MOSQUITO ATTRACTANTS
20210298297 · 2021-09-30
Assignee
Inventors
- Jetske Gudrun de Boer (Wageningen, NL)
- Ailie Robinson (London, GB)
- James George Logan (Hertford, GB)
- Joseph Johannes Antonius van Loon (Bennekom, NL)
- Willem Takken (Wageningen, NL)
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A01M1/023
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
International classification
A01N35/02
HUMAN NECESSITIES
A01M1/02
HUMAN NECESSITIES
Abstract
A mosquito attractant composition is described comprising heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal. More particularly a composition wherein the compounds are present in the following proportions: nonanal 1.00, octanal 0.32±0.16, heptanal 0.06±0.03, (E)-2-octenal 0.04±0.02 and (E)-2-decenal 0.13±0.065.
Claims
1. A mosquito attractant composition comprising heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal.
2. The mosquito attractant composition of claim 1, comprising the following ratios of heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal: TABLE-US-00016 Nonanal 1.00 Octanal 0.32 ± 0.16 Heptanal 0.06 ± 0.03 (E)-2-octenal 0.04 ± 0.02 (E)-2-decenal 0.13 ± 0.065
3. The mosquito attractant composition of claim 1, having the following concentrations of heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal: TABLE-US-00017 Concentration (μg/ml) Nonanal 47.6 ± 11.9 Octanal 15.429 ± 3.8575 Heptanal 2.857 ± 0.71425 2-octenal 1.871 ± 0.46775 2-decenal 6.136 ± 1.534
4. The mosquito attractant composition of claim 1, further comprising hexanal.
5. The mosquito attractant composition of claim 1, further comprising 1-octen-3-one.
6. The mosquito attractant composition of claim 1, not comprising 2-octanone.
7. The mosquito attractant composition of claim 1, further comprising an organic solvent.
8. An admixture comprising the mosquito attractant composition of claim 1, and a synthetic human or animal mosquito attractant blend.
9. The admixture of claim 8, wherein the mosquito attractant composition present in an amount of at least 0.5% (v/v) with the synthetic attractant blend.
10. The mosquito attractant composition of claim 1, wherein the heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal are present in combination as more than 15% v/v of total volatiles in the composition, and/or the heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal are present in combination as not more than 60% v/v of total volatiles in the composition.
11. (canceled)
12. A mosquito attractant composition comprising a natural human or animal odour source plus added heptanal.
13. The mosquito attractant composition of claim 12, wherein the added heptanal is present providing a total amount of heptanal which is about 10% greater (v/v) than the amount of heptanal in a natural human or animal odour obtained from Plasmodium-free human(s) or animals(s).
14. A composition comprising, a natural or synthetic mosquito attractant, and heptanal, wherein when the composition is volatilised, heptanal is present in an amount of at least 0.7% v/v of all volatile odour compounds present.
15. The composition of claim 14, wherein heptanal is present in an amount of at least 0.9% v/v of all volatile odour compounds present.
16. The composition of claim 1, wherein heptanal is present in an amount of not more than 1.5% v/v of all volatile odour compounds present.
17. The mosquito attractant composition of claim 1, wherein the composition is in gaseous form.
18. A mosquito trapping composition comprising an adhesive substance and the mosquito attractant composition of claim 1.
19. The mosquito trapping composition of claim 18, further comprising an insecticide.
20. An apparatus for trapping or killing mosquitoes comprising: (i) paper comprising the attractant composition of claim 1; and a CO2 emitting device comprising the paper; (ii) a container or device comprising a porous medium or wax-like medium within the container or device, wherein the porous medium or wax-like medium comprises the attractant composition of claim 1; (iii) a jar trap comprising the attractant composition of claim 1, wherein the jar trap dispenses carbon dioxide as an attractant; (iv) a bug zapping device comprising the attractant composition of claim 1; (v) an atomization device comprising the attractant composition of claim 1; or (vi) an ionic dispersing device comprising the attractant composition of claim 1.
21. A method of detecting Plasmodium infection in a subject comprising: (a) collecting a sample of odour emanated from the subject, (b) detecting and measuring amounts of one or more indicative volatile compounds in the odour, the indicative volatile compound(s) selected from: heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal, 2-octanone, hexanal and 1-octen-3-one, (c) comparing the measured amounts of the indicative volatile compounds with i) the amounts of the same compounds in a reference sample of body odour from an uninfected subject or subjects; and/or ii) predetermined reference amounts, wherein an increase in the indicative volatile compound(s) indicates the subject has a Plasmodium infection.
