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As malaria transmission wanes, an increasing number of individuals harbor asymptomatic infections that are not typically detected by conventional rapid diagnostic methods. This presents distinct challenges as interventions shift from malaria control to elimination in endemic settings. To identify these asymptomatic carriers and to better understand transmission patterns on the individual human level, it is important to identify individual Plasmodium parasites in both humans and mosquitoes. Although previous work has identified individual parasites in human blood samples, this has not yet been replicated in mosquitoes. In this proof of concept experiment, we focused on genetic identification of individual Plasmodium ring and trophozoite stage parasites in blood-fed Anopheles mosquito midguts. We established in vitro levels of detection of individual Plasmodium parasites in human blood samples using four different parasitemias (10000, 1000, 100, and 10 parasites/µL) and four mixed ratios of (80:20, 20:80, 90:10, and 10:90) of both the standard laboratory Plasmodium strain 3D7 and a clinical isolate at the ring and trophozoite stages using qPCR. We then conducted Anopheles mosquito membrane feeds using the same four levels of Plasmodium parasitemias and four mixed ratios of the genetically distinct parasites to determine a pragmatic level of monoclonal and multiclonal detection specificity within the mosquito midgut. Fed mosquitoes were dissected and DNA extracted from the abdomens. qPCR for Plasmodium falciparum L-lactate dehydrogenase (PfLDH) DNA was used to determine presence of Plasmodium. Samples positive for PfLDH were then characterized at six previously identified single nucleotide polymorphisms. Data regarding sensitivity will be presented; preliminary results indicate that although in vitro detection is quite sensitive (to the 100 parasites/µL level), in vivo detection at low parasitemia (

Additional file 1: Table S1. Household indices and brand nets from the collection sites.

Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarray-based transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome-wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region.

Additional file 2: Figure S1. Resistance (%) of An. funestus populations to Deltamethrin from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot. Figure S2. Resistance (%) of An. funestus populations to Permethrin from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot. Figure S3. Resistance (%) of An. funestus populations to Bedniocarb from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot. Figure S4. Resistance (%) of An. arabiensis populations to Deltamethrin from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot. Figure S5. Resistance (%) of An. arabiensis populations to Permethrin from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot. Figure S6. Resistance (%) of An. arabiensis populations to Deltamethrin from 2011 to 2015 from various sampling areas across Malawi. Highest levels of phenotypic resistance are denoted by a large open circle and the lowest by the smallest dark dot.

Laboratory based experiments were conducted using Bacillus thuringiensis var israelinsis (Bti) to establish the efficacy of Bti on Anopheles and Culex mosquito larvae from Zomba. The study evaluated two formulations of Bti namely VectoBac® WG and VectobaBac® 12AS against selected species of mosquito larvae. During this study, six different concentrations of Bti were set and 360 mosquito larvae were exposed to these different concentrations and results were observed hourly for 10 hours, then 24 hours and 48 hours. The experiment was replicated three times. Results show that the lower effective dosage that can be used to control Culex mosquito larvae in Zomba after 48hours of exposure is 47.73g/ha. The LT50 and LT90 being 7.5hrs and 24.3 hrs respectively. On the other hand, Anopheles mosquito larvae require 103.41g/ha of Bti which is almost double as much as that required by Culex. Anopheles LT50 is 6.2 hrs and LT90 is 18.5 hrs. In addition, it was observed that when Culex and Anopheles mosquito larvae were exposed to the same dosage of liquid formulation of Bti (0.001ml/L) there was no significant difference in their mortalities. Following the successful results of Bti in controlling mosquito larvae at laboratory level it is our recommendation to ask the Government of Malawi to come up with a policy to allow the use of Bti in controlling mosquito larvae in Malawi

Additional file 3: Table S2. Summary statistics for polymorphism at the sodium channel gene in susceptible and resistant permethrin and DDT Anopheles funestus in Chikwawa, Malawi.

Additional file 1: Table S1. List of primers used for Taqman A296S assay and VGSC sequencing.

Additional file 3: Table S2. Summary statistics for polymorphism at the sodium channel gene in susceptible and resistant permethrin and DDT Anopheles funestus in Chikwawa, Malawi.

Additional file 1: Table S1. Approximate locations of villages sampled from 2011 to 2015. Asterisks indicate latitude and longitude were estimated as the average of other villages in the same district or were estimated as the centroid of the district. Table S2. Mortality of An. funestus in WHO susceptibility tests against deltamethrin. 95% confidence limits and sample size are given in parentheses. Table S3. Mortality of An. funestus in WHO susceptibility tests against permethrin. 95% confidence limits and sample size are given in parentheses. Table S4. Mortality of An. funestus in WHO susceptibility tests against bendiocarb. 95% confidence limits and sample size are given in parentheses. Table S5. Mortality of An. funestus in WHO susceptibility tests against propoxur. 95% confidence limits and sample size are given in parentheses. Table S6. Mortality of An. funestus in WHO susceptibility tests against DDT. 95% confidence limits and sample size are given in parentheses. Table S7. Mortality of An. funestus in WHO susceptibility tests against malathion or pirimiphos-methyl (Chikwawa, Ntwana, 2015 only). 95% confidence limits and sample size are given in parentheses. Table S8. Mortality of An. arabiensis in WHO susceptibility tests against deltamethrin. 95% confidence limits and sample size are given in parentheses. Table S9. Mortality of An. arabiensis in WHO susceptibility tests against permethrin. 95% confidence limits and sample size are given in parentheses. Table S10. Mortality of An. arabiensis in WHO susceptibility tests against bendiocarb. 95% confidence limits and sample size are given in parentheses.

Additional file 1: Table S1. Approximate locations of villages sampled from 2011 to 2015. Asterisks indicate latitude and longitude were estimated as the average of other villages in the same district or were estimated as the centroid of the district. Table S2. Mortality of An. funestus in WHO susceptibility tests against deltamethrin. 95% confidence limits and sample size are given in parentheses. Table S3. Mortality of An. funestus in WHO susceptibility tests against permethrin. 95% confidence limits and sample size are given in parentheses. Table S4. Mortality of An. funestus in WHO susceptibility tests against bendiocarb. 95% confidence limits and sample size are given in parentheses. Table S5. Mortality of An. funestus in WHO susceptibility tests against propoxur. 95% confidence limits and sample size are given in parentheses. Table S6. Mortality of An. funestus in WHO susceptibility tests against DDT. 95% confidence limits and sample size are given in parentheses. Table S7. Mortality of An. funestus in WHO susceptibility tests against malathion or pirimiphos-methyl (Chikwawa, Ntwana, 2015 only). 95% confidence limits and sample size are given in parentheses. Table S8. Mortality of An. arabiensis in WHO susceptibility tests against deltamethrin. 95% confidence limits and sample size are given in parentheses. Table S9. Mortality of An. arabiensis in WHO susceptibility tests against permethrin. 95% confidence limits and sample size are given in parentheses. Table S10. Mortality of An. arabiensis in WHO susceptibility tests against bendiocarb. 95% confidence limits and sample size are given in parentheses.

Additional file 1: Table S1. List of primers used for Taqman A296S assay and VGSC sequencing.