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1.  Integrating the Public in Mosquito Management: Active Education by Community Peers Can Lead to Significant Reduction in Peridomestic Container Mosquito Habitats 
PLoS ONE  2014;9(9):e108504.
Mosquito species that utilize peridomestic containers for immature development are commonly aggressive human biters, and because they often reach high abundance, create significant nuisance. One of these species, the Asian tiger mosquito Aedes albopictus, is an important vector of emerging infectious diseases, such as dengue, chikungunya, and Zika fevers. Integrated mosquito management (IMM) of Ae. albopictus is particularly difficult because it requires access to private yards in urban and suburban residences. It has become apparent that in the event of a public health concern due to this species, homeowners will have to be active participants in the control process by reducing mosquito habitats in their properties, an activity known as source reduction. However, limited attempts at quantifying the effect of source reduction by homeowners have had mixed results. Of note, many mosquito control programs in the US have some form of education outreach, however the primary approach is often passive focusing on the distribution of education materials as flyers. In 2010, we evaluated the use of active community peer education in a source reduction program, using AmeriCorps volunteers. The volunteers were mobilized over a 4-week period, in two areas with approximately 1,000 residences each in urban Mercer and suburban Monmouth counties in New Jersey, USA. The volunteers were first provided training on peridomestic mosquitoes and on basic approaches to reducing the number of container habitats for mosquito larvae in backyards. Within the two treatment areas the volunteers successfully engaged 758 separate homes. Repeated measures analysis of variance showed a significant reduction in container habitats in the sites where the volunteers actively engaged the community compared to untreated control areas in both counties. Our results suggest that active education using community peer educators can be an effective means of source reduction, and a critical tool in the arsenal against peridomestic mosquitoes.
doi:10.1371/journal.pone.0108504
PMCID: PMC4177891  PMID: 25255027
2.  The Impact of Pyrethroid Resistance on the Efficacy of Insecticide-Treated Bed Nets against African Anopheline Mosquitoes: Systematic Review and Meta-Analysis 
PLoS Medicine  2014;11(3):e1001619.
In a systematic review and meta-analysis, Clare Strode and colleagues assess whether insecticide resistance in African Anopheline mosquitoes affects the efficacy of insecticide-treated bed nets.
Please see later in the article for the Editors' Summary
Background
Pyrethroid insecticide-treated bed nets (ITNs) help contribute to reducing malaria deaths in Africa, but their efficacy is threatened by insecticide resistance in some malaria mosquito vectors. We therefore assessed the evidence that resistance is attenuating the effect of ITNs on entomological outcomes.
Methods and Findings
We included laboratory and field studies of African malaria vectors that measured resistance at the time of the study and used World Health Organization–recommended impregnation regimens. We reported mosquito mortality, blood feeding, induced exophily (premature exit of mosquitoes from the hut), deterrence, time to 50% or 95% knock-down, and percentage knock-down at 60 min. Publications were searched from 1 January 1980 to 31 December 2013 using MEDLINE, Cochrane Central Register of Controlled Trials, Science Citation Index Expanded, Social Sciences Citation Index, African Index Medicus, and CAB Abstracts. We stratified studies into three levels of insecticide resistance, and ITNs were compared with untreated bed nets (UTNs) using the risk difference (RD). Heterogeneity was explored visually and statistically. Included were 36 laboratory and 24 field studies, reported in 25 records. Studies tested and reported resistance inconsistently. Based on the meta-analytic results, the difference in mosquito mortality risk for ITNs compared to UTNs was lower in higher resistance categories. However, mortality risk was significantly higher for ITNs compared to UTNs regardless of resistance. For cone tests: low resistance, risk difference (RD) 0.86 (95% CI 0.72 to 1.01); moderate resistance, RD 0.71 (95% CI 0.53 to 0.88); high resistance, RD 0.56 (95% CI 0.17 to 0.95). For tunnel tests: low resistance, RD 0.74 (95% CI 0.61 to 0.87); moderate resistance, RD 0.50 (95% CI 0.40 to 0.60); high resistance, RD 0.39 (95% CI 0.24 to 0.54). For hut studies: low resistance, RD 0.56 (95% CI 0.43 to 0.68); moderate resistance, RD 0.39 (95% CI 0.16 to 0.61); high resistance, RD 0.35 (95% CI 0.27 to 0.43). However, with the exception of the moderate resistance category for tunnel tests, there was extremely high heterogeneity across studies in each resistance category (chi-squared test, p<0.00001, I2 varied from 95% to 100%).
Conclusions
This meta-analysis found that ITNs are more effective than UTNs regardless of resistance. There appears to be a relationship between resistance and the RD for mosquito mortality in laboratory and field studies. However, the substantive heterogeneity in the studies' results and design may mask the true relationship between resistance and the RD, and the results need to be interpreted with caution. Our analysis suggests the potential for cumulative meta-analysis in entomological trials, but further field research in this area will require specialists in the field to work together to improve the quality of trials, and to standardise designs, assessment, and reporting of both resistance and entomological outcomes.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year more than 200 million cases of malaria occur worldwide, and more than 600,000 people, mostly children living in sub-Saharan Africa, die from this parasitic infection. Malaria is transmitted to people through the bites of night-flying mosquitoes. Soon after entering the human body, the parasite begins to replicate in red blood cells, bursting out every 2–3 days and infecting more red blood cells. The presence of the parasite in the bloodstream causes malaria's recurring flu-like symptoms, which need to be treated promptly with antimalarial drugs to prevent anemia (a reduction in red blood cell numbers) and life-threatening organ damage. Malaria can be prevented by using insecticides to control the mosquitoes (vectors) that spread the parasite and by sleeping under insecticide-treated bed nets (ITNs) to avoid mosquito bites. High levels of ITN use reduce malaria-related deaths among children by about 20%. Consequently, the widespread provision of ITNs is a mainstay of global efforts to control malaria.
Why Was This Study Done?
About 50% of African households now possess an ITN. However, the emergence of resistance to pyrethroid insecticides—the insecticide class recommended by the World Health Organization for use in ITNs—in some mosquitoes potentially threatens the efficacy of ITNs. Pyrethroids kill Anopheles mosquitoes (the main malaria vectors in sub-Saharan Africa) but also prevent mosquitoes entering houses (deterrence), disrupt feeding, and encourage mosquitoes to leave homes prematurely (“induced exophily”; Anopheles mosquitoes usually rest inside for a while after feeding). Worryingly, 27 countries in sub-Saharan Africa have already reported resistance to pyrethroids in Anopheles mosquitoes. In this systematic review and meta-analysis, the researchers assess the impact of pyrethroid resistance on the efficacy of ITNs against African anopheline mosquitoes in terms of entomological outcomes. A systematic review identifies all the research on a given topic using predefined criteria, meta-analysis uses statistical methods to combine the results of several studies, and entomological outcomes are measures of mosquito behavior and survival.
What Did the Researchers Do and Find?