22. The method of detecting Plasmodium infection of claim 21, wherein the indicative volatile compounds are (E)-2-octenal and (E)-2-decenal and an increase in (E)-2-octenal and/or (E)-2-decenal compared to reference sample and/or reference amounts indicates that the subject is Plasmodium positive; wherein the indicative volatile compounds are heptanal, octanal and nonanal, and the amount of increase in heptanal, octanal and nonanal compared to reference sample and/or reference amounts is proportional to the Plasmodium infection density in the subject; and/or wherein the indicative volatile compound is selected from one or more of 2-octanone, hexanal and 1-octen-3-one and an increase in the indicative compound(s) compared to reference sample(s) thereof, and/or reference amount(s) is indicative of the presence of microscopic gametocytes in the subject.
23.-24. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE INVENTION
[0065] The invention will now be described in more particular detail and with reference to Examples.
[0066] Alternative mosquito attractant compositions of the invention may have the following ratios of heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal, based on a reference of 1 part nonanal as follows:
TABLE-US-00003 nonanal 1.00 octanal 0.32 ± 0.08 heptanal 0.06 ± 0.015 (E)-2-octenal 0.04 ± 0.01 (E)-2-decenal 0.13 ± 0.0235
TABLE-US-00004 nonanal 1.00 octanal 0.32 ± 0.04 heptanal 0.06 ± 0.0075 (E)-2-octenal 0.04 ± 0.005 (E)-2-decenal 0.13 ± 0.0118
TABLE-US-00005 nonanal 1.00 octanal 0.32 heptanal 0.06 (E)-2-octenal 0.04 (E)-2-decenal 0.13
[0067] Attractant blends in accordance with the invention are defined in the table below as follows, whereby the variation (±) is the acceptable range of concentration Wimp of each component compound.
TABLE-US-00006 Optional Optional Optional Optional Optional Optional Optional Optional Concentration Variation variation variation variation variation variation variation variation variation (μg/ml) (±) I (±) II (±) III (±) IV (±) V (±) VI (±) VII (±) VIII (±) Nonanal 47.6 11.9 9.52 7.14 4.76 2.38 0.1904 0.1428 0.0952 0.0476 Octanal 15.429 3.8575 3.086 2.3145 1.5429 0.7715 0.61716 0.46287 0.30858 0.15429 Heptanal 2.857 0.71425 0.5714 0.42855 0.2857 0.14285 0.11428 0.08571 0.05714 0.02857 2-octenal 1.871 0.46775 0.3742 0.28065 0.1871 0.09355 0.07484 0.05613 0.03742 0.01871 2-decenal 6.136 1.534 1.2272 0.9204 0.6136 0.3068 0.24544 0.18408 0.12272 0.06136
[0068] Regarding hexanal, the amount or concentration of this may be about 0.16 to 0.17 of the respective amount or concentration of nonanal in a composition.
[0069] A mosquito attractant composition as described herein may have heptanal, octanal, nonanal, (E)-2-octenal and (E)-2-decenal present in combination as not more than 60% v/v of total volatiles in the composition. Optionally, not more than a percentage value selected from 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%.
[0070] However, what is disclosed herein is the possibility that for one of the compounds a certain optional variation in concentration may be chosen, whereas for another compound, a different optional variation concentration may be chosen. That is to say, whilst all compounds may vary in concentration according to the same variation column, this is not necessarily essential. A person of average skill can choose a concentration variation column for each compound in the blend independently of the other, so for example the following is possible where the variations denoted with an asterisk (*) for each compound are chosen:
TABLE-US-00007 Optional Optional Optional Optional Optional Optional Optional Optional Concentration Variation variation variation variation variation variation variation variation variation (μg/ml) (±) I (±) II (±) III (±) IV (±) V (±) VI (±) VII (±) VIII (±) Nonanal 47.6 11.9 9.52 7.14 4.76 2.38 0.1904 0.1428 0.0952 0.0476 Octanal 15.429 3.8575 3.086 2.3145 1.5429 0.7715 0.61716 0.46287 0.30858 0.15429 Heptanal 2.857 0.71425 0.5714 0.42855 0.2857 0.14285 0.11428 0.08571 0.05714 0.02857 2-octenal 1.871 0.46775 0.3742 0.28065 0.1871 0.09355 0.07484 0.05613 0.03742 0.01871 2-decenal 6.136 1.534 1.2272 0.9204 0.6136 0.3068 0.24544 0.18408 0.12272 0.06136
[0071] Any other variations denoted with an asterisk (*) like the above to exemplify compositions of the invention are possible.
