Insecticidal roof barriers mounted on untreated bed nets can be as effective against Anopheles gambiae as regular insecticide-treated bed nets

Barrier bednets (BBnets), regular bednets with a vertical insecticidal panel to target mosquitoes above the bednet roof, where they are most active, have the potential to improve existing Insecticidal Treated Bednets (ITNs), by reducing the quantity of insecticide required per net, reducing the toxic risks to those using the net, potentially increasing insecticide choice. We evaluated the performance of PermaNet 3.0 (P3) and untreated (Ut) bed nets with and without pyrethroid and piperonyl butoxide roof barriers in killing pyrethroid-resistant and susceptible Anopheles gambiae, simultaneously video-recording mosquito flight tracks. Bioassay results showed that treated roof barriers, particularly the longitudinal P3 barrier (P3L) could be an effective addition to a bed net: P3 + P3L were consistently significantly more effective than the reference P3 bednet while performance of untreated nets could be raised to equal that of the reference P3 following the addition of a P3 barrier. The BBnet’s potential to augment existing bednets and enhance their performance is considered.

intercept mosquitoes above the bednet roof, where An. gambiae s.l. are most active 10 and where the insecticidetreatment cannot contact the sleeper (Fig. 1).The possibility that deploying a more effective insecticide on the barrier could result in a net capable of performances that are equal to, or even better than, those of a standard ITN is an attractive prospect.Such a BBNet could potentially increase the range of candidate insecticides, by making newer expensive insecticides affordable, allowing higher concentrations of the a.i. or permitting the use of some insecticides that would be considered unsafe or unsuitable for direct skin contact if the sides and roof of the bed net were treated.In addition to increasing treatment choice, BBnets would require less insecticide per net, which translates as safer cheaper nets, less toxic waste, and fewer impacts on non-target fauna.
Following the initial demonstration of barrier bednet efficacy against a wild pyrethroid resistant population in Burkina Faso 9 we report here on laboratory tests investigating the potential of different roof barrier designs of PBO-treated netting.We used the same infra-red video tracking system as earlier studies 9,10 to video-record the interactions between An. gambiae s.l.from a laboratory colony as they flew freely in a large climate-controlled room, responding to a human volunteer host within different BBnet variants.This allowed considerable control over the experimental setup without interfering with the spatial behavior of the mosquitoes as they interacted with the ITN while responding to the host.

Results
The study started in late 2019 in UK and was forced to stop as the first Covid lockdown measures were implemented in March 2020, necessitating a reassessment of experimental plans to ensure we could complete enough bioassays to compare the range of BBnet variants.Consequently, the resistant Tiassalé strain was tested in six repeat assays with all BBnet variants, but the susceptible Kisumu strain was tested only with transverse BBNet types, in six repeat assays for all variants except P3 + P3T, for which only 4 repeat assays were completed.
Following completion of the assays, susceptibility to deltamethrin and permethrin of the colonies of both strains were tested in May 2020 by LITE using standard WHO tube tests to determine levels of pyrethroid susceptibility: Kisumu was fully susceptible to both, with 100% mortality recorded in all tests.The Tiassale strain recorded mortality rates post exposure of 13% and 8% to deltamethrin and permethrin, respectively, and the colony underwent re-selection, beginning in May 2000 from which time it was no longer available.

Performance of the BBnet variants against pyrethroid resistant mosquitoes
The mortality and knockdown rates of tests with Tiassale strain mosquitoes at transverse and longitudinal BBnets are summarized in Fig. 3.
Four of the five variants with efficacy comparable to the P3 (knockdown > 70%; mortality > 50`%) comprised a barrier mounted on a complete P3 base.The fifth variant, Ut + P3L, was an exception, as it achieved mortality rates comparable to the other four variants and the P3 reference although its bed net base was untreated.The P3L barrier on a P3 bed net base performed very well, exceeding mortality rates of the P3 in all assay repeats, a difference that was significant despite wide confidence intervals (OR 2.09; 95% CI 1.21, 3.64; p = 0.0086).No significant differences were found between the other nets and the P3 net.

Contact rate and duration of contact
The mean total number of contacts (and CI) made and the duration of that contact for each BBnet variant are given for Tiassale strain mosquitoes in Fig. 4, which shows the total number and duration of contacts on any part of the BBnet (Fig. 4a), on the treated sections only (Fig. 4b) and per cm 2 of treated net (Fig. 4c).See also data and results, summarised in Tables S1 and S2).The duration of contact with the total net surface (all untreated and treated net surfaces, including barrier, Fig. 4a) during exposure was longer (at least 1465.66s) in all net variants compared to P3.However, these differences were significant only with P3 + P3L, P3 + UtL and Ut + P3L).

