Sub-lethal aquatic doses of pyriproxyfen may increase pyrethroid resistance in malaria mosquitoes.

BACKGROUND: Pyriproxyfen (PPF), an insect growth hormone mimic is widely used as a larvicide and in some second-generation bed nets, where it is combined with pyrethroids to improve impact. It has also been evaluated as a candidate for auto-dissemination by adult mosquitoes to control Aedes and Anopheles species. We examined whether PPF added to larval habitats of pyrethroid-resistant malaria vectors can modulate levels of resistance among emergent adult mosquitoes. METHODOLOGY: Third-instar larvae of pyrethroid-resistant Anopheles arabiensis (both laboratory-reared and field-collected) were reared in different PPF concentrations, between 1×10-9 milligrams active ingredient per litre of water (mgAI/L) and 1×10-4 mgAI/L, or no PPF at all. Emergent adults escaping these sub-lethal exposures were tested using WHO-standard susceptibility assays on pyrethroids (0.75% permethrin and 0.05% deltamethrin), carbamates (0.1% bendiocarb) and organochlorides (4% DDT). Biochemical basis of pyrethroid resistance was investigated by pre-exposure to 4% PBO. Bio-efficacies of long-lasting insecticide-treated nets, Olyset® and PermaNet 2.0 were also examined against adult mosquitoes with or without previous aquatic exposure to PPF. RESULTS: Addition of sub-lethal doses of PPF to larval habitats of pyrethroid-resistant An. arabiensis, consistently resulted in significantly reduced mortalities of emergent adults when exposed to pyrethroids, but not to bendiocarb or DDT. Mortality rates after exposure to Olyset® nets, but not PermaNet 2.0 were also reduced following aquatic exposures to PPF. Pre-exposure to PBO followed by permethrin or deltamethrin resulted in significant increases in mortality, compared to either insecticide alone. CONCLUSIONS: Partially-resistant mosquitoes exposed to sub-lethal aquatic concentrations of PPF may become more resistant to pyrethroids than they already are without such pre-exposures. Studies should be conducted to examine whether field applications of PPF, either by larviciding or other means actually exacerbates pyrethroid-resistance in areas where signs of such resistance already exist in wild the vector populations. The studies should also investigate mechanisms underlying such magnification of resistance, and how this may impact the potential of PPF-based interventions in areas with pyrethroid resistance

Evaluation of a simple polytetrafluoroethylene (PTFE)-based membrane for blood-feeding of malaria and dengue fever vectors in the laboratory

BACKGROUND: Controlled blood-feeding is essential for maintaining laboratory colonies of disease-transmitting mosquitoes and investigating pathogen transmission. We evaluated a low-cost artificial feeding (AF) method, as an alternative to direct human feeding (DHF), commonly used in mosquito laboratories. METHODS: We applied thinly-stretched pieces of polytetrafluoroethylene (PTFE) membranes cut from locally available seal tape (i.e. plumbers tape, commonly used for sealing pipe threads in gasworks or waterworks). Approximately 4 ml of bovine blood was placed on the bottom surfaces of inverted Styrofoam cups and then the PTFE membranes were thinly stretched over the surfaces. The cups were filled with boiled water to keep the blood warm (~37 degrees C), and held over netting cages containing 3-4 day-old inseminated adults of female Aedes aegypti, Anopheles gambiae (s.s.) or Anopheles arabiensis. Blood-feeding success, fecundity and survival of mosquitoes maintained by this system were compared against DHF. RESULTS: Aedes aegypti achieved 100% feeding success on both AF and DHF, and also similar fecundity rates (13.1 +/- 1.7 and 12.8 +/- 1.0 eggs/mosquito respectively; P > 0.05). An. arabiensis had slightly lower feeding success on AF (85.83 +/- 16.28%) than DHF (98.83 +/- 2.29%) though these were not statistically different (P > 0.05), and also comparable fecundity between AF (8.82 +/- 7.02) and DHF (8.02 +/- 5.81). Similarly, for An. gambiae (s.s.), we observed a marginal difference in feeding success between AF (86.00 +/- 10.86%) and DHF (98.92 +/- 2.65%), but similar fecundity by either method. Compared to DHF, mosquitoes fed using AF survived a similar number of days [Hazard Ratios (HR) for Ae. aegypti = 0.99 (0.75-1.34), P > 0.05; An. arabiensis = 0.96 (0.75-1.22), P > 0.05; and An. gambiae (s.s.) = 1.03 (0.79-1.35), P > 0.05]. CONCLUSIONS: Mosquitoes fed via this simple AF method had similar feeding success, fecundity and longevity. The method could potentially be used for laboratory colonization of mosquitoes, where DHF is unfeasible. If improved (e.g. minimizing temperature fluctuations), the approach could possibly also support studies where vectors are artificially infected with blood-borne pathogens