The researchers identified 25 reports of laboratory and field studies of the impact of ITNs on African malaria vectors that measured the mosquitoes' resistance to pyrethroid insecticides at the time of the study. The laboratory studies used two assays to measure entomological outcomes. The cone test measured mosquito mortality (death), percent of mosquitoes knocked down (immobilized) after 60 minutes, and the time to knock down 50% or 95% of the mosquitoes after brief exposure to an ITN or untreated bed net (UTN). In the tunnel test, mosquitoes had to pass through a holed ITN or UTN to reach animal baits; counts of live and dead mosquitoes, and fed and unfed mosquitoes on both sides of the net measured deterrence, blood feeding, and mosquito mortality. In the field studies, volunteers slept under an ITN or UTN in an experimental hut. Subsequent counts of live and dead mosquitoes and fed and unfed mosquitoes inside the huts and in exit traps measured deterrence, blood feeding, mosquito mortality, and induced exophily. The researchers report that the measurement of insecticide resistance was inconsistent across the identified studies. Nevertheless, their analysis found that ITNs are more effective than UTNs in relation to mosquito mortality, regardless of resistance. There was a relationship between resistance and the risk difference for mosquito mortality in laboratory and field studies, but the substantive variation between studies means that the findings should be interpreted with caution.
What Do These Findings Mean?
These findings show that pyrethroid resistance clearly affects entomological outcomes in laboratory studies, and suggests that this pattern may also be observed in field trials. However, ITNs remained at least somewhat effective despite insecticide resistance in terms of personal protection. The researchers note that there was considerable variability (heterogeneity) among the results obtained in the field trials and suggest that poorly standardized methods and reporting might have masked the true relationship between insecticide resistance and ITN efficacy in these studies. Thus, although ITNs continue to have a substantive effect in many laboratory studies in the face of insecticide resistance, whether ITNs are likely to remain effective against insecticide-resistant mosquitoes in the real world cannot be definitively concluded. Malaria experts and vector biologists need to work together to improve the quality of field trials and to standardize the measurement of insecticide resistance and entomological outcomes, suggest the researchers. Such collaborations, they conclude, are essential to provide the data that policy makers need to plan malaria control strategies.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001619.
Information is available from the World Health Organization on malaria (in several languages); the World Malaria Report 2013 provides details of the current global malaria situation
Information is available from the World Health Organization on a call for action to tackle the growing threat of insecticide resistance and to facilitate the development of innovative vector control tools and strategies (in English, French and Spanish)
The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish) and on insecticide-treated bed nets; it also provides a selection of personal stories about malaria
Information is available from the Roll Back Malaria Partnership on the global control of malaria and on the Global Malaria Action Plan (in English and French); its website includes fact sheets about malaria in Africa and about insecticide-treated bed nets
MedlinePlus provides links to additional information on malaria (in English and Spanish)
doi:10.1371/journal.pmed.1001619
PMCID: PMC3958359  PMID: 24642791
3.  Using a new odour-baited device to explore options for luring and killing outdoor-biting malaria vectors: a report on design and field evaluation of the Mosquito Landing Box 
Parasites & Vectors  2013;6:137.
Background
Mosquitoes that bite people outdoors can sustain malaria transmission even where effective indoor interventions such as bednets or indoor residual spraying are already widely used. Outdoor tools may therefore complement current indoor measures and improve control. We developed and evaluated a prototype mosquito control device, the ‘Mosquito Landing Box’ (MLB), which is baited with human odours and treated with mosquitocidal agents. The findings are used to explore technical options and challenges relevant to luring and killing outdoor-biting malaria vectors in endemic settings.
Methods
Field experiments were conducted in Tanzania to assess if wild host-seeking mosquitoes 1) visited the MLBs, 2) stayed long or left shortly after arrival at the device, 3) visited the devices at times when humans were also outdoors, and 4) could be killed by contaminants applied on the devices. Odours suctioned from volunteer-occupied tents were also evaluated as a potential low-cost bait, by comparing baited and unbaited MLBs.
Results
There were significantly more Anopheles arabiensis, An. funestus, Culex and Mansonia mosquitoes visiting baited MLB than unbaited controls (P≤0.028). Increasing sampling frequency from every 120 min to 60 and 30 min led to an increase in vector catches of up to 3.6 fold (P≤0.002), indicating that many mosquitoes visited the device but left shortly afterwards. Outdoor host-seeking activity of malaria vectors peaked between 7:30 and 10:30pm, and between 4:30 and 6:00am, matching durations when locals were also outdoors. Maximum mortality of mosquitoes visiting MLBs sprayed or painted with formulations of candidate mosquitocidal agent (pirimiphos-methyl) was 51%. Odours from volunteer occupied tents attracted significantly more mosquitoes to MLBs than controls (P<0.001).
Conclusion
While odour-baited devices such as the MLBs clearly have potential against outdoor-biting mosquitoes in communities where LLINs are used, candidate contaminants must be those that are effective at ultra-low doses even after short contact periods, since important vector species such as An. arabiensis make only brief visits to such devices. Natural human odours suctioned from occupied dwellings could constitute affordable sources of attractants to supplement odour baits for the devices. The killing agents used should be environmentally safe, long lasting, and have different modes of action (other than pyrethroids as used on LLINs), to curb the risk of physiological insecticide resistance.
doi:10.1186/1756-3305-6-137
PMCID: PMC3655910  PMID: 23642306
4.  Beer Consumption Increases Human Attractiveness to Malaria Mosquitoes 
PLoS ONE  2010;5(3):e9546.
Background
Malaria and alcohol consumption both represent major public health problems. Alcohol consumption is rising in developing countries and, as efforts to manage malaria are expanded, understanding the links between malaria and alcohol consumption becomes crucial. Our aim was to ascertain the effect of beer consumption on human attractiveness to malaria mosquitoes in semi field conditions in Burkina Faso.
Methodology/Principal Findings
We used a Y tube-olfactometer designed to take advantage of the whole body odour (breath and skin emanations) as a stimulus to gauge human attractiveness to Anopheles gambiae (the primary African malaria vector) before and after volunteers consumed either beer (n = 25 volunteers and a total of 2500 mosquitoes tested) or water (n = 18 volunteers and a total of 1800 mosquitoes). Water consumption had no effect on human attractiveness to An. gambiae mosquitoes, but beer consumption increased volunteer attractiveness. Body odours of volunteers who consumed beer increased mosquito activation (proportion of mosquitoes engaging in take-off and up-wind flight) and orientation (proportion of mosquitoes flying towards volunteers' odours). The level of exhaled carbon dioxide and body temperature had no effect on human attractiveness to mosquitoes. Despite individual volunteer variation, beer consumption consistently increased attractiveness to mosquitoes.
Conclusions/Significance
These results suggest that beer consumption is a risk factor for malaria and needs to be integrated into public health policies for the design of control measures.
doi:10.1371/journal.pone.0009546
PMCID: PMC2832015  PMID: 20209056
5.  Door to Door Survey and Community Participation to Implement a New County Mosquito Control Program in Wayne County, North Carolina, USA 
Community involvement in mosquito management programs provides more sustainable and effective organization and service. A door to door survey in Wayne County, NC carried out by student volunteers, resulted in 60 household responses. Residents had not previously experienced outreach from the county (88%), and 95% of them thought the student door to door survey was an effective form of outreach. One third of the residents thought mosquitoes were severe where they lived, but only 9% thought they had any containers in their yard that might breed mosquitoes. Only 15% of the residents were concerned about mosquito borne diseases. These responses provide evidence that outreach and education on mosquito control and diseases were necessary steps for future mosquito control community planning.
doi:10.3390/ijerph6082150
PMCID: PMC2738879  PMID: 19742152
community health; mosquito control; community survey
6.  Attraction of Anopheles gambiae to odour baits augmented with heat and moisture 
Malaria Journal  2010;9:6.
Background
The search for a standard human surrogate in the form of a synthetic mosquito attractant has been the goal of many laboratories around the world. Besides alleviating the occupational risk subjected to volunteers participating in vector surveillance and control, discovery of potent attractants underpins the development and deployment of mass trapping devices for controlling mosquito-borne diseases.
