Project a long-lasting insecticide treated net (LLIN) containing both

  Project Proposal   Trop 936; Research Methods in parasitology and vector Biology   Supervisors: Dr Lisa Reimer and Dr Geraldine Foster       Word count: 2481         Title: Laboratory Assessments of Behavioural and Physiological Responses of Anopheles gambie to the P450 synergist, Piperonyl Butoxide                   Exam number: 17-90938 Laboratory Assessments of Behavioural and Physiological Responses of Anopheles gambie to the P450 synergist, Piperonyl Butoxide. Summary Malaria occurrence has decreased remarkably across sub-Saharan Africa. This is in large part due to the mass distribution of bed nets which contain a class of insecticides called pyrethroids. Mosquitoes that come into contact with the net are killed by the insecticide, reducing the population of mosquitoes that can transmit malaria. However, mosquitoes have acquired resistance to pyrethroids and this threatens the future effectiveness of bed nets, therefore alternative solutions and compounds are urgently needed. One solution is a long-lasting insecticide treated net (LLIN) containing both a pyrethroid and a compound known as Piperonyl Butoxide (PBO). PBO is a synergist that enhances the pyrethroid activity by inhibiting the P450 enzymes that break down the insecticide inside the mosquito, however little is currently known regarding whether exposure to this compound in an LLIN affects the way in which mosquitoes interact with the net.

Information regarding this behaviour is urgently required, as changes in behaviour such as a decreased amount of net contact time may limit the effectiveness of the PBO LLIN when deployed in community settings. Therefore, this project will assess the impacts of exposure to PBO-LLINs on Anopheles gambiae longevity, behaviour, host seeking and reproductive capacity. Background and rationale Long-lasting insecticide treated nets (LLINs) have made a considerable contribution toreductions in malaria across Africa. Widespread resistance to pyrethroids, which are the onlyinsecticide class available for use in LLINs, is threatening the future effectiveness of LLINs (Ranson et al, 2016) and alternatives to pyrethroids are urgently needed (Hemingway et al, 2016). One potential solution is a ‘combination of LLIN’ incorporating a pyrethroid with the synergist Piperonyl butoxide (PBO). PBO inhibits a group of enzymes (P450s) that cause insecticide resistance by metabolising pyrethroids (Feyereisen R, 2015). Pyrethroids are a very important tool for malaria control.

Resistance to pyrethroids is emerging and the P450 enzymes seem to play an important role. Not only does PBO increase the efficacy of current tools(LLINs) but exposure to PBO may limit other aspects of mosquito physiology (WHO 2015). Five LLINs that include both a pyrethroid insecticide and the synergist have become available (WHO, 2017). As PBO is a synergist that acts by inhibiting certain metabolic enzymes (e.g., mixed-function oxidases) within the mosquito that detoxify or sequester insecticides before they can have a toxic effect on the mosquito, compared to a pyrethroid-only net, a pyrethroid-PBO net should, in theory, have an increased killing effect on malaria vectors that express such resistance mechanisms. However, the entomological and epidemiological impact of pyrethroid-PBO nets may vary depending on the bioavailability and retention of PBO in the net, and on the design of the net (i.e.

, whether only some or all panels are treated with PBO) (WHO,2015). Laboratory bioassays and experimental hut trials have shown increased efficacy of PBO-LLINs against resistant mosquitoes compared to pyrethroid-only nets (Churcher et al, 2016). Furthermore, PBO may exert additional sub-lethal effects beyond conventional LLINs as they inhibit a large group of enzymes that catalyse a range of physiological reactions in insects (Daimon et al, 2012). However, at present, a knowledge gap exists regarding both whether the incorporation of PBO into an LLIN changes the way in which mosquitoes interact with the net, and which, if any, sub-lethal effects are triggered by exposure to a pyrethroid-PBO net (WHO, 2017).

 According to Parker et al, 2015 delivering the ‘next generation’ of LLINs or similar tools will require a thorough understanding of how LLINs function, yet remarkably little is known of the mode of action or of precisely how mosquitoes behave at the LLIN interface. Consequently, behavioural resistance to insecticides remains poorly understood and rarely reported in mosquitoes, though the risk of vector populations switching blood-feeding times, locations or host preferences in order to avoid LLINs is recognized and closely monitored today (Russell et al, 2011). However, additional but less apparent or detectable behavioural changes also might exist, potentially conferring partial or complete insecticide resistance (e.g. changes in sensitivity to repellents, attractants, or modified flight or resting behaviours). In the absence of definitions or quantifications of the basic behavioural events likely to be affected (Rivero et al, 2010), these changes cannot be investigated, let alone monitored.

