtained from a pilot program using a new approach, the Monitoring System and Population Control of Aedes aegypti (SMCPAedes), based on spatial information technology utilizing data on the spatial and temporal distribution of Aedes. The SMCP-Aedes data was continuously generated by a set of sentinel-ovitraps (OVT-S) in Santa Cruz do Capibaribe and Ipojuca urban areas, two municipalities of Pernambuco, Brazil, which have different patterns of settlement and biogeography. The first results, recorded in June 2008, showed that both areas were highly infested by A. aegypti, with more than 90% of OVT-S positive for A. aegypti, and detected densities of 1223 eggs/OVT-S/month in Sta Cruz and 561.2 eggs/OVT-S/month in Ipojuca. A significant reduction in population densities to 363.5 eggs/OVT-S/month and 138.6 eggs/OVT-S/month, respectively, was observed after six rounds of control intervention consisting of massive egg elimination using 8,000 control-ovitraps (OVT-C). Over this period, approximately 5 million eggs and 2,700 adult mosquitoes were removed from those areas. The strategies and tools used in the SMCP-Aedes allowed us to quantify the active population of A. aegypti, indicating periods and places of higher human exposure to the mosquito. The instruments used for mechanical removal of eggs and adults are effective and can be integrated in a sustainable control program.

Background Dengue is a disease of great complexity, due to interactions between humans, mosquitoes and various virus serotypes as well as efficient vector survival strategies. Thus, understanding the factors influencing the persistence of the disease has been a challenge for scientists and policy makers. The aim of this study is to investigate the influence of various factors related to humans and vectors in the maintenance of viral transmission during extended periods. Methodology/Principal Findings We developed a stochastic cellular automata model to simulate the spread of dengue fever in a dense community. Each cell can correspond to a built area, and human and mosquito populations are individually monitored during the simulations. Human mobility and renewal, as well as vector infestation, are taken into consideration. To investigate the factors influencing the maintenance of viral circulation, two sets of simulations were performed: (1st) varying human renewal rates and human population sizes and (2nd) varying the house index (fraction of infested buildings) and vector per human ratio. We found that viral transmission is inhibited with the combination of small human populations with low renewal rates. It is also shown that maintenance of viral circulation for extended periods is possible at low values of house index. Based on the results of the model and on a study conducted in the city of Recife, Brazil, which associates vector infestation with Aedes aegytpi egg counts, we question the current methodology used in calculating the house index, based on larval survey. Conclusions/Significance This study contributed to a better understanding of the dynamics of dengue subsistence. Using basic concepts of metapopulations, we concluded that low infestation rates in a few neighborhoods ensure the persistence of dengue in large cities and suggested that better strategies should be implemented to obtain measures of house index values, in order to improve the dengue monitoring and control system.

Entomological indices based on larval surveillance methods are not sensitive enough to accurately monitor Aedes aegypti population densities in areas differing in levels of infestation. Here, we present and discuss results obtained from a pilot program using a new approach, the Monitoring System and Population Control of Aedes aegypti (SMCPAedes), based on spatial information technology utilizing data on the spatial and temporal distribution of Aedes. The SMCP-Aedes data was continuously generated by a set of sentinel-ovitraps (OVT-S) in Santa Cruz do Capibaribe and Ipojuca urban areas, two municipalities of Pernambuco, Brazil, which have different patterns of settlement and biogeography. The first results, recorded in June 2008, showed that both areas were highly infested by A. aegypti, with more than 90% of OVT-S positive for A. aegypti, and detected densities of 1223 eggs/OVT-S/month in Sta Cruz and 561.2 eggs/OVT-S/month in Ipojuca. A significant reduction in population densities to 363.5 eggs/OVT-S/month and 138.6 eggs/OVT-S/month, respectively, was observed after six rounds of control intervention consisting of massive egg elimination using 8,000 control-ovitraps (OVT-C). Over this period, approximately 5 million eggs and 2,700 adult mosquitoes were removed from those areas. The strategies and tools used in the SMCP-Aedes allowed us to quantify the active population of A. aegypti, indicating periods and places of higher human exposure to the mosquito. The instruments used for mechanical removal of eggs and adults are effective and can be integrated in a sustainable control program.