Methods
A dual-port olfactometer was used to assess behavioural responses of female Anopheles gambiae mosquitoes towards synthetic versus natural (whole human emanations and worn socks) attractants. The synthetic attractants included a standard blend consisting of ammonia, carbon dioxide and water; and Ifakara blend 1 (IB1) consisting of various aliphatic carboxylic acids. Natural attractants were obtained from two males known to be less and highly attractive (LA and HA, respectively) to the mosquitoes. Mosquito responses to the volunteers' worn socks were also investigated. The effect of heat (25-27°C) and moisture (75-85%) on the mosquito behavioural responses was determined.
Results
A significantly higher proportion of mosquitoes was attracted to each volunteer when compared to the standard blend. Whereas the proportion of mosquitoes attracted to person LA versus IB1 (49% versus 51%, respectively; P = 0.417) or his worn socks did not differ (61% versus 39%, respectively; P = 0.163), far more mosquitoes were attracted to person HA relative to IB1 (96% versus 4%; P = 0.001) or his worn socks (91% versus 9%; P = 0.001). Person HA attracted a significantly higher proportion of mosquitoes than his worn socks, the standard blend and IB1 when these were augmented with heat, moisture or both (P = 0.001). Similar results were obtained with person LA except that the proportion of mosquitoes attracted to him versus his worn sock augmented with heat (P = 0.65) or IB1 augmented with heat and moisture (P = 0.416) did not differ significantly.
Conclusions
These findings indicate that olfactory cues are key mediators of the mosquito host-seeking process and that heat and moisture play a minor role. The need for a standard, highly stringent positive control for screening synthetic attractants is strongly highlighted.
doi:10.1186/1475-2875-9-6
PMCID: PMC2822790  PMID: 20051143
7.  Ethnobotanical study of some of mosquito repellent plants in north-eastern Tanzania 
Malaria Journal  2008;7:152.
Background
The use of plant repellents against nuisance biting insects is common and its potential for malaria vector control requires evaluation in areas with different level of malaria endemicity. The essential oils of Ocimum suave and Ocimum kilimandscharicum were evaluated against malaria vectors in north-eastern Tanzania.
Methodology
An ethnobotanical study was conducted at Moshi in Kilimanjaro region north-eastern Tanzania, through interviews, to investigate the range of species of plants used as insect repellents. Also, bioassays were used to evaluate the protective potential of selected plants extracts against mosquitoes.
Results
The plant species mostly used as repellent at night are: fresh or smoke of the leaves of O. suave and O. kilimandscharicum (Lamiaceae), Azadirachta indica (Meliaceae), Eucalyptus globules (Myrtaceae) and Lantana camara (Verbenaceae). The most popular repellents were O. kilimandscharicum (OK) and O. suave (OS) used by 67% out of 120 households interviewed. Bioassay of essential oils of the two Ocimum plants was compared with citronella and DEET to study the repellence and feeding inhibition of untreated and treated arms of volunteers. Using filter papers impregnated with Ocimum extracts, knockdown effects and mortality was investigated on malaria mosquito Anopheles arabiensis and Anopheles gambiae, including a nuisance mosquito, Culex quinquefasciatus. High biting protection (83% to 91%) and feeding inhibition (71.2% to 92.5%) was observed against three species of mosquitoes. Likewise the extracts of Ocimum plants induced KD90 of longer time in mosquitoes than citronella, a standard botanical repellent. Mortality induced by standard dosage of 30 mg/m2 on filter papers, scored after 24 hours was 47.3% for OK and 57% for OS, compared with 67.7% for citronella.
Conclusion
The use of whole plants and their products as insect repellents is common among village communities of north-eastern Tanzania and the results indicate that the use of O. suave and O. kilimandscharicum as a repellent would be beneficial in reducing vector biting. The widespread use of this approach has a potential to complement other control measures.
doi:10.1186/1475-2875-7-152
PMCID: PMC2519077  PMID: 18687119
8.  Preventing Childhood Malaria in Africa by Protecting Adults from Mosquitoes with Insecticide-Treated Nets 
PLoS Medicine  2007;4(7):e229.
Background
Malaria prevention in Africa merits particular attention as the world strives toward a better life for the poorest. Insecticide-treated nets (ITNs) represent a practical means to prevent malaria in Africa, so scaling up coverage to at least 80% of young children and pregnant women by 2010 is integral to the Millennium Development Goals (MDG). Targeting individual protection to vulnerable groups is an accepted priority, but community-level impacts of broader population coverage are largely ignored even though they may be just as important. We therefore estimated coverage thresholds for entire populations at which individual- and community-level protection are equivalent, representing rational targets for ITN coverage beyond vulnerable groups.
Methods and Findings
Using field-parameterized malaria transmission models, we show that high (80% use) but exclusively targeted coverage of young children and pregnant women (representing <20% of the population) will deliver limited protection and equity for these vulnerable groups. In contrast, relatively modest coverage (35%–65% use, with this threshold depending on ecological scenario and net quality) of all adults and children, rather than just vulnerable groups, can achieve equitable community-wide benefits equivalent to or greater than personal protection.
Conclusions
Coverage of entire populations will be required to accomplish large reductions of the malaria burden in Africa. While coverage of vulnerable groups should still be prioritized, the equitable and communal benefits of wide-scale ITN use by older children and adults should be explicitly promoted and evaluated by national malaria control programmes. ITN use by the majority of entire populations could protect all children in such communities, even those not actually covered by achieving existing personal protection targets of the MDG, Roll Back Malaria Partnership, or the US President's Malaria Initiative.
Based on malaria transmission models, Gerry Killeen and colleagues suggest that, while coverage of pregnant women and children should still be prioritized, wide-scale communal use of insecticide-treated bed nets would provide considerable benefit to vulnerable groups and should be promoted and evaluated in the field.
Editors' Summary
Background.
Malaria—a parasitic disease common in tropical and subtropical countries—causes about a million deaths every year, mainly among young children and pregnant women living in sub-Saharan Africa. The parasite responsible for most of these deaths is Plasmodium falciparum. Like all malaria parasites, it has a complex life cycle, part of which takes place inside mosquitoes. When a malaria-carrying mosquito bites a person (usually at night), parasites enter the human blood stream and travel to the liver where they reproduce before invading red blood cells. Here, they multiply again before bursting out and infecting more red blood cells as well as causing a high fever and sometimes damaging the nervous system, liver, and kidneys. When a mosquito bites someone who is infected, it often picks up parasites in its blood meal (mosquitoes need mammalian blood for successful reproduction), thus completing the malarial transmission cycle.
Why Was This Study Done?
One way to break this cycle and reduce malarial transmission is to decrease the number of contacts between people and mosquitoes by encouraging people to sleep under insecticide-treated nets (ITNs). Field studies have shown that ITN use reduces deaths from malaria, so the Roll Back Malaria Partnership, the United Nations Millennium Development Goals, and the US President's Malaria Initiative have set a target of at least 80% use of ITNs by young children and pregnant women (the people most vulnerable to malaria) by 2010. But would broader population coverage with ITNs interrupt the malarial transmission cycle more effectively? Might the wider use of ITNs (which both directly kill mosquitoes and repel them so that mosquitoes have to travel farther to get the blood they need to reproduce) be a more effective way to reduce local mosquito numbers and, consequently, human–mosquito contacts and malarial transmission? In this study, the researchers used mathematical modeling to ask how much community-wide coverage with ITNs is needed to protect vulnerable individuals against malaria without them personally using an ITN.