The incorporation of PBO onto LLINs may exert fitness penalties for mosquitoes. PBOsynergises pyrethroids by inhibiting P450 enzymes which have many functions beyondinsecticide detoxification including juvenile hormone biosynthesis (Daimon et al, 2012), a key hormone in developmental and reproductive physiology. It is conceivable that PBO may disrupt the reproductive fitness of Anopheles mosquitoes affecting their population dynamics.While there is strong evidence for improved efficacy of PBO-LLINs against resistantAnopheles under laboratory settings, evidence gaps on performance under field conditions isproving an impediment to decision making on the use of PBO nets. Despite extensive research focussing on the survival rate of mosquitos exposed to the PBO LLIN, there is limited knowledge about the effect PBO has on the vector behaviour and fitness costs.

 While there is strong evidence for improved efficacy of PBO-LLINs against resistantAnopheles under laboratory settings, evidence gaps on performance under field conditions isproving an impediment to decision making by the WHO on the use of PBO nets. Despite extensive research focussing on the survival rate of mosquitoes exposed to the PBO LLIN, there is limited knowledge about the effect PBO has on the vector behaviour and fitness costs (WHO,2017) Consequently, there is need to understand mosquito behaviour using assays designed to more closely replicate ‘natural’ conditions than standard laboratory tests, thereby providing data that can be more easily extrapolated to field settings; using tracking assays is a way in which this can be achieved. Additionally, understanding vector behaviour is important in determining the type of vector control that is most likely to be efficacious. The WHO 2017 report states that the mortality rate for PBO LLINs is slightly higher than the LLINs but because of the higher cost of a PBO net it is essential to know if there are other physiological changes that exposure to PBO may have on the mosquito (WHO 2017), as this is important prior to implementation in a control programme. The resting behaviour, oviposition are all factors for consideration in introducing a control programme (WHO 2013)  I will look more closely at the entomological impact of PBO-LLINs, using video tracking techniques to quantify differences in mosquito behaviour when exposed to untreated, PBO-only and PBO-pyrethroid bednets.

Following exposure in the tracking assay, each mosquito will be monitored for sub-lethal effects of insecticide exposure. Mosquito survival is key to malaria transmission dynamics as the lengthy incubation period of the parasite (approximately nine days’ post blood meal (Beier J, 1998) means that few females survive long enough to transmit the disease. Even if insecticides do not directly kill the insect they may induce fitness costs related to survival, reproduction and development. This may explain why, despite widespread insecticide resistance (Ranson et al,2016), insecticide-based interventions continue to remain effective.

 The study aims to show if exposure to PBO affects mosquito host-seeking and blood-feeding behaviours, reproductive fitness, and survival. mosquitoes will be bio-assayed and the results will aid comparison of responses to the different treatments.  Aim:To determine immediate and sub-lethal impacts of exposure to a PBO-LLIN in a resistant Anopheles vector through characterisation of mosquito host seeking and blood feeding behaviour and measurement of reproductive capacity and longevity.

 Objectives:·         Assess mosquito behaviour in response to exposure to an untreated net, and PBO-only and PBO-pyrethroid LLINs using the video tracked assay the ‘thumb test’. It is estimated that achieving this objective will take approximately 5-6 weeks of laboratory time, depending upon the rate of mosquito responsiveness.·         Apply the sub-lethal effects pipeline currently implemented in the McCall group to the mosquitoes used in the thumb test and quantify any sub-lethal effects that occur post-PBO exposure, including effects on blood-feeding and reproductive capacity.  ·         Quantify the survival rate of Anopheles vectors exposed to untreated nets, PBO-pyrethroid LLINs and PBO-only nets.

  MethodsThis project will take place at the Liverpool School of Tropical Medicine (LSTM) under the supervision of Dr Geraldine Foster and Dr Lisa Reimer. Mosquito strains Laboratory reared Banfora Anopheles gambiae, which is a strain of An. gambiae resistant to pyrethroid insecticides, will be obtained from the McCall group vector biology colonies at LSTM. Female mosquitoes, 2-5 days of age will be used in the assay.

Thumb TestMosquitoes will be exposed to different nets using the thumb test method. This method allows quantification of the effects of net treatments on mosquito behaviour during host seeking and blood feeding, and on survival and a range of sub-lethal effects post-exposure it is a replica of the experiment by Parker et al, 2015 Fig 4.Sample sizeThe mosquitoes will be tested individually, and the number of mosquitoes per treatment is 25. According to Abe et al, 2012, 25 is the number of mosquitoes needed to detect differences in mosquito behaviour in response to different nets.

  1. Assess mosquito behaviour in response to exposure to an untreated net, and PBO-only and PBO-pyrethroid LLINs using the video tracked assay the ‘thumb test’. The mosquito is placed in the tube and allowed to acclimatize for 1 minute, the host thumb is placed against the netting in the net tube. After one minute, recording begins and the gate is opened, the mosquito, following the odour of the host, moves into the 10 by 10cm box as in fig 2.Fig 2.