Discussion
The results of these tests, although not fully conclusive, demonstrate the potential of simple roof mounted bednet barriers for malaria vector control.The bioassays clearly demonstrate that treated longitudinal barriers are likely to greatly improve the performance of the bednet beneath them.This ability could extend as far as 'converting' an untreated net into an effective ITN or 'restoring' an intact aged net simply by the addition of a treated barrier.
The key to the barriers' ability to kill so efficiently is almost certainly their position on the roof: Here they are located directly above the supine human inside the bednet, where activity by incoming mosquitoes is greatest 10 A comparison of the total duration of contact at the treated sections on a Ut + P3L BBnet and the reference P3 bednet (Fig. 4b), shows that despite the disparity in insecticidal surface area the longitudinal P3 barrier accumulated 1374 s duration from 1273 contacts, greatly exceeding the 792 s duration, from 8049 contacts, accumulated by all the treated net surface area at the Permanet 3.0.This was equivalent to contacts rates at the L barrier with a total duration of 0.9 s/cm 2 compared with the P3 ITN which was 0.11 s/cm 2 (Fig. 4c).The duration rates are remarkably similar, despite the difference in area.
The ITN has 8 times greater surface area loaded with insecticide, most of which does not contribute to its impact (Fig. 4).Moreover, mosquitoes orienting towards the bednet roof probably arrive first at the barrier/roof and may already have picked up a lethal dose of insecticide before they visit any other part of the net.This begs the question of whether an effective barrier on an untreated bednet base could kill mosquitoes as well as a regular treated bednet or put another way, could an untreated net reproduce the killing effect of a standard ITN simply by the addition of a treated barrier?These results certainly suggest it is possible, with an appropriate insecticide treatment.As Fig. 4a shows, the duration of contact with the total net surface (all untreated and treated net surfaces, including barrier) during exposure was longer in all net variants compared to P3, but the differences were significant only with P3 + P3L, P3 + UtL, Ut + P3L.The killing performance of the untreated bednet with a long P3 barrier (Ut + P3L) was comparable to a standard treated Permanet 3 ITN (Fig. 3).
To date, the BBnet has been tested mainly with insecticides that would be permissible on a standard bednet, except for the use of fenitrothion in Burkina Faso study, when the BBnet (insecticidal barrier with untreated bed net base) performed very well, increasing lethality without compromising personal protection 9 .
The pattern of movement around a bednet where most mosquito activity occurs on or above the roof, is a response to olfactory and thermal cues rising from the host below, and Sutcliffe and Yin 11 reported that the pattern disappeared when a breeze was blown across the host, dissipating any rising attractants, and eliminating the focus of activity on the net roof.This is true and it is widely known that sleeping with a steady low continuous flow of air from a reliable electric fan prevents mosquitoes from landing to the extent that it is possible to sleep comfortably without any bednet.However, at present few communities in endemic malaria zones are likely to have access to affordable power needed to run an electric fan all night every night, while security concerns are likely to continue to overrule any desire for comfort when deciding to open a window.Hence, until such basic improvements are widespread, this is unlikely to be a factor affecting the performance of BBnets.
The poor performance of the transverse barriers against resistant mosquitoes was unexpected.As already mentioned, in our earlier field study, we found that a transverse barrier treated with the organophosphate fenitrothion was highly effective against a wild population of pyrethroid-resistant An. gambiae s.l., in Burkina Faso increasing killing approximately 34% more than a standard bednet 9 .Here, it performed well against susceptible mosquitoes (Fig. 2) but did not improve the performance of bednets against resistant mosquitoes compared to the negative controls, despite the high duration of contact at the treated barrier in the Ut + P3T Fig. 4c).Differences in the insecticides used could explain this at least in part.In the field study where the transverse barriers were effective, they carried a highly effective insecticide, either deltamethrin against susceptible mosquitoes, or www.nature.com/scientificreports/an organophosphate, fenitrothion against the wild highly resistant vector population in Burkina Faso 9 .In both cases, the insecticide's effect was rapid following brief contact with a treated net.In studies where the transverse barriers performed poorly, as with Tiassale in the present study, impact depended on the synergist piperonyl butoxide (PBO) disabling the resistant mosquito's p450 resistance mechanism before sufficient insecticide could impact.Although the minimum contact times needed to pick up a lethal dose for each of these treatments are not known, it is conceivable that the faster acting insecticides were more suited to delivery on a barrier, as the threshold for a lethal level of contact is brief (< 50 s) 10 .The loss of the highly resistant Tiassale colony when it was too late to repeat the experiments of was most unfortunate, given the importance of demonstrating an impact with resistant mosquitoes.However, as mentioned above, the Burkina Faso study where fenitrothion-loaded T-barriers were effective against highly resistant wild/natural vector population serves to demonstrate that with an appropriate insecticide, BB-nets can be effective against highly resistant vectors 9 .
An estimated 79% of malaria cases are transmitted when people are in bed 12 , making the value and importance of ITNs in malaria prevention very clear.With careful management, ITNs can remain the primary means of reducing indoor malaria transmission in Africa for many more years.Standard bednet shapes can safely deliver only a very limited range of insecticides, seriously limiting options for management of insecticide resistance.Simplicity has been integral to ITN's success, but nets do not need much sophistication to improve them.They employ the sleeper's attractants to lure potentially infectious mosquitoes to the net surface where they are rapidly killed on contact while the sleeper is protected from bites behind a protective insecticidal screen 10 .Requiring only minimal change to the basic design, a roof barrier exploits this further.Of note here is the improvement in performance that a treated barrier contributes to the base bed net's efficacy.This is apparent in Fig. 3b where the long P3 barrier raised the untreated bed net's mortality rate from minimal to a rate equivalent to that of a standard P3, and the standard P3 from a mean mortality rate of 54% to 70%.If insecticide were to be delivered only on the roof and barrier it would reduce the risk of the occupant's skin becoming irritated by insecticide picked up during entry and exit from the protective net, further enhancing its advantages.
Given the importance of the net roof, and the region above it to the lethality of any ITN, it would make sense if attempts to improve ITN killing efficiency were to focus on this region.As Fig. 5 shows, none of the BBnets altered the preference for the bed net roof.The low levels of mosquito activity at the vertical sides and ends of the bednet 10 suggest that the best we can do at these net locations, where most physical damage occurs, is to improve the quality of the material used in manufacturing the net to make stronger more durable nets.Interest in improving nets has increased such that most of those involved in iTN deployment and development recognize the need to make nets that are more durable not only in use but also when in storage prior to use [13][14][15][16] , nets that produce fewer toxic residues when discarded and are generally more recyclable 17 .Some have prioritized durability www.nature.com/scientificreports/over insecticide delivery 18 .This is the first study to demonstrate how an insecticidal barrier can improve ITN performance, and how it can increase the options for different ITN designs.Moreover, we have shown that this can be achieved without the use of an additional active ingredient.The BBnet uses our knowledge of the habits of Anopheles around an occupied bed net by placing treated netting precisely where the mosquitoes are most likely to occur when they first arrive at the net.Existing bed nets do not go far enough to achieve this.Better bednets should mean a lot more than simply bednets with active ingredients that are effective against the target vector population, as this is surely the least any bednet should be.It is often a shock when the delicate fibers comprising the flimsy mesh that an ITN is made from are first seen.Sufficient knowledge of the entomological mode of action of ITNs already exists to enable us to identify the location of regions of the net where a number of important vector mosquito species are most likely to arrive [19][20][21] and where the insecticide is best positioned to maximise contact with mosquitoes 11 , the areas most vulnerable to physical damage that would benefit from tougher net fibre, and areas that are rarely visited by mosquitoes.Recent research suggests that most bed nets, including the new bi-treated nets, have similar entomological modes of action 22 .Can this range of knowledge not be used to design better bed nets, durable ITNs that are optimised to kill mosquitoes and prevent malaria transmission in rural Africa, rather than simply the cheapest designed products that tick the boxes required to complete a WHO form?