'We spray and walk away': wall modifications decrease the impact of indoor residual spray campaigns through reductions in post-spray coverage

Malaria prevalence has significantly reduced since 2000, largely due to the scale-up of vector control interventions, mainly indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs). Given their success, these tools remain the frontline interventions in the fight against malaria. Their effectiveness relies on three key ingredients: the intervention, the mosquito vector and the end-user. Regarding the intervention, factors such as the insecticide active ingredient(s) used and the durability and/or bio-efficacy of the tool over time are critical. For the vectors, these factors include biting and resting behaviours and the susceptibility to insecticides. Finally, the end-users need to accept and properly use the intervention. Whilst human attitude and behaviour towards LLINs are well-documented both during and after distribution, only initial coverage is monitored for IRS and in a few geographic settings the residual efficacy of the used product. Here, the historical evidence on end-users modifying their wall surfaces post-spraying is presented, a behaviour that has the potential to reduce actual IRS coverage, effectiveness and impact, as fewer people are truly protected. Therefore, clear guidelines on how to monitor IRS acceptability and/or coverage, both before, during and after spraying, are urgently needed as part of the Monitoring and Evaluation of malaria programmes

Field evaluation of the BG-Malaria trap for monitoring malaria vectors in rural Tanzanian villages.

BG-Malaria (BGM) trap is a simple adaptation of the widely-used BG-Sentinel trap (BGS). It is proven to be highly effective for trapping the Brazilian malaria vector, Anopheles darlingi, in field conditions, and the African vector, Anopheles arabiensis, under controlled semi-field environments, but has not been field-tested in Africa. Here, we validated the BGM for field sampling of malaria vectors in south-eastern Tanzania. Using a series of Latin-Square experiments conducted nightly (6pm-7am) in rural villages, we compared mosquito catches between BGM, BGS and human landing catches (HLC). We also compared BGMs baited with different attractants (Ifakara-blend, Mbita-blend, BG-Lure and CO2). Lastly, we tested BGMs baited with Ifakara-blend from three odour-dispensing methods (BG-Cartridge, BG-Sachet and Nylon strips). One-tenth of the field-collected female Anopheles gambiae s.l. and Anopheles funestus were dissected to assess parity. BGM captured more An. gambiae s.l. than BGS (p < 0.001), but HLC caught more than either trap (p < 0.001). However, BGM captured more An. funestus than HLC. Proportions of parous An. gambiae s.l. and An. funestus consistently exceeded 50%, with no significant difference between methods. While the dominant species caught by HLC was An. gambiae s.l. (56.0%), followed by Culex spp. (33.1%) and Mansonia spp. (6.0%), the BGM caught mostly Culex (81.6%), followed by An. gambiae s.l. (10.6%) and Mansonia (5.8%). The attractant-baited BGMs were all significantly superior to un-baited controls (p < 0.001), although no difference was found between the specific attractants. The BG-Sachet was the most efficient dispenser for capturing An. gambiae s.l. (14.5(2.75-42.50) mosquitoes/trap/night), followed by BG-Cartridge (7.5(1.75-26.25)). The BGM caught more mosquitoes than BGS in field-settings, but sampled similar species diversity and physiological states as BGS. The physiological states of malaria vectors caught in BGM and BGS were similar to those naturally attempting to bite humans (HLC). The BGM was most efficient when baited with Ifakara blend, dispensed from BG-Sachet. We conclude that though BGM traps have potential for field-sampling of host-seeking African malaria vectors with representative physiological states, both BGM and BGS predominantly caught more culicines than Anopheles, compared to HLC, which caught mostly An. gambiae s.l

Evaluation of a simple polytetrafluoroethylene (PTFE)-based membrane for blood-feeding of malaria and dengue fever vectors in the laboratory