The Southern house mosquito, Culex quinquefasciatus, is an important human health pest as a vector of several pathogens, including agents of lymphatic filariasis and arboviruses like West Nile virus. We conducted preliminary experiments in Recife, Brazil, to explore applications of Culex oviposition attractants in combination with Bacillus thuringiensis variety israelensis (Bti) in an attract-and-kill approach. Simple, cost-effective oviposition traps, BR-OVT, loaded with Bti and baited with or without attractant, were deployed in 10 homes for 30 d in 2 consecutive yr. Significantly higher numbers of egg rafts were deposited in traps baited with skatole or infusion than the control water traps. In the first year, 2006, significantly higher numbers of eggs were deposited in infusion-baited traps, particularly in the first 15 d of the experiment, than in skatole traps, but in the following year no significant difference was observed between synthetic and natural attractants. The tests strongly demonstrate that skatole or infusion can be used to enhance the number of egg rafts deposited on Bti-treated oviposition traps.

BACKGROUND & OBJECTIVESDengue is currently one of the most important arthropod-borne diseases and may be caused by four different dengue virus serotypes (DENV-1 to DENV-4), transmitted mainly by Aedes aegypti (Diptera: Culicidae) mosquitoes. With the lack of a dengue vaccine, vector control strategies constitute a crucial mode to prevent or reduce disease transmission. In this context, DENV detection in natural Ae. aegypti populations may serve as a potential additional tool for early prediction systems of dengue outbreaks, leading to an intensification of vector control measures, aimed at reducing disease transmission. In Brazil, this type of surveillance has been performed sporadically by a few groups and has not been incorporated as a routine activity in control programs. This study aimed at detecting DENV in natural Ae. aegypti from Recife, Pernambuco, to check the circulating serotypes and the occurrence of transovarial transmission in local mosquito populations.METHODSFrom January 2005 to June 2006, mosquitoes (adults and eggs) were collected in houses where people with clinical suspicion of dengue infection lived at. RNA was extracted from pooled mosquitoes and RT-PCR was performed in these samples for detection of the four DENV serotypes.RESULTS & CONCLUSIONOut of 83 pools of adult mosquitoes collected in the field, nine were positive for DENV: five for DENV-1, two for DENV-2 and two for DENV-3. From 139 pools of adult mosquitoes reared from collected eggs, there were 17 positive pools: three for DENV-1, 10 for DENV-2, and four for DENV-3. These results are discussed in the paper in regard to the local dengue epidemiological data. The conclusions clearly point to the informative power and sensitivity of DENV entomological surveillance and to the importance of including mosquito immature forms in this strategy.

A synthetic mixture of an oviposition-stimulating kairomone for the yellow fever mosquito, Aedes aegypti, comprising of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (NTT, in short) was tested in a dengue endemic area in Recife, Brazil. Gravid female mosquitoes confined to a cage under semi-field conditions deposited significantly higher numbers of eggs in traps baited with NTT at doses ranging from 0.6 to 600 ng/microl than in control (water) traps. When tested in homes, egg-laying in traps baited with 60 ng NTT/microl (final concentration in trap, approximately 3.33 ng/ml) and in control traps was not significantly different, but egg deposited in traps with lower dosage (6 ng NTT/microl; final concentration in trap, approximately 0.33 ng/ml) was significantly higher than in control traps. In subsequent trials, the numbers of eggs laid in traps baited with 0.6 ng NTT/microl (final concentration in trap, approximately 0.033 ng/ml) were not significantly different from the numbers deposited in trap loaded with 6 ng NTT/microl. Egg-laying was significantly higher in these treatments than in control traps.

Aedes aegypti is a very efficient disseminator of human pathogens. This condition is the result of evolutionary adaptations to frequent haematophagy, as well as to the colonization of countless types of habitats associated with environmental and cultural factors that favor the proliferation of this mosquito in urban ecosystems. Studies using sensitive methods of monitoring demonstrate that the methods of surveillance used in the Brazilian program do not show the high degrees of the infestation of cities by this vector. To increase the capacity of the health sector, new tools are needed to the practice of surveillance, which incorporate aspects of the vector, place and human population. We describe here the SMCP-Aedes - Monitoring System and Population Control of Aedes aegypti, aiming to provide an entomological surveillance framework as a basis for epidemiological surveillance of dengue. The SMCP-Aedes is uphold in the space technology information, supported by the intensive use of the web and free software to collect, store, analyze and disseminate information on the spatial-temporal distribution of the estimated density for the population of Aedes, based on data systematically collected with the use of ovitraps. Planned control interventions, intensified where and when indicated by the entomological surveillance, are agreed with the communities, relying on the permanent social mobilization.