Mosquitoes and bioassays
Mosquitoes used in all tests were obtained from LITE All colonies were maintained, and bioassays were performed in a climate-controlled unit at LSTM (27 ± 2 °C, relative humidity 70 ± 10%) measuring 5.6 m × 3.6 m in area and 2.3 m high, using 2-7-day old unfed adult females from two An.gambiae s.l colonized strains.The Kisumu strain originated in Kenya in 1975 and is susceptible to pyrethroids and all other insecticides.The Tiassalé strain was established at LSTM in 2013, from material collected in Côte d'Ivoire.It is pyrethroid resistant and resistant to DDT and carbamates, conferred by a combination of target site mutations and P450 enzymes 23 .The colonies are maintained under a standardised rearing regime and Tiassalé strain undergoes continual selection with deltamethrin by Liverpool Insect Testing Establishment (LITE).Mosquitoes were obtained from this central facility as required (https:// lite.lstmed.ac.uk/ mosqu ito-colon ies).
Mosquitoes used in bioassays were deprived of sugar and water for 24 h and 4 h respectively before bioassays and transferred to a 300 ml paper cup to the bioassay room to acclimatize for 1 h prior to the start.Tests began 1-3 h after the start of the scotophase with the release of 25 test mosquitoes into the room (5.6 m × 3.6 m in area, 2.3 m high) and ran for 2 h, after which time the room was searched thoroughly to count and collect live, dead, or knocked down mosquitoes.All live mosquitoes were aspirated into a cup and provided with 10% sugar water in a separate room where mortality was recorded at 1 h and 24 h after the end of the experiment.
We used 27 volunteers (11 male, 16 female) aged between 22 and 61 years, all recruited from within LSTM and representing a range of ethnicities.Each person volunteered twice or more, acting as host for a different net on the second or subsequent occasion.The bioassay room was cleaned on Friday and an untreated net was tested on the next day (Monday) to ensure the room was clean.If the mortality was unexpectedly high, the bioassay room would be cleaned again.
Volunteers lay without shoes but clothed in their own trousers and t-shirt and uncovered within the bednet.They were requested to eschew strongly aromatic or spiced foods and scented personal hygiene products for 24 h prior to each experiment.Since the tests were also video recorded for tracking, volunteers were requested to remain as still as possible for the duration of the experiment.