What Did the Researchers Do and Find?
The researchers developed a model for the transmission of the malaria parasite using data collected in Tanzanian villages where malaria is common, and used it to investigate how different patterns of ITN use might affect the individual protection of ITN users and the communal protection of nonusers. High (80%) coverage targeted at young children and pregnant women (less than 20% of the population) provided limited but valuable protection to these vulnerable individuals. However, the model predicted that a similar degree of community-wide protection would result if 35% of the whole population slept under ITNs provided there was no nonhuman blood supply for the mosquitoes. In other words, the vulnerable individuals in the population received the same degree of protection from this intervention as they would have got from personally using an ITN. If an alternative blood supply for the mosquitoes (for example, cattle) was included in the model, just over half of the population needed to sleep under ITNs to provide the same degree of community-wide protection as targeted ITN use.
What Do These Findings Mean?
Although the use of ITNs by vulnerable groups should remain a priority, these findings suggest that the wide-scale ITN use by the entire population should also be promoted. The use of ITNs by about half the population, predict the researchers, could protect all the young children in that population, even those who did not sleep under a net. As with all mathematical models, the accuracy of this prediction depends on the assumptions and data incorporated into the model. So before recommending community-wide use of ITNs, the actual level of communal protection provided by increased ITN coverage must be measured by rigorously evaluating ongoing national programmes. If such surveillance data confirm this model's predictions, wide coverage with ITNs might do more for public health in Africa than previously thought, suggest the researchers, provided the financial and logistical challenges associated with achieving high ITN coverage in this poor region of the world can be solved.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040229.
The US Centers for Disease Control and Prevention provide information on malaria and on insecticide-treated nets (in English and Spanish)
The MedlinePlus encyclopedia contains a page on malaria (in English and Spanish)
Information is available from the World Health Organization on malaria (in English, Spanish, French, Russian, Arabic, and Chinese) and from the Roll Back Malaria Partnership on the use of insecticide-treated nets
Information about the US President's Malaria Initiative is available, including a fact sheet on the use of ITNs
doi:10.1371/journal.pmed.0040229
PMCID: PMC1904465  PMID: 17608562
9.  Heterogeneous Feeding Patterns of the Dengue Vector, Aedes aegypti, on Individual Human Hosts in Rural Thailand 
Background
Mosquito biting frequency and how bites are distributed among different people can have significant epidemiologic effects. An improved understanding of mosquito vector-human interactions would refine knowledge of the entomological processes supporting pathogen transmission and could reveal targets for minimizing risk and breaking pathogen transmission cycles.
Methodology and principal findings
We used human DNA blood meal profiling of the dengue virus (DENV) vector, Aedes aegypti, to quantify its contact with human hosts and to infer epidemiologic implications of its blood feeding behavior. We determined the number of different people bitten, biting frequency by host age, size, mosquito age, and the number of times each person was bitten. Of 3,677 engorged mosquitoes collected and 1,186 complete DNA profiles, only 420 meals matched people from the study area, indicating that Ae. aegypti feed on people moving transiently through communities to conduct daily business. 10–13% of engorged mosquitoes fed on more than one person. No biting rate differences were detected between high- and low-dengue transmission seasons. We estimate that 43–46% of engorged mosquitoes bit more than one person within each gonotrophic cycle. Most multiple meals were from residents of the mosquito collection house or neighbors. People ≤25 years old were bitten less often than older people. Some hosts were fed on frequently, with three hosts bitten nine times. Interaction networks for mosquitoes and humans revealed biologically significant blood feeding hotspots, including community marketplaces.
Conclusion and significance
High multiple-feeding rates and feeding on community visitors are likely important features in the efficient transmission and rapid spread of DENV. These results help explain why reducing vector populations alone is difficult for dengue prevention and support the argument for additional studies of mosquito feeding behavior, which when integrated with a greater understanding of human behavior will refine estimates of risk and strategies for dengue control.
Author Summary
Dengue, a potentially lethal infection impacting hundreds of millions of human lives annually, is caused by viruses transmitted during mosquito blood feeding. With no vaccine or treatment commercially available, understanding the underlying factors linked to virus exposure is critical for developing more effective dengue interventions. We conducted a study in an endemic region of Thailand where transmission is high and children are expected to be the non-immune, amplifying portion of the host population. We examined Ae. aegypti feeding patterns and risk by matching human DNA profiles in blood-fed mosquitoes to study area residents. A small number of meals matched people from the study area, suggesting that mosquitoes feed on people moving transiently through communities. People under 25 years of age were bitten less frequently than older people. We constructed network models to explore the presence of mosquito feeding “hotspots” and detected a local market “hotspot” in one study village during the high dengue transmission season. Our results provide new details on dengue vector feeding patterns and highlight the need to conduct integrated studies of vector feeding and human behavior, and virus transmission patterns in order to better understand the dengue transmission efficiency and spread.
doi:10.1371/journal.pntd.0003048
PMCID: PMC4125296  PMID: 25102306
10.  Comparison of mosquito control programs in seven urban sites in Africa, the Middle East, and the Americas 
Mosquito control programs at seven urban sites in Kenya, Egypt, Israel, Costa Rica, and Trinidad are described and compared. Site-specific urban and disease characteristics, organizational diagrams, and strengths, weaknesses, obstacles and threats (SWOT) analysis tools are used to provide a descriptive assessment of each mosquito control program, and provide a comparison of the factors affecting mosquito abatement. The information for SWOT analysis is collected from surveys, focus group discussions, and personal communication. SWOT analysis identified various issues affecting the efficiency and sustainability of mosquito control operations. The main outcome of our work was the description and comparison of mosquito control operations within the context of each study site’s biological, social, political, management, and economic conditions. The issues identified in this study ranged from lack of inter-sector collaboration to operational issues of mosquito control efforts. A lack of sustainable funding for mosquito control was a common problem for most sites. Many unique problems were also identified, which included lack of mosquito surveillance, lack of law enforcement, and negative consequences of human behavior. Identifying common virtues and shortcomings of mosquito control operations is useful in identifying “best practices” for mosquito control operations, thus leading to better control of mosquito biting and mosquito-borne disease transmission.
doi:10.1016/j.healthpol.2007.01.009
PMCID: PMC2048658  PMID: 17316882
11.  Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae 
PLoS Pathogens  2013;9(1):e1003145.
Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
Author Summary
The mosquito immune system relies on innate humoral and cellular reactions to fight infections, including those by malaria parasites that must pass through mosquitoes before they can infect humans. Therefore, a detailed molecular understanding of these reactions could assist the design of new ways to control the spread of malaria and other mosquito-borne diseases. Here we use a technique to silence in mosquito cultured cells genes that are highly and/or specifically expressed in mosquito hemocytes, the equivalent of human white blood cells, as a means to investigate their function in reactions of the mosquito immune system. Our study identifies several novel regulators of immune reactions including phagocytosis, the engulfment and subsequent destruction of bacteria and other pathogens by hemocytes, the production of antimicrobial peptides, which directly kill or inhibit the proliferation of microbes, and the basal and induced production of an important complement regulator. Complement is a robust reaction of mosquitoes against malaria parasites and bacteria through phagocytosis, lysis or melanization (the enclosure of pathogens in a melanin capsule). We also reveal intriguing molecular connections between these reactions such as phagocytosis and regulation of complement. Our study provides novel insights into mosquito immune system and its reactions against infections.
doi:10.1371/journal.ppat.1003145
PMCID: PMC3561300  PMID: 23382679
12.  Perspectives of people in Mali toward genetically-modified mosquitoes for malaria control 
Malaria Journal  2010;9:128.