Insecticide resistance is one of the main problems in vector control programs. Because insects have developed resistance to all classes of available chemical insecticides, a proper surveillance and management of resistance in areas where these compounds are being utilized is crucial for the success of control programs. Since the mechanisms and molecular bases of resistance are various, they must be characterized to allow efficient monitoring strategies. Here we report the establishment of an Aedes aegypti strain resistant to temephos, named RecR, selected under laboratory conditions. The parental A. aegypti population was obtained from eggs collected in an area where temephos had been used for 8 years, and presented a baseline resistance ratio (RR) of 7. After 17 generations under selective pressure, the RR has increased to 180. Biochemical assays indicate that metabolic mechanisms are involved on temephos resistance in the selected strain. These experiments showed that, compared to the susceptible colony Rockefeller, RecR present higher activity of glutathione S-transferases (GSTs), alpha- and beta-esterases, and, to a lesser degree, mixed function oxidases (MFO). At the 14th or 17th generations, there was no cross resistance of these insects to deltamethrin, cypermethrin and malathion, while a low resistance level (RR=3) was observed for pyriproxyfen, a juvenile hormone analogue. Experiments on resistance reversal, performed through three different field simulated schemes using the resistant strain, showed that temephos susceptibility can be recovered. The establishment of an A. aegypti colony resistant to temephos is extremely valuable for a deeper understanding of resistance mechanisms and thus for further improvements in control strategies against this vector. With the urgent need on improving methodologies to monitor resistance, molecular studies such as microarrays, and resistant colonies such as RecR will certainly hasten such studies.

The performance of two experimental formulations of Bti, tablet and technical powder, was evaluated against Aedes aegypti larvae. The influence of larval density, and presence of active or inactivated spores upon Bti persistence and bacterial recycling was analyzed. The microbiological viability and toxicity of technical powder and irradiated-technical powder was analyzed under laboratory bioassays as well as under simulated field conditions (SFC). The SFC experiments were performed in plastic containers filled with 50L of tap water, placed under shade and colonized with 50L1 weekly or monthly. The initial efficacy of the product was estimated through mortality of L4 within 48h after treatment. The persistence was defined as the period in which larval mortality was 80%, estimated by the number of live pupae. Water samples were collected from the containers throughout the experiments to verify spores concentration. Results showed that in SFC the tablet, technical powder and irradiated-technical powder eliminated 90–100% of larvae in 48h, and this efficiency persisted for 6 months. There were no differences in the residual activity between technical powder or irradiated-technical powder, nor among containers colonized with different amount of larvae. Significant differences in bacteria concentration were observed along time between containers treated with technical powder or irradiated-technical powder, however, Bti concentration after a single treatment warranted high larvicidal activity for 6 months, under all experimental conditions, indicating the occurrence of bacterial recycling.

A new approach to dengue vector surveillance based on permanent egg- collection using a modified ovitrap and Bacillus thuringiensis israelensis ( Bti) was evaluated in different urban landscapes in Recife, Northeast Brazil. From April 2004 to April 2005, 13 egg- collection cycles of four weeks were carried out. Geo- referenced ovitraps containing grass infusion, Bti and three paddles were placed at fixed sampling stations distributed over five selected sites. Continuous egg- collections yielded more than four million eggs laid into 464 sentinel- ovitraps over one year. The overall positive ovitrap index was 98.5% ( over 5,616 trap observations). The egg density index ranged from 100 to 2,500 eggs per trap- cycle, indicating a wide spread and high density of Aedes aegypti ( Diptera: Culicidae) breeding populations in all sites. Fluctuations in population density over time were observed, particularly a marked increase from January on, or later, according to site. Massive egg- collection carried out at one of the sites prevented such a population outbreak. At intra- site level, egg counts made it possible to identify spots where the vector population is consistently concentrated over the time, pinpointing areas that should be considered high priority for control activities. The results indicate that these could be promising strategies for detecting and preventing Ae. aegypti population outbreaks.