Thumb test box (thumb test sop, 2017)Once it is in the box behaviour is video recorded for subsequent analysis. The mosquito will approach the thumb and begin feeding. If a mosquito does not approach the thumb within 3 minutes (or begin probing within 10 minutes) then the mosquito is regarded as a non -responder and discarded. Fig 3 Thumb test Procedure (thumb test SOP, 2017)Recording continues until the mosquito completes the blood meal and rests post-feeding for 3 minutes. All mosquito behaviours are observed in the video and the observer simultaneously inputs this data by pressing the keys in table 1.

This method is repeated on all 3 net types.  Key Code Point/State Description A Arriving State Mosquito arrives into view T Touching State Mosquito touches the net for the first time L Landing State Mosquito lands on the net P Probing State Mosquito probes I Inserting State Mosquito inserts proboscis and begins to blood feed B Blood feeding State Blood feeding becomes visible D Defecating State Mosquito blood feeds with defaecation W Withdrawing State Mosquito completes blood feeding and withdraws proboscis R Resting State Mosquito rests post-blood feeding E Ending Point End of observations  Table 1 thumb test video analysis (Thumb test SOP ,2017)  Figure 4: Flight activity of Anopheles gambiae at unbaited, baited and insecticide-treated bed nets. (method where the thumb test originated from)    2.Quantify any sub-lethal effects that occur post-PBO exposure, including effects on blood-feeding and reproductive capacity 2a. Quantify the blood meal concentration for each mosquitoTo determine if the different treatments affect the duration of the bloodmeal as well as the volume of the ingested blood; following exposure in the thumb test placed in a Falcon tube and allowed to digest the blood meal for 72 hours, excreting haematin. The haematin is mixed with 1% Lithium Carbonate and then placed in a spectrophotometer and read at 397nm.

OD readings are converted to a blood meal size in µg/ml using a standard curve. 2b. Assess the impact of PBO on the reproductive fitness of female Anopheles.After the mosquito has digested its meal and excreted haematin, it is placed in another tube with a piece of moistened filter paper, allowing the mosquito to lay eggs. Following egg laying, the eggs are floated in a container, and the number of hatched larvae are recorded for five days.  3. Quantify the survival rate of Anopheles vectors exposed to PBO-pyrethroid LLINs and PBO-only nets Mosquitoes are maintained in individual Falcon tubes with an untreated net lid with a cotton wool pad moistened with 10% sugar water.

After every 24 hrs, the mosquito is observed until it dies.Data analysisObjective one For the behavioural assays, the mosquito’s behaviours in table 1 will be analysed using an application called Behavioural Observation Research Interactive Software (BORIS). A defined set of point and state behaviours are used to compile an ethogram, which is used to classify observed behaviour during the length of the thumb test (thumb test SOP, 2017) These will then be extracted to excel and then analysed on the TraMiner package in R. Objective twoHaematin concentration, egg and larval counts will be imported into R and linear models will be plotted to express the comparisons of reproductive capacity to different net exposures as well as consider any impacts on blood meal size, egg and larval counts simultaneously. Objective threeKaplan-Meier plots will be used to determine the survival probabilities for each treatment, these will be considered in terms of the mosquito’s ability to transmit infection post-net exposure. The results between each treatment type will be compared. Quality AssuranceExperimental proceduresPrior to the experiments, I will have done some reading on the experimental designs and standard operating procedures, and will be trained on all assays and procedures used in the study by LSTM laboratory staff and my supervisors.

Sample sizeA sample size of 25 mosquitoes will be used, individually per turn for each net type (PBO-only, PBO + insecticide and Control. The tests will be done in rotation (swapping net types) rather than completing one net type and moving on to the next, this will be done so that the results are not affected by the time of day, hence removing any potential bias that may occur from environmental conditions.Evaluation of resultsIn order to reduce errors in the results, a separate controlled exposure to PBO will be done in WHO cone assays, and a video recorded variation of the assays will be used to make sure there either exists an effect or not. If there is an effect these should display similar results to the Thumb test result. Additionally, the thumb test videos will be reanalysed by a member of the McCall group experienced in performing thumb tests in order to ensure that there is consistency in the recording of the observed behavioursEthical IssuesEthical issues in this study will be centred on the candidate (Rosheen Mthawanji) will be performing thumb tests, on which the mosquitoes may bite.

All mosquitoes will be reared from pupae and so there will be no risk of malarial or filarial infection, however the candidate will provide written consent.  REFERENCES ·         Balabanidou V, Riga M, Chalepakis G, Paine M, Ranson H, Lycett G, Vontas J (2013). A potential role for cytochrome P450s in conferring insecticide resistance in Anopheles gambiae by altering cuticule structure. Pathogens and Global Health 2013, 107(8):435.·         Beier JC (1998). Malaria parasite development in mosquitoes.

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