Background
Genetically-modified (GM) mosquitoes have been proposed as part of an integrated vector control strategy for malaria control. Public acceptance is essential prior to field trials, particularly since mosquitoes are a vector of human disease and genetically modified organisms (GMOs) face strong scepticism in developed and developing nations. Despite this, in sub-Saharan Africa, where the GM mosquito effort is primarily directed, very little data is available on perspectives to GMOs. Here, results are presented of a qualitative survey of public attitudes to GM mosquitoes for malaria control in rural and urban areas of Mali, West Africa between the months of October 2008 and June 2009.
Methods
The sample consisted of 80 individuals - 30 living in rural communities, 30 living in urban suburbs of Bamako, and 20 Western-trained and traditional health professionals working in Bamako and Bandiagara. Questions were asked about the cause of malaria, heredity and selective breeding. This led to questions about genetic alterations, and acceptable conditions for a release of pest-resistant GM corn and malaria-refractory GM mosquitoes. Finally, participants were asked about the decision-making process in their community. Interviews were transcribed and responses were categorized according to general themes.
Results
Most participants cited mosquitoes as one of several causes of malaria. The concept of the gene was not widely understood; however selective breeding was understood, allowing limited communication of the concept of genetic modification. Participants were open to a release of pest-resistant GM corn, often wanting to conduct a trial themselves. The concept of a trial was reapplied to GM mosquitoes, although less frequently. Participants wanted to see evidence that GM mosquitoes can reduce malaria prevalence without negative consequences for human health and the environment. For several participants, a mosquito control programme was preferred; however a transgenic release that satisfied certain requirements was usually acceptable.
Conclusions
Although there were some dissenters, the majority of participants were pragmatic towards a release of GM mosquitoes. An array of social and cultural issues associated with malaria, mosquitoes and genetic engineering became apparent. If these can be successfully addressed, then social acceptance among the populations surveyed seems promising.
doi:10.1186/1475-2875-9-128
PMCID: PMC2881074  PMID: 20470410
13.  Implications of low-density microfilariae carriers in Anopheles transmission areas: molecular forms of Anopheles gambiae and Anopheles funestus populations in perspective 
Parasites & Vectors  2014;7:157.
Background
Previous studies have shown a general reduction in annual transmission potential (ATP) of Anopheles species after mass drug administration (MDA) in lymphatic filariasis endemic communities. Whereas results obtained from a monitoring programme after three years of MDA revealed a decrease in ATP of Anopheles funestus this was not the same for An. gambiae s.s. in Ghana. In this study, the ability of these vectors in transmitting Wuchereria bancrofti in nine lymphatic filariasis endemic communities in Gomoa District of Ghana after four rounds of MDA with ivermectin and albendazole was investigated.
Methods
After mass screening of inhabitants in these communities, twelve consenting volunteers with different intensities of microfilariae (mf) slept under partly opened mosquito nets as sources of mf blood meal. Hourly collection of mosquitoes and finger-pricked blood were taken from 21.00 to 06.00 hours the following day. For each hour, half of the mosquitoes collected were immediately killed and dissected for mf. The remaining half were maintained up to 13 days for parasite maturation. Parasitaemia and infection rates in the mosquitoes were determined by microscopy. The mosquitoes were identified by microscopy and molecular techniques.
Results
A total of 1,083 participants were screened and the overall parasite prevalence was 1.6% with mf intensities ranging from 0 to 59 per 100 μl and geometric mean intensity of 1.1 mf per ml of blood. Of the 564 mosquitoes collected, 350 (62.1%) were Anopheles spp., from which 310 (88.6%) were An. funestus and 32 (9.1%) An. gambiae. Six anopheline mosquitoes (1.7%) were found infected with L1, but no larva was observed in any of the mosquitoes maintained up to 13 days. Molecular studies showed all An. gambiae s.l. to be An. gambiae s.s., of which 21 (70%) were of the M molecular form.
Conclusion
At low-level parasitaemia after 4 rounds of MDA, there was no recovery of infective stage larvae of W. bancrofti in An. funestus s.l. as well as M and S forms of An. gambiae.
doi:10.1186/1756-3305-7-157
PMCID: PMC3974918  PMID: 24690378
Mass drug administration; Low-density microfilariae carriers; Wuchereria bancrofti; Anopheles gambiae; Anopheles funestus
14.  Mosquitoes Associated with Ditch-Plugged and Control Tidal Salt Marshes on the Delmarva Peninsula 
A study was conducted during the summer of 2009 (from July to September) to characterize mosquito communities among different habitats in five historically ditched tidal salt marshes and three adjacent wooded areas in the E.A. Vaughn Wetland Management Area on the Maryland Delmarva Peninsula, USA. Study marshes are characteristic of Atlantic coastal salt marshes that had undergone grid ditching from the 1930s to 1950s. In the autumn of 2008 (October and November) ditches were plugged near their outlets in two (‘experimental’) marshes with the aim to restore their natural tidal hydrology. The three other marshes were not plugged. Marshes were sampled from July to September in 2009 by using standard dip count method. A total of 2,457 mosquito larvae representing six species were collected on 15.4% (86/557) of all sample occasions and 399 adults representing four mosquito species were collected from landing counts. Aedes sollicitans, Anopheles bradleyi and Culex salinarius were the most common species collected in larval habitats, and Ae. sollicitans was the most common adult collected. Wooded habitats had more total mosquitoes, were also more frequently occupied by mosquitoes and had higher densities of mosquitoes than marsh habitats. Almost all larvae collected from marshes were from one experimental and one control site. The majority of larvae at the control site were Ae. sollicitans in marsh pannes while Cx. salinarius, An. bradleyi, Ae. cantator, and Ae. sollicitans were collected in high numbers from ditches at the experimental site. We found a difference in the proportion of marsh pannes occupied by Ae. sollicitans but not total mosquitoes sampled 4–5 days after spring tide events than on other occasions. Salinity measures of 42 larval habitats showed lower median salinity in mosquito-occupied habitats (11.5 ppt) than unoccupied habitats (20.1 ppt), and in habitats in wooded areas followed by ditches and pannes in marsh areas. The results of this study suggest that wooded areas adjacent to salt marshes may be a substantial source of biting adult mosquitoes usually associated with salt marsh habitats and that ditch plugging may alter the productivity of mosquitoes on some marshes. We recommend future studies consider mosquito productivity from habitats surrounding salt marshes, and if assessments of marsh alterations are a goal, compare multiple experimental and control areas before and after treatments to determine if alterations have a consistent impact on regional mosquito production.
doi:10.3390/ijerph8083099
PMCID: PMC3166729  PMID: 21909293
ditch plugging; Delmarva Peninsula; tidal salt marsh; restoration; salinity
15.  Indoor Application of Attractive Toxic Sugar Bait (ATSB) in Combination with Mosquito Nets for Control of Pyrethroid-Resistant Mosquitoes 
PLoS ONE  2013;8(12):e84168.
Background
Attractive toxic sugar bait (ATSB) sprayed onto vegetation has been successful in controlling Anopheles mosquitoes outdoors. Indoor application of ATSB has yet to be explored. The purpose of this study was to determine whether ATSB stations positioned indoors have the potential to kill host-seeking mosquitoes and constitute a new approach to control of mosquito-borne diseases.