[0072] A mosquito attractant composition of the invention may be part of, or added to a synthetic human or animal mosquito attractant blend. An example of a suitable synthetic attractant blend is BG-Lure mixture of ammonia, lactic acid and caproic acid (Biogents AG, Regensburg, Germany). Also Mbita Blend 5 (MB5) which is described in more detail in Menger D. J., Otieno B., de Rijk M., Mukabana W. R., van Loon J. J., Takken W. (2014) A push-pull system to reduce house entry of malaria mosquitoes. Malar J. 13:119. MB5 includes ammonia (2.5%), lactic acid (85%), tetradecanoic acid (0.00025%), 3-methyl-1-butanol (0.000001%) and 1-butylamine (0.001%).
[0073] In the aspects of the invention which provide a mosquito attractant composition comprising a natural human or animal odour source plus added heptanal, the added heptanal may be present providing a total amount of heptanal which is at least about 7% more, 8% more, 9% more, 10% more, 11% more, 12% more, 13% more, 14% more, 15% more, 16% more, 17% more, 18% more, 19% more, 20% more, 21% more, 22% more, 23% more, 24% more, 25% more, 26% more, 27% more, 28% more, 29% more or 30% more than the amount of heptanal in a natural human or animal odour obtained from Plasmodium-free human(s) or animal(s).
[0074] A “mosquito attractant” as described herein, is a substance or more particularly a blend of substance molecules which when interact with the sensory apparatus of mosquitoes causing them to move towards a site or area, and usually the source of the substance molecules in air would take the form of a vapour concentration gradient.
[0075] The formulations of the invention can be placed in any suitable container or device for dispensing the attractant compound and attracting or trapping mosquitoes.
[0076] The attractant compound(s) of this invention may be employed in any formulation suitable for dispensing attractant effective amounts of the compounds. The compounds will generally be employed in formulations comprising a suitable vehicle or carrier containing the attractant compounds.
[0077] An attractant composition of the present invention may be applied with a carrier component or carrier (e.g., biologically or agronomically acceptable carrier). The carrier component can be a liquid or a solid material. As is known in the art, the vehicle or carrier to be used refers to a substrate such as a membrane, hollow fiber, microcapsule, cigarette filter, gel, polymers, septa, or the like. All of these substrates have been used to release insect attractants in general and are well known in the art. Suitable carriers are well-known in the art and are selected in accordance with the ultimate application of interest. Solid carriers such as clays, cellulose-based and rubber materials and synthetic polymers may be used.
[0078] For example, an attractant composition can be formulated into a waxy medium or vehicle engineered to release desired amounts of vaporous attractant compound at ambient temperatures. Exemplary waxy media are available from Koster Keunen of Watertown, Conn., U.S.A., e.g. Insect Repellent Wax Bar No. 9. This is made of fatty acids ranging in carbon chain length of from C.sub.16 to C.sub.22, fatty alcohols ranging in carbon chain length of from C.sub.16 to C.sub.22, paraffinic hydrocarbons ranging in carbon chain length of from C.sub.19 to C.sub.47, branched hydrocarbons ranging in carbon chain length of from C.sub.23 to C.sub.69, beeswax and other natural waxes such as candelilla and carnauba. The wax formulations together with the compositions of the invention can be made to have a congealing point in the range from about 75° C. to about 45° C.
[0079] Other suitable carriers may include existing mosquito or other insect attractant compositions. The attractant composition of the invention may therefore be mixed with the other attractant composition or compositions. For example, the attractant composition of the invention may be mixed with the art known MB5 attractant composition. The percentage by volume of the attractant composition of the invention to the other attractant composition, e.g. MB5, may be in the range 0.5% (v/v)-99% (v/v). Optionally in the range 1% (v/v) to 50% (v/v). For example, percentage by volume of the attractant composition of the invention to the other attractant composition may be (in v/v) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%; or any range of percentage between any selected lower % and higher % number in the aforementioned list.
[0080] The compositions of the invention may be placed in any suitable container or device for dispensing the attractant compound and attracting or trapping mosquitoes. A “lure and kill” format of device for example. The formulations can be placed in a suitable device so that one can obtain, for example, evaporation of the attractant compound from a porous medium or wax-like medium containing the attractant compound positioned within the dispensing device. As examples of such devices, there can be mentioned the devices disclosed in U.S. Pat. Nos. 5,205,064, 5,799,436 and 6,055,706 of BioSensory Insect Control Corporation and James Nolen & Company. The formulations can also be placed in jar traps such as those that dispense carbon dioxide as an attractant. The formulations can also be placed in “bug zapping” devices for electrocuting the mosquitoes attracted to the device containing the attractant-containing formulation.