BBnet variants
In all tests, rectangular bednets measuring 1.9 m × 0.8 m × ~ 1.0 m tall were used as the standard bednet.Treated nets were Permanet 3.0 (Side walls of 75 denier polyester, deltamethrin 2.1 g/kg ± 25%; roof of 100 denier polyethylene, deltamethrin 4.0 g/kg ± 25% and PBO 25 g/kg ± 25%; Vestergaard, Lausanne).Barriers comprised sections cut to size from the roof of a Permanet 3.0.Untreated polyester nets were used as untreated controls, and matched Permanet 3.0 nets as closely as possible fiber thickness and hole size.Before being used in experiments, untreated nets were first tested by WHO cone test to confirm the absence of any insecticidal effect, and all nets were hung for 4 weeks before use to allow evaporation of any potentially repellent or attractant volatile odors.
Two styles of barrier were investigated, a transverse (from elbow to elbow of the supine sleeper beneath) and a longitudinal barrier (from head to toe; see Fig. 1), designs derived from earlier bioassay in the field or from mathematical models comparing their potential 9 .Transverse barriers were positioned off-center on the roof above the sleeper's stomach or torso at the 30:70 division of the bednet's length, where mosquito activity is known to be greatest 10 .
To facilitate image capture on the top of the bednet, the net roof was tilted on its long axis when facing the cameras to ensure all mosquito activity on the roof was visible 9,10 .Hence, the height of the roof above the mattress was 0.80 m at the front and 1 m at the rear (when viewed/recorded from the front).To ensure the top edge was horizontal when mounted on this roof, the transverse barrier was 40 cm tall at the rear and 60 cm at the front of the net.The longitudinal barrier was 0.4 m higher than the roof and ran the length of the bednet in the center of the roof.
The basic structure of transverse and longitudinal barriers was similar in all tests as illustrated in Figs.2a and 3a.BBnets were coded according to the identity of the treatment on the bed net followed by the treatment on the barrier, and the style of barrier (Fig. 3a) as follows: • Untreated net + untreated long barrier Ut + UtL

Figure 1 .
Figure 1.Example of a longitudinal Permanet 3.0 polyethylene barrier bednet.The blue netting on the barrier and roof is 100 denier polyethylene net with deltamethrin incorporated at 120 mg/m 2 and PBO at 750 mg/m 2 .The white netting on the sides is 75 denier polyethylene with deltamethrin incorporated at 84 mg/m 2 (Photo by PJ McCall).

Figure 2 .
Figure 2. Mortality and knockdown rates of pyrethroid susceptible Anopheles gambiae Kisumu strain at transverse barrier bednet variants.(a) schematic illustrating the composition of each of the BBnets tested; (b) Mortality and (c) knockdown rates following the room-scale tests, as determined from videos recorded during tests and final counts at the termination of the 120 min assay (mean and CI; n = 6 repeat tests /treatment, except P3 + P3T (n = 4)).All images were created by the authors.

Figure 3 .
Figure 3. Mortality and knockdown rates of pyrethroid resistant Anopheles gambiae Tiassale strain at transverse and longitudinal barrier bednet variants.(a) schematic of the composition of the BBnet variants tested.1.(b) Mortality and (c) knockdown rates following the room-scale tests, as observed in videos recorded during the 120 min tests and final counts at the termination of the 120 min assay (mean and CI; n = 6 repeat tests / treatment).All images were created by the authors.

Figure 4 .
Figure 4. Mean total numbers and durations of contact by Anopheles gambiae Tiassalé strain with netting during the 120 min assay for (a) the entire surface area of netting (treated and untreated) on the BBnet; (b) the entire treated net surface area only (c) per cm 2 of the entire area of treated netting.

Figure 5 .
Figure 5. Examples of 120-min composite images showing all flight tracks of An. gambiae Tiassale strain at each of the BBnet variants.In all images, the sleeper is lying on their back, with their head at the left.Each colored track is the path of a single mosquito flight event, (25 mosquitoes released simultaneously) in all tests and color-coded according to time of appearance as shown in the key: blue tracks at the start through to red at the end of the 120-min test.