Methods
Insecticides were mixed with dyed sugar solution and tested as toxic baits against Anopheles arabiensis, An. Gambiae s.s. and Culex quinquefasciatus in feeding bioassay tests to identify suitable attractant-insecticide combinations. The most promising ATSB candidates were then trialed in experimental huts in Moshi, Tanzania. ATSB stations were hung in huts next to untreated mosquito nets occupied by human volunteers. The proportions of mosquitoes killed in huts with ATSB treatments relative to huts with non-insecticide control treatments huts were recorded, noting evidence of dye in mosquito abdomens.
Results
In feeding bioassays, chlorfenapyr 0.5% v/v, boric acid 2% w/v, and tolfenpyrad 1% v/v, mixed in a guava juice-based bait, each killed more than 90% of pyrethroid-susceptible An. Gambiae s.s. and pyrethroid-resistant An. arabiensis and Cx. quinquefasciatus. In the hut trial, mortality rates of the three ATSB treatments ranged from 41-48% against An. arabiensis and 36-43% against Cx. quinquefasciatus and all were significantly greater than the control mortalities: 18% for An. arabiensis, 7% for Cx. quinquefasciatus (p<0.05). Mortality rates with ATSB were comparable to those with long lasting insecticidal nets previously tested against the same species in this area.
Conclusions
Indoor ATSB shows promise as a supplement to mosquito nets for controlling mosquitoes. Indoor ATSB constitute a novel application method for insecticide classes that act as stomach poisons and have not hitherto been exploited for mosquito control. Combined with LLIN, indoor use of ATSB has the potential to serve as a strategy for managing insecticide resistance.
doi:10.1371/journal.pone.0084168
PMCID: PMC3868566  PMID: 24367638
16.  Anti-mosquito plants as an alternative or incremental method for malaria vector control among rural communities of Bagamoyo District, Tanzania 
Background
Plants represent one of the most accessible resources available for mosquito control by communities in Tanzania. However, no documented statistics exist for their contribution in the management of mosquitoes and other insects except through verbal and some publications. This study aimed at assessing communities’ knowledge, attitudes and practices of using plants as an alternative method for mosquito control among selected communities in a malaria-prone area in Tanzania.
Methods
Questionnaires were administered to 202 respondents from four villages of Bagamoyo District, Pwani Region, in Tanzania followed by participatory rural appraisal with village health workers. Secondary data collection for plants mentioned by the communities was undertaken using different search engines such as googlescholar, PubMED and NAPRALERT.
Results
Results showed about 40.3% of respondents used plants to manage insects, including mosquitoes. A broad profile of plants are used, including “mwarobaini” (Azadirachta indica) (22.5%), “mtopetope” (Annona spp) (20.8%), “mchungwa/mlimau” (Citrus spp) (8.3%), “mvumbashi/uvumbati” (Ocimum spp) (7.4%), “mkorosho” (Anacadium occidentale) (7.1%), “mwembe” (5.4%) (Mangifera indica), “mpera” (4.1%) (Psidium spp) and “maganda ya nazi” (4.1%) (Cocos nucifera). Majority of respondents collected these plants from the wild (54.2%), farms (28.9%) and/or home gardens (6%). The roles played by these plants in fighting mosquitoes is reflected by the majority that deploy them with or without bed-nets (p > 0.55) or insecticidal sprays (p >0.22). Most respondents were aware that mosquitoes transmit malaria (90.6%) while few respondents associated elephantiasis/hydrocele (46.5%) and yellow fever (24.3%) with mosquitoes. Most of the ethnobotanical uses mentioned by the communities were consistent with scientific information gathered from the literature, except for Psidium guajava, which is reported for the first time in insect control.
Conclusion
This survey has indicated some knowledge gap among community members in managing mosquito vectors using plant. The communities need a basic health education and sensitization for effective exploitation of this valuable tool for reducing mosquitoes and associated disease burdens. On the other hand, the government of Tanzania should strengthen advocacy of botanical pesticides development, registration and regulation for public health benefits because they are source of pest control tools people rely on them.
doi:10.1186/1746-4269-10-56
PMCID: PMC4131773  PMID: 25015092
Mosquitoes; Vector control; Ethno-knowledge; Medicinal plants; Azadirachta indica; Annona species
17.  Heterogeneity, Mixing, and the Spatial Scales of Mosquito-Borne Pathogen Transmission 
PLoS Computational Biology  2013;9(12):e1003327.
The Ross-Macdonald model has dominated theory for mosquito-borne pathogen transmission dynamics and control for over a century. The model, like many other basic population models, makes the mathematically convenient assumption that populations are well mixed; i.e., that each mosquito is equally likely to bite any vertebrate host. This assumption raises questions about the validity and utility of current theory because it is in conflict with preponderant empirical evidence that transmission is heterogeneous. Here, we propose a new dynamic framework that is realistic enough to describe biological causes of heterogeneous transmission of mosquito-borne pathogens of humans, yet tractable enough to provide a basis for developing and improving general theory. The framework is based on the ecological context of mosquito blood meals and the fine-scale movements of individual mosquitoes and human hosts that give rise to heterogeneous transmission. Using this framework, we describe pathogen dispersion in terms of individual-level analogues of two classical quantities: vectorial capacity and the basic reproductive number, . Importantly, this framework explicitly accounts for three key components of overall heterogeneity in transmission: heterogeneous exposure, poor mixing, and finite host numbers. Using these tools, we propose two ways of characterizing the spatial scales of transmission—pathogen dispersion kernels and the evenness of mixing across scales of aggregation—and demonstrate the consequences of a model's choice of spatial scale for epidemic dynamics and for estimation of , both by a priori model formulas and by inference of the force of infection from time-series data.
Author Summary
Pathogens transmitted by mosquitoes, such as malaria and dengue, are notorious for the biological complexity associated with how they are transmitted within local communities. Yet mathematical models for these pathogens, which are critical tools for making recommendations for control policy, are based around concepts originally designed to describe how molecules interact in chemical systems. To provide those interested in mosquito-borne diseases a more appropriate tool for modeling their transmission, we introduce a mathematical framework that is based on the spatial locations where mosquitoes lay eggs and feed on blood and how mosquitoes and hosts move about those locations. Analysis of this framework shows that the transmission contributions of different hosts and locations can be calculated, and that overall potential for transmission in a community depends on three concepts: heterogeneous exposure (some people bitten by mosquitoes more than others), poor mixing (non-random contacts between hosts and mosquitoes), and finite population sizes (each host can contribute at most one new infection towards the population total). Together, these factors determine critical levels of vaccination coverage to eliminate a pathogen and the spatial areas over which transmission should be modeled and studied in the field.
doi:10.1371/journal.pcbi.1003327
PMCID: PMC3861021  PMID: 24348223
18.  Competency of Anopheles stephensi mysorensis strain for Plasmodium vivax and the role of inhibitory carbohydrates to block its sporogonic cycle 
Malaria Journal  2008;7:131.
Background
Despite the abundance of studies conducted on the role of mosquitoes in malaria transmission, the biology and interaction of Plasmodium with its insect host still holds many mysteries. This paper provides the first study to follow the sporogonic cycle of Plasmodium vivax in a wild insecticide-resistant mysorensis strain of Anopheles stephensi, a major vector of vivax malaria in south-eastern Iran. The study subsequently demonstrates that host-parasite sugar binding interactions are critical to the development of this parasite in the salivary glands of its mosquito host. The identity of the receptors or sugars involved was revealed by a receptor "pre-saturation" strategy in which sugars fed to the mosquitoes inhibited normal host-parasite interactions.