[0081] Other suitable means of dispensing the attractant compositions of the invention may be by atomization and/or ionic dispersion of the compound as suitable-sized, positively-charged droplets from a suitable atomization or ionic dispersing apparatus, such as the Ionic Wind™ device, available from Brandenburg, Ltd. of Brierery Hill, United Kingdom used in connection with any suitable mosquito trapping device or apparatus.
Example 1
[0082] Materials and Methods
[0083] Ethics
[0084] Study participants were five- to twelve-year-old children local to the Thomas Odhiambo Campus of icipe in Western Kenya (000251S, 34° 131E), including Rusinga Island, in Suba District, Homabay County. Participants were recruited after obtaining signed consent. The study protocol (NON SSC 389) was approved by the Scientific and Ethical Review Committee of the Kenya Medical Research Institute (KEMRI/RES/7/3/1). Subsequent analyses were conducted at the London School of Hygiene & Tropical Medicine (LSHTM) (ethics reference 8510).
[0085] Attractiveness of ‘Infected Odour’ (Socks) by Cage Assays
[0086] A cohort of Plasmodium-infected, asymptomatic (tympanic temperature <37.5° C.), individuals that participated in an olfactometer study.sup.7 was studied for the attractiveness of their skin odour to Anopheles gambiae s.s. Forty-five children were included, of which there were: 23 with microscopic gametocytes or an estimated gametocyte density above 50 gametocytes/μL blood by QT-NASBA, 10 positive for asexual parasites by microscopy, and 12 that tested Plasmodium-parasite free by 18S-qPCR.sup.23. Samples were collected at two time points: within 24 hours of antimalarial treatment but while children still harboured parasites.sup.22 (time point one [T1] samples), and 21 days later (time point two [T2] samples). Antimalarial treatment with artemether-lumefantrine (AL) was administered to Plasmodium positive individuals according to manufacturer's instructions (20 mg artemether/120 mg lumefantrine per tablet, Coartem™; Novartis, Basel), and socks were put on within one hour of treatment. Age, haemoglobin (Hb), weight and temperature were measured as covariates.sup.7. At day 21 (‘after’), both parasitological testing and participant covariate measurements were repeated.
[0087] Procedures for Collection of Body Odour
[0088] Body odours were collected for 20 hours on nylon socks (97% polyamide, 3% elastane, 20 denier, Hema, The Netherlands), which were washed using 70% ethanol and dried at 70° C. for two hours before use. Surgical gloves were worn throughout collection procedures. Children were assisted in putting on and removing the socks. These were stored in clean glass jars at −20° C. until use in cage assay experiments. Children were asked not to bathe during this time but had no other behavioural restrictions.
[0089] Behavioural Assays for Attractiveness of Odour Samples
[0090] A dual choice cage assay was modified.sup.47 to determine the relative attractiveness of odour samples from T1 and T2. Three WHO bioassay tubes (12.5 cm long, 5 cm wide).sup.48 were connected with sliding units between the inner and outer tubes (
[0091] Six- to eight- day old, non blood-fed, female An. gambiae s.s. mosquitoes (Mbita strain, with published rearing methods.sup.49) were collected prior to the experiment and allowed eight hours acclimatisation. Ten mosquitoes were released into the central tube per bioassay, and the gates of the tubes opened for 15 minutes to allow mosquitoes to make a choice of odour source. Experiments were conducted between 18:30-22:30 under ambient conditions, in a red fluorescent-lit room (average temperature, 24.1° C.) with the dual cage covered by black cotton cloth. After 15 minutes mosquito choice was recorded. All sock pairs were tested simultaneously on the same nights, and in total each pair (child) was tested six times, replicating over experimental nights, dual cage set-ups and between cages. All disposable equipment was changed, and cages cleaned (70% ethanol), between experiments/replicates.
[0092] Statistical Analysis
[0093] Per child, the number of mosquitoes that chose the T1 or T2 odour sample was summed over six replicates, and the relative attractiveness of samples determined as the proportion of mosquitoes that selected a sample over the total number of mosquitoes that made a choice. A generalized linear model (GLM; Binomial distribution, logit link function and dispersion estimated) was used to test the effect of parasitological status (parasite free, asexual or gametocytes) on the relative attractiveness. The number of mosquitoes caught in the cage with the T1 sample was used as the response variable, and all mosquitoes caught in both cages as the binomial total. Covariates associated with participants (age, sex, Hb and tympanic temperature measured at T1) were tested, but removed from the model because they were not significant (P>0.05, F-tests). Per parasitological group, we used the 95 CI of the predicted proportion of mosquitoes choosing T1 odour samples, derived from the GLM, to assess whether mosquito choice differed significantly from a 50:50 distribution over the two odour samples. SPSS® (2016, version 24, IBM) was used for the analyses.