BACKGROUND: Controlled blood-feeding is essential for maintaining laboratory colonies of disease-transmitting mosquitoes and investigating pathogen transmission. We evaluated a low-cost artificial feeding (AF) method, as an alternative to direct human feeding (DHF), commonly used in mosquito laboratories. METHODS: We applied thinly-stretched pieces of polytetrafluoroethylene (PTFE) membranes cut from locally available seal tape (i.e. plumbers tape, commonly used for sealing pipe threads in gasworks or waterworks). Approximately 4 ml of bovine blood was placed on the bottom surfaces of inverted Styrofoam cups and then the PTFE membranes were thinly stretched over the surfaces. The cups were filled with boiled water to keep the blood warm (~37 degrees C), and held over netting cages containing 3-4 day-old inseminated adults of female Aedes aegypti, Anopheles gambiae (s.s.) or Anopheles arabiensis. Blood-feeding success, fecundity and survival of mosquitoes maintained by this system were compared against DHF. RESULTS: Aedes aegypti achieved 100% feeding success on both AF and DHF, and also similar fecundity rates (13.1 +/- 1.7 and 12.8 +/- 1.0 eggs/mosquito respectively; P > 0.05). An. arabiensis had slightly lower feeding success on AF (85.83 +/- 16.28%) than DHF (98.83 +/- 2.29%) though these were not statistically different (P > 0.05), and also comparable fecundity between AF (8.82 +/- 7.02) and DHF (8.02 +/- 5.81). Similarly, for An. gambiae (s.s.), we observed a marginal difference in feeding success between AF (86.00 +/- 10.86%) and DHF (98.92 +/- 2.65%), but similar fecundity by either method. Compared to DHF, mosquitoes fed using AF survived a similar number of days [Hazard Ratios (HR) for Ae. aegypti = 0.99 (0.75-1.34), P > 0.05; An. arabiensis = 0.96 (0.75-1.22), P > 0.05; and An. gambiae (s.s.) = 1.03 (0.79-1.35), P > 0.05]. CONCLUSIONS: Mosquitoes fed via this simple AF method had similar feeding success, fecundity and longevity. The method could potentially be used for laboratory colonization of mosquitoes, where DHF is unfeasible. If improved (e.g. minimizing temperature fluctuations), the approach could possibly also support studies where vectors are artificially infected with blood-borne pathogens

Illustration of how the mean isotopic ratios of δ¹⁵N and δ¹³C change when different quantities of mosquitoes from enriched versus control basins are included in the pools for analysis.

<p>Mean (CI: 95%) δ¹⁵N and δ¹³C for mosquitoes obtained from basins that were either enriched using the stable isotopes (enriched mosquitoes) or control basins that were not enriched (unenriched mosquitoes). We analysed adult mosquitoes in pools containing a total of four mosquitoes, but variable ratios of enriched to unenriched mosquitoes as indicated by the x-axis label (i.e. 0/4, 1/4, 2/4, ¾ or 4/4). Panel A represents females for <i>Aedes aegypti</i> and <i>Anopheles gambiae</i> sensu lato, while panel B represents males of the same species. All values referenced against international standards (nitrogen = air; carbon = Vienna Pee Dee Belemnite (VPDB)).</p

Comparison of isotopic ratios between mosquitoes obtained from enriched pools, and those obtained from control basins: Standardized isotopic ratios δ¹⁵N and δ¹³C for adult male and female <i>Anopheles gambiae</i> sensu lato and <i>Aedes aegypti</i>, and the pupae collected from control and enriched basins.

<p>Figure panels A, C and E represent results for mosquitoes collected from basins enriched with ¹⁵N-labelled potassium nitrate, and the respective controls, while figure panels B, D and F represent results of mosquitoes collected from basins enriched with ¹³C-labelled glucose, and the respective controls. All values referenced against international standards (nitrogen = air; carbon = Vienna Pee Dee Belemnite (VPDB).</p

Results showing δ¹⁵N and δ¹³C mean (±SE) of adult male and female pooled mosquitoes regardless of species from habitats enriched using ¹⁵N-labelled potassium nitrate, ¹³C-labelled carbon and un-enriched habitats (controls).

<p>Results showing δ¹⁵N and δ¹³C mean (±SE) of adult male and female pooled mosquitoes regardless of species from habitats enriched using ¹⁵N-labelled potassium nitrate, ¹³C-labelled carbon and un-enriched habitats (controls).</p

Mean (±SE) isotopic ratio (δ) of mosquitoes obtained from habitats enriched using ¹⁵N-labelled potassium nitrate and ¹³C-labelled glucose.

<p>All values referenced against international standards (nitrogen = air; carbon = Vienna Pee Dee Belemnite (VPDB)). Data grouped by species and sex for adult mosquitoes and combined pupae.</p

Results of pair-wise post hoc comparison using Tukey’s honestly significance tests (Tukey’s HSD).

<p>Howing similarities and differences between number of mosquitoes caught in traps baited with different lures (Panel A) and number of mosquitoes caught in traps baited with different lures dispensed from different media (Panel B).</p