Methods
Anopheles stephensi mysorensis mosquitoes were artificially infected with P. vivax by feeding on the blood of gametocytaemic volunteers reporting to local malaria clinics in the Sistan-Baluchistan province of south-eastern Iran. In order to determine the inhibitory effect of carbohydrates on sporogonic development, vector mosquitoes were allowed to ingest blood meals containing both gametocytes and added carbohydrates. The carbohydrates tested were GlcNAc, GalNAc, arabinose, fucose, mannose, lactose, glucose and galactose. Sporogonic development was assessed by survival of the parasite at both the oocyst and sporozoite stages.
Results
Oocyst development was observed among nearly 6% of the fed control mosquitoes but the overall number of mosquitoes exhibiting sporozoite invasion of the salivary glands was 47.5% lower than the number supporting oocysts in their midgut. Of the tested carbohydrates, only arabinose and fucose slightly perturbed the development of P. vivax oocysts at the basal side of the mosquito midgut, and the remaining sugars caused no reductions in oocyst development. Strikingly however, sporozoites were completely absent from the salivary glands of mosquitoes treated with mannose, GalNAc, and lactose.
Conclusion
The study indicates that An. stephensi in southern Iran has the potential to survive long enough to be re-infected and transmit vivax malaria several times, based on the average adult female longevity (about 30 days) and its gonotrophic cycle (2–3 days) during the malaria transmission season. Certain sugar binding interactions are important for the development of P. vivax sporozoites, and this information may be instrumental for the development of transmission blocking strategies.
doi:10.1186/1475-2875-7-131
PMCID: PMC2500038  PMID: 18627630
19.  Community Knowledge and Experience of Mosquitoes and Personal Prevention and Control Practices in Lhasa, Tibet 
Since 2009, great public attention has been paid in Lhasa City (Tibet, China) to mosquito bites and accompanying inflammatory complications. However, the potential contribution of knowledge levels, experiences, disease control and preventive practices (KEP) towards mosquitoes has not received much attention. To investigate community KEP concerning mosquitoes in Lhasa, a cross-sectional survey was undertaken in four sub-districts of urban Lhasa in 2012. Questionnaires were designed to collect information regarding socio-demographics and KEP concerning the harmful effects of mosquitoes on participants. The scoring for KEP was developed after consultation of literature. A total of 591 eligible questionnaires were examined. The majority of respondents were female (61.8%) with a mean age of 46 years. Nearly all of the respondents were of Tibetan nationality (97.4%) and living in registered native households (92.7%), who have less than primary school education. The averages of overall score, knowledge score, experience score, and practice score were 9.23, 4.53, 1.80, 2.90, respectively. The registered household with the highest overall score, knowledge score and practice score was non-native. Female subjects with monthly incomes between 1000 and 3000 RMB had higher experience scores. The correlation analysis revealed that significant positive linear correlations existed between knowledge and experience, knowledge and practices, and experience and practices towards mosquitoes. Past experiences with mosquitoes can result in a better knowledge of effective mosquito control practices in the present and the future. Though the average of overall scores related to mosquitoes is high among the participants in Lhasa, however, the knowledge about the ecological habits of mosquitoes should be strengthened. The findings in this study may help to develop strategies and measures of mosquito and mosquito-borne diseases in the future, not only in Lhasa, but also in similar altitude, latitude and longitude regions worldwide.
doi:10.3390/ijerph110909919
PMCID: PMC4199058  PMID: 25250847
knowledge; experience; prevention and control practice; mosquito; public health; Tibet
20.  Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems 
PLoS Pathogens  2012;8(11):e1003058.
Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect's open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.
Author Summary
Mosquitoes transmit diseases such as malaria, dengue fever, West Nile virus and lymphatic filariasis. A mosquito initially acquires a pathogen when she ingests a blood meal from an infected person or animal. Then, after a period of development and/or replication in the mosquito gut, the pathogen enters the hemocoel (body cavity) and undergoes an obligate migration to the salivary glands (the destination for viruses and protozoans) or the mouthparts (the destination for larger worms). During this migration, pathogens are subject to two potentially antagonistic mosquito forces: immune responses and circulatory currents. In this study, we examined the physiological interactions between the mosquito immune and circulatory systems. We show that when mosquitoes are infected with bacteria or malaria parasites, mosquito immune cells (hemocytes) migrate to the areas surrounding the valves of the heart. At these areas of rapid and dynamic hemolymph (mosquito blood) flow, hemocytes swiftly phagocytose and kill pathogens. These experiments describe a novel and basal insect immune response that fundamentally relies on the physiological interaction between the mosquito circulatory and immune system. Furthermore, because traversal of the hemocoel is required for pathogen transmission, this new knowledge could be used in the development of novel disease control strategies.
doi:10.1371/journal.ppat.1003058
PMCID: PMC3510235  PMID: 23209421
21.  Bacterial Communities Associated with Culex Mosquito Larvae and Two Emergent Aquatic Plants of Bioremediation Importance 
PLoS ONE  2013;8(8):e72522.
Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito (Culextarsalis), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectuscalifornicus and alkali bulrush, Schoenoplectusmaritimus), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus Thorsellia (Enterobacteriaceae), was the most abundant phylum recovered from C. tarsalis larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within C. tarsalis larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although Thorsellia spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.
doi:10.1371/journal.pone.0072522
PMCID: PMC3744470  PMID: 23967314
22.  Target product profile choices for intra-domiciliary malaria vector control pesticide products: repel or kill? 
Malaria Journal  2011;10:207.
Background
The most common pesticide products for controlling malaria-transmitting mosquitoes combine two distinct modes of action: 1) conventional insecticidal activity which kills mosquitoes exposed to the pesticide and 2) deterrence of mosquitoes away from protected humans. While deterrence enhances personal or household protection of long-lasting insecticidal nets and indoor residual sprays, it may also attenuate or even reverse communal protection if it diverts mosquitoes to non-users rather than killing them outright.
Methods
A process-explicit model of malaria transmission is described which captures the sequential interaction between deterrent and toxic actions of vector control pesticides and accounts for the distinctive impacts of toxic activities which kill mosquitoes before or after they have fed upon the occupant of a covered house or sleeping space.
Results
Increasing deterrency increases personal protection but consistently reduces communal protection because deterrent sub-lethal exposure inevitably reduces the proportion subsequently exposed to higher lethal doses. If the high coverage targets of the World Health Organization are achieved, purely toxic products with no deterrence are predicted to generally provide superior protection to non-users and even users, especially where vectors feed exclusively on humans and a substantial amount of transmission occurs outdoors. Remarkably, this is even the case if that product confers no personal protection and only kills mosquitoes after they have fed.
Conclusions
Products with purely mosquito-toxic profiles may, therefore, be preferable for programmes with universal coverage targets, rather than those with equivalent toxicity but which also have higher deterrence. However, if purely mosquito-toxic products confer little personal protection because they do not deter mosquitoes and only kill them after they have fed, then they will require aggressive "catch up" campaigns, with behaviour change communication strategies that emphasize the communal nature of protection, to achieve high coverage rapidly.
doi:10.1186/1475-2875-10-207
PMCID: PMC3199905  PMID: 21798023
23.  Comparative assessment of the bacterial communities associated with Aedes aegypti larvae and water from domestic water storage containers 
Parasites & Vectors  2014;7(1):391.