[0094] Collection of Volatile Odour Samples
[0095] In the same locality, a separate cohort of schoolchildren, of varying Plasmodium infection status, were sampled for foot odour (
[0096] For each child, one foot was placed in a prepared bag (Fresh and Eazy oven bags, 45×50 cm, Meda-Pak, Uithoorn, The Netherlands), clipped shut around the calf. At each sampling round (R1-R3), a control (empty) bag was tightly closed and sampled in the same manner. Bags were fitted with Swagelok fittings at opposing corners, allowing connection to polytetrafluoroethylene (PTFE) tubing for air flow. Air (charcoal-filtered) was pumped into the top of the bag and vacuumed from the bottom (both at 500 mL/min), with a 30-minute purge prior to fitting the polymer filters, to ensure system cleanliness. Porapak filters were connected (Porapak Q, mesh size 50/80, Supelco Analytical, Bellefonte, Pa., USA) and sampled for 100 minutes, then stored in stoppered glass vials in a cool box before sealing under filtered nitrogen on the same day. Ampoules were stored at −20° C. until shipping to LSHTM. Prior to use, all PTFE tubing, Swagelok fittings and glassware were cleaned with 70% ethanol, then baked in an oven at 150° C. for two hours. Sampling bags and charcoal filters were baked in the same manner. Cotton gloves were worn by the investigators throughout.
[0097] Infection Status
[0098] Odour sampling was informed by RDT (One Step malaria HRPII and pLDH antigen rapid test [SD BIOLINE, Cat no 05FK60]), performed as per manufacturer's guidelines, and thick and thin blood films made using peripheral blood from a finger prick. Whole blood (50 μL) was stored in RNAprotect (250 μL; QIAGEN, Germany). Retrospectively, DNA/RNA extraction was performed using Total Nucleic Acid Isolation Kit (with methods as published previously.sup.50) and P. falciparum parasite density, and stage V gametocyte density, determined by 18S qPCR.sup.23 and QT-NASBA.sup.51. Additionally, dried blood stored on both Whatman No. 3 filter paper (Whatman, Maidstone, United Kingdom [wDBS]) and used RDTs (air dried and stored in sealed plastic bags containing the desiccant silica gel [uRDT]) was used as a DNA template. DNA was extracted from circles (3 mm) punched from the wDBS, and sections (3×2 mm) cut from the central section of the nitrocellulose strips in the uRDTs.sup.52. Extraction was performed in a deep well plate using an automated extraction system (QIAsymphony), with the QIAsymphony DSP DNA mini kit (QIAGEN, Germany) and according to the manufacturer's instructions, and a Plasmodium tRNA methionine-based duplex qPCR was used to measure Plasmodium density.sup.22. Good correlation in parasite density was obtained between duplex qPCR using wDBS or uRDT whole blood template.sup.53. Where available, the same DNA extracts were used for species specific (P. falciparum, P. ovale spp. and P. malariae) nPCR.sup.54, with some P. ovale spp. identifications confirmed by the P. ovale spp. tryptophan-rich antigen (PoTRA) assay.sup.55.
[0099] Gas Chromatography-Electroantennography (GC-EAG) of Pooled Odour Samples
[0100] GC-EAG Odour Sample Blends
[0101] Porapak filters were eluted using re-distilled diethyl ether (750 μL), and, to approximate an “average” odour per category, extracts were pooled according to the individual's parasitological status: (1) Plasmodium infection, no gametocytes (2) high-density P. falciparum gametocytes (3) parasite-free individuals, (4) Plasmodium infection, sub-microscopic P. falciparum gametocytes. Aliquots (400 μL) of extracts were mixed, then concentrated (to 60 μL) under a stream of nitrogen (charcoal-filtered). Glassware, charcoal filters and PTFE tubing were cleaned as before.
[0102] Experimental set-up
[0103] GC-EAG was conducted during the scotophase, using four- to eight- day-old, unfed female Anopheles coluzzii (N′gousso strain.sup.56). Adults were maintained at 70% RH, with a 12 h light/dark cycle (scotophase 09:00-21:00) and access to 50% glucose solution. The order of testing blends was determined by a 5×5 Latin square (including control blend). The mosquito head was dissected, and the palps, proboscis, and half of the terminal (13.sup.th) antennal flagellomere cut off. The indifferent electrode was inserted into the back of the head and the antennal tips guided into the recording electrode to complete the circuit (
[0104] Analysis of Odour Profiles by GC
[0105] Instruments used for GC analysis were 7890A, 6890N and HP6890 (Agilent Technologies, Stockport, UK). Each was fitted with a cool-on-column injector, flame ionization detector, used hydrogen carrier gas, and 1 μL injections were performed. All were fitted with an HP1 column, 50 m×0.32 mm, film thickness 0.52 μm, and the following programme was used: oven temperature maintained at 40° C. for 0.5 minutes, increased by 5° C. per minute to 150° C., held for 0.1 minute, raised by 10° C. per minute to 230° C., held for 40 minutes. Traces were analysed using the R package MALDIquant.sup.57 (R version 3.3.0, 2016, The R Foundation for Statistical Computing©). In brief, raw x,y co-ordinates for GC traces were exported from Agilent ChemStation (C.01.04) and the y value (height, for 1 μL) multiplied by total extract to represent actual amount per sample (ng). Following baseline removal, traces were visually inspected for consistent differences between parasitological groupings. Compounds of interest (COI) were then compared quantitatively, by integrating peaks in ChemStation, and calculating retention index and amount relative to a standard series of n-alkanes (C7-C25), using Equation 1.
[0106] Equation 1. Retention index (RI) calculation
RI=100((log.sub.10RtX−log.sub.10Rtn)/(log.sub.10Rtn+1−log.sub.10Rtn))+100n [0107] RtX=Retention time for compound of interest [0108] Rtn=Retention time for alkane before compound of interest [0109] Rtn+1=Retention time for alkane after compound of interest [0110] n=number of carbons in alkane before compound of interest
[0111] Following statistical analysis (below), IAC were identified by gas chromatography-mass spectrometry (GC-MS), using either a Micromass Autospec Ultima (a magnetic sector mass spectrometer equipped with a Programmed Temperature Vaporising inlet (GL Sciences B.V., Eindhoven, The Netherlands) and Agilent 6890N GC), or a Mass Selective Detector (quad GC-MS). Peaks were compared with MS databases (National Institute of Standards and Technology, NIST). For confirmation of identification, authentic standards were injected onto two GC columns (HP1 and DB wax) simultaneously with samples containing those compounds. Standards were: heptanal (Sigma-Aldrich), octanal (Sigma-Aldrich), nonanal (Sigma-Aldrich), (E)-2-octenal (Acros Organics), (E)-2-decenal (SAFC), 2-octanone (Sigma-Aldrich). Identifications were considered certain when the resultant peak increased in height without increasing in width. Co-injections were conducted for all IAC.
[0112] Statistical Analysis
[0113] Any sample that had detectable parasite DNA at amounts greater than published limits of detection (LOD) for the assays (0.02 p/μL for 18S.sup.23 and 5 p/μL for duplex qPCR.sup.22) was considered positive, and those with DNA amounts beneath these thresholds were excluded. Only samples that were negative by all measures, including at least one molecular diagnostic measure, were taken to be negative, other than RDTs for which positivity was acceptable (on an assumption of positivity due to circulating HRP-2 protein).sup.58. Individuals with Plasmodium parasitaemia, but without microscopic gametocytes, were divided into higher and lower parasite density categories: ‘higher density’ with greater than 50 p/μL, and ‘lower density’ with between the LOD and 50 p/μL (
[0114] The association between the production of COI (variate: percentage of total entrainment) and parasitological category was assessed by linear mixed models fitted using the method of residual maximum likelihood, REML. This modelling allowed for unequal sample sizes (per parasitological category) and repeated measures on the same individuals. We tested (F-tests) for the main effects of covariates (age, Hb, day of the year, weight) before the treatment (parasitological status) term, and for factors (sex, round) after the treatment term, in a forward selection, parallel-lines, regression analysis approach. Pairwise comparisons between groups of most biological interest were made using the LSD at the 5% level, and COI demonstrating significant (REML, LSD, 5%) differences between groups were termed ‘infection-associated compounds’. Data analysis was conducted using Genstat (2013, 16.sup.th edition, VSN International, Hemel Hempstead, UK).
[0115] Behavioural Testing of Candidate Compounds
[0116] Testing IAC Individually
[0117] Six IAC (heptanal, octanal, nonanal, (E)-2-octenal, (E)-2-decenal and 2-octanone) were tested in a background of odour from the worn nylon socks of twelve parasite-free children (18S qPCR confirmed). Each sock pair was cut into twelve strips after removing the foot part, then 12 bundles were made, each containing a strip from each individual. Bundles were stored at −20° C. until, and between, experiments. IAC were positioned downwind and separated from sock bundles by a metal grid, ensuring no contact. Parasite-free odour (bundles) was tested with or without individual IAC (in 10 μL hexane on filter paper) and against the same but with hexane alone. For each IAC, a decimal dilution series was made (in hexane) and two/three concentrations chosen, to bracket the differential amount between significantly different groups (LSD 5%, REML), adjusted to represent 15 minutes of compound release (test duration).