Background
Domestic water storage containers constitute major Aedes aegypti breeding sites. We present for the first time a comparative analysis of the bacterial communities associated with Ae. aegypti larvae and water from domestic water containers.
Methods
The 16S rRNA-temporal temperature gradient gel electrophoresis (TTGE) was used to identify and compare bacterial communities in fourth-instar Ae. aegypti larvae and water from larvae positive and negative domestic containers in a rural village in northeastern Thailand. Water samples were cultured for enteric bacteria in addition to TTGE. Sequences obtained from TTGE and bacterial cultures were clustered into operational taxonomic units (OTUs) for analyses.
Results
Significantly lower OTU abundance was found in fourth-instar Ae. aegypti larvae compared to mosquito positive water samples. There was no significant difference in OTU abundance between larvae and mosquito negative water samples or between mosquito positive and negative water samples. Larval samples had significantly different OTU diversity compared to mosquito positive and negative water samples, with no significant difference between mosquito positive and negative water samples. The TTGE identified 24 bacterial taxa, belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and TM7 (candidate phylum). Seven of these taxa were identified in larval samples, 16 in mosquito positive and 13 in mosquito negative water samples. Only two taxa, belonging to the phyla Firmicutes and Actinobacteria, were common to both larvae and water samples. Bacilli was the most abundant bacterial class identified from Ae. aegypti larvae, Gammaproteobacteria from mosquito positive water samples, and Flavobacteria from mosquito negative water samples. Enteric bacteria belonging to the class Gammaproteobacteria were sparsely represented in TTGE, but were isolated from both mosquito positive and negative water samples by selective culture.
Conclusions
Few bacteria from water samples were identified in fourth-instar Ae. aegypti larvae, suggesting that established larval bacteria, most likely acquired at earlier stages of development, control the larval microbiota. Further studies at all larval stages are needed to fully understand the dynamics involved. Isolation of enteric bacteria from water samples supports earlier outcomes of E. coli contamination in Ae. aegypti infested domestic containers, suggesting the need to further explore the role of enteric bacteria in Ae. aegypti infestation.
doi:10.1186/1756-3305-7-391
PMCID: PMC4156648  PMID: 25151134
Aedes aegypti; Bacterial diversity; Enteric bacteria; E. coli; Domestic water storage containers; 16S rRNA-TTGE; Thailand
24.  Anopheles gambiae PGRPLC-Mediated Defense against Bacteria Modulates Infections with Malaria Parasites 
PLoS Pathogens  2009;5(8):e1000542.
Recognition of peptidoglycan (PGN) is paramount for insect antibacterial defenses. In the fruit fly Drosophila melanogaster, the transmembrane PGN Recognition Protein LC (PGRP-LC) is a receptor of the Imd signaling pathway that is activated after infection with bacteria, mainly Gram-negative (Gram−). Here we demonstrate that bacterial infections of the malaria mosquito Anopheles gambiae are sensed by the orthologous PGRPLC protein which then activates a signaling pathway that involves the Rel/NF-κB transcription factor REL2. PGRPLC signaling leads to transcriptional induction of antimicrobial peptides at early stages of hemolymph infections with the Gram-positive (Gram+) bacterium Staphylococcus aureus, but a different signaling pathway might be used in infections with the Gram− bacterium Escherichia coli. The size of mosquito symbiotic bacteria populations and their dramatic proliferation after a bloodmeal, as well as intestinal bacterial infections, are also controlled by PGRPLC signaling. We show that this defense response modulates mosquito infection intensities with malaria parasites, both the rodent model parasite, Plasmodium berghei, and field isolates of the human parasite, Plasmodium falciparum. We propose that the tripartite interaction between mosquito microbial communities, PGRPLC-mediated antibacterial defense and infections with Plasmodium can be exploited in future interventions aiming to control malaria transmission. Molecular analysis and structural modeling provided mechanistic insights for the function of PGRPLC. Alternative splicing of PGRPLC transcripts produces three main isoforms, of which PGRPLC3 appears to have a key role in the resistance to bacteria and modulation of Plasmodium infections. Structural modeling indicates that PGRPLC3 is capable of binding monomeric PGN muropeptides but unable to initiate dimerization with other isoforms. A dual role of this isoform is hypothesized: it sequesters monomeric PGN dampening weak signals and locks other PGRPLC isoforms in binary immunostimulatory complexes further enhancing strong signals.
Author Summary
Recognition of peptidoglycan on the bacteria cell wall triggers insect immune responses. The fruit fly PGRPLC receptor protein senses the presence of peptidoglycan and activates a pathway that mediates resistance to bacterial infections, mainly Gram-negative. We show that the PGRPLC receptor of the malaria vector mosquito Anopheles gambiae can also sense infections of the hemolymph (the mosquito blood) or the gut with bacteria of both Gram types and thereby activate a pathway that confers resistance to these infections. PGRPLC and its downstream responses also control the numbers of symbiotic bacteria that are mostly found in the mosquito gut where they drastically proliferate after a female mosquito takes a bloodmeal. Importantly, when the bloodmeal is infected with malaria parasites, the defense reaction that the mosquito mounts against proliferating bacteria also eliminate a large number of parasites. These mechanisms are largely elucidated using a rodent malaria parasite, but we also show that they significantly affect the intensities of mosquito infections with Plasmodium falciparum parasites found in the blood of children in sub-Saharan Africa.
doi:10.1371/journal.ppat.1000542
PMCID: PMC2715215  PMID: 19662170
25.  An experimental hut study to quantify the effect of DDT and airborne pyrethroids on entomological parameters of malaria transmission 
Malaria Journal  2014;13:131.
Background
Current malaria vector control programmes rely on insecticides with rapid contact toxicity. However, spatial repellents can also be applied to reduce man-vector contact, which might ultimately impact malaria transmission. The aim of this study was to quantify effects of airborne pyrethroids from coils and DDT used an indoor residual spray (IRS) on entomological parameters that influence malaria transmission.
Methods
The effect of Transfluthrin and Metofluthrin coils compared to DDT on house entry, exit and indoor feeding behaviour of Anopheles gambiae sensu lato were measured in experimental huts in the field and in the semi-field. Outcomes were deterrence - reduction in house entry of mosquitoes; irritancy or excito-repellency – induced premature exit of mosquitoes; blood feeding inhibition and effect on mosquito fecundity.
Results
Transfluthrin coils, Metofluthrin coils and DDT reduced human vector contact through deterrence by 38%, 30% and 8%, respectively and induced half of the mosquitoes to leave huts before feeding (56%, 55% and 48%, respectively). Almost all mosquitoes inside huts with Metofluthrin and Transfluthrin coils and more than three quarters of mosquitoes in the DDT hut did not feed, almost none laid eggs and 67%, 72% and 70% of all mosquitoes collected from Transfluthrin, Metofluthrin and DDT huts, respectively had died after 24 hours.
Conclusion
This study highlights that airborne pyrethroids and DDT affect a range of anopheline mosquito behaviours that are important parameters in malaria transmission, namely deterrence, irritancy/excito-repellency and blood-feeding inhibition. These effects are in addition to significant toxicity and reduced mosquito fecundity that affect mosquito densities and, therefore, provide community protection against diseases for both users and non-users. Airborne insecticides and freshly applied DDT had similar effects on deterrence, irritancy and feeding inhibition. Therefore, it is suggested that airborne pyrethroids, if delivered in suitable formats, may complement existing mainstream vector control tools.
doi:10.1186/1475-2875-13-131
PMCID: PMC4230423  PMID: 24693934

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