[0118] Improving a Mosquito Lure
[0119] Next, we verified whether the IAC could improve a mosquito lure for monitoring or mass trapping of Anopheles. Heptanal, the most promising candidate from the above experiment, was tested as well as two blends: Plas 5 contained the IAC that were associated with parasitological positivity (nonanal, heptanal, octanal, (E)-2-decenal and (E)-2-octenal), and Plas 6 additionally contained the gametocyte-associated 2-octanone (
TABLE-US-00008 Plas5 Concentration Plas6 Concentration IAC Plas5 % proportion.sup.(d) (μg/ml) Plas6 % proportion.sup.(d) (μg/ml) Nonanal.sup.(a) 16.36 1.00 47.60 16.36 1.00 47.60 Octanal.sup.(a) 5.30 0.32 15.429 5.30 0.32 15.429 Heptanal.sup.(a) 0.98 0.06 2.857 0.98 0.06 2.857 (E)-2-octenal.sup.(b) 0.64 0.04 1.871 0.64 0.04 1.871 (E)-2-decenal.sup.(b) 2.11 0.13 6.136 2.11 0.13 6.136 2-octanone.sup.(c) 0.09 0.01 0.256 .sup.(a)The predicted proportion of each compound found in the ‘higher’ density group (‘total density’ categorisation, REML) was used to generate values for octanal, nonanal and heptanal as the production of these compounds was upregulated in these groups .sup.(b)The predicted proportion found in the ‘positive’ group (‘positive vs. negative’ categorisation, REML) was used .sup.(c)The predicted proportion was taken from the ‘gametocyte’ group, ‘total density’ categorisation .sup.(d)Ratios were derived from these proportions, then all were normalised to the actual mean amount of nonanal found in the ‘higher’ density category (476 ng) in 100 min.
[0120] MB5 was presented on nylon strips as described in Menger et al. (2014).sup.36 (except the five compounds were incubated onto a single 5×26.5 cm nylon strip instead of five narrower strips). 10 μl of the Plas5 or Plas6 blend at 100% or serial dilution was pipetted onto a filter paper and added in the same olfactometer trap.
[0121] Assay
[0122] A triple chamber dual-port olfactometer.sup.59 was used to test the preference of 30 five- to eight-day-old female, non blood-fed Anopheles coluzzii (Suokoko strain.sup.21, rearing procedures as published previously.sup.21) for parasite free odour or MB5, supplemented with IAC or IAC blends, against background odour alone (parasite-free odour or MB5). Mosquitoes were maintained in a release cage prior to testing (for 16-22 hours). Experiments took place during the last four hours of the scotophase under near-dark conditions (<1 lux). Mosquitoes were allowed to fly for 15 minutes, then those that had entered the traps with test/control odours were counted. Each IAC/concentration combination was tested eight-nine times on different days, and each Plas concentration 10-11 times on different days, rotating treatments between the left and right port of the olfactometer. Climatic data (R.H., temperature and air pressure) were recorded in the flight chambers and in the surrounding room.
[0123] Statistical Analyses
[0124] Generalized linear models (GLMs) were used to test the effect of odours (individual IAC/heptanal/Plas blends) on relative attractiveness (the proportion of mosquitoes selecting the test odour). GLMs were run as described above (statistical analysis, attractiveness of ‘infected odour’ (socks) by cage assays), testing parameters associated with the set-up as additional factors or covariates in the model, and retaining when significant (P<0.05, F-test). Sets of compounds were run in separate models. SPSS® was used for the analyses.
[0125] Plasmodium Manipulation of Odour Profile?
[0126] Parasite transmission often constitutes a population bottleneck: of the many parasites within one host, only a few are successfully transmitted to the next.sup.1. Hence, parasites often evolve to exert influence over these transmission events. The malaria parasite Plasmodium would benefit from increasing its infected vertebrate host's attractiveness to susceptible Anopheles mosquito vectors, if this resulted in increased contact rates between the two hosts. Such changes in attractiveness in both animal.sup.2-6 and human.sup.7-9 malaria systems have previously been demonstrated. Changes in vertebrate host attractiveness in response to infection have also been documented in other vector-borne disease systems.sup.10-13, possibly indicating evolutionary convergence, which supports parasite manipulation underlying these phenomena.sup.1. Body odour, comprising the volatile compounds emitted from the skin of vertebrates, is the most important cue used by Anopheles for host location.sup.14. It has been shown that differences in the composition of body odour are responsible for the variation in attractiveness to biting insects known to exist between people.sup.15,16, and these differences may be influenced by body weight and/or surface area, hormones or genetic factors.sup.17-19. Human body odour can also be influenced by disease, including metabolic disorders, genetic disorders, and infections.sup.20. A study of Plasmodium infection in mice found such changes in body odour to be associated with changes in attractiveness to mosquitoes.sup.5, and another found compositional changes in body odour during controlled human malaria infection (CHMI), with a variable effect on attractiveness.sup.21. While increased attractiveness of Plasmodium-infected individuals has been demonstrated in a malaria-endemic setting.sup.7, remarkably, no study has yet investigated the skin chemistry underlying this phenomenon. Given the crucial importance of body odour to mosquito host location, and the proposition that body odour can be altered during disease, here, we hypothesise that Plasmodium parasites manipulate the odour of infected humans, and that this influences attractiveness of humans to mosquitoes. To test this hypothesis, we first confirmed that asymptomatic children in Western Kenya were more attractive to mosquitoes when harbouring Plasmodium parasites, before comparing skin odour composition between Plasmodium-infected and parasite-free children from the same population. Using analytical chemistry, and the antennal and behavioural responses of Anopheles mosquitoes, we identified and established the role of Plasmodium infection-associated compounds in human body odour.
[0127] Attractiveness and Plasmodium Infection
[0128] To assess whether Plasmodium infection changes the attractiveness of human hosts to mosquitoes, we measured the behavioural response of Anopheles gambiae sensu stricto (s.s.) to foot odour of 5-12 year-old school children at two sampling time points. At time point one (T1), foot odour of asymptomatic Plasmodium falciparum-infected, and uninfected, children was collected on socks for 20 hours. For infected individuals, this occurred immediately after administration of the first dose of treatment with artemether-lumefantrine (AL), which is known to allow residual parasitaemia during this time period.sup.22.
[0129] Odour samples were collected in the same manner from the same children 21 days later, following confirmed parasite clearance (time point two, T2). Odour samples from participants with malaria parasites were categorized by those who harboured transmissible gametocyte stages (n=23), as identified either by microscopy or by the molecular diagnostic QT-NASBA that detects female gametocyte Pfs25 mRNA, or those with microscopically detected asexual stage parasites but no gametocytes (n=10). Samples were considered parasite-free (n=12) when no parasites were detected by microscopy and 18S qPCR.sup.23. Anopheles gambiae s.s. mosquitoes were offered the choice of either T1 or T2 odour samples from the same child, in a dual choice cage assay (
[0130] Mosquitoes were more attracted to odours collected at T1 from children harbouring asexual or gametocyte stage parasites relative to T2 odour samples (GLM, 95% confidence intervals (95 CI) 0.55-0.62 and 0.59-0.63 respectively,
[0131] Antennal Response to Malaria Odour
[0132] We analysed air entrainment odour extracts using coupled gas chromatography-electroantennography (GC-EAG).sup.15. A change in the electric potential across the antenna resulting from stimulated neuropsychological activity, i.e. the EAG response, is caused during olfactory nerve cell response. This allows detection of compounds that are important to the mosquito (
[0133] Point-of-care malaria diagnostics (rapid diagnostic test (RDT) and microscopy), used to inform odour sampling from asymptomatic individuals, were retrospectively confirmed using molecular diagnostics. Infected children were treated after odour sampling, and repeat sampling of all individuals was attempted one and three weeks later alongside repeat parasitological diagnoses (
[0134] A further group, (4) Plasmodium infection, sub-microscopic gametocytes, was included due to the frequency of sub-microscopic gametocytaemia in endemic infections. Plasmodium falciparum gametocyte densities were determined by Pfs25 mRNA QT-NASBA, while 18S qPCR and duplex qPCR were used to determine P. falciparum and Plasmodium densities respectively. Twenty-two analytes (Table 2) were found to elicit antennal response in Anopheles coluzzii (formerly the M-form of An. gambiae s.s. Giles), including the aldehydes heptanal, octanal and nonanal.
TABLE-US-00009 TABLE 2 Analytes (compounds eluting during gas chromatography, sometimes at the same time) within odour samples that were found to induce antennal response in Anopheles coluzzii. The identification of analytes in bold font was subsequently confirmed by co-injection with an authentic standard. RI.sup.(a) Tentative identification 716 Methyl cyclohexane 750 Unknown 781 Dimethyl sulfide 795 Solvent 801 Octane 853 1-Hexanol (unsure identification), dimethyl sulfone 880 Heptanal 889 Solvent 933 Benzaldehyde 958 Phenol/1-octen-3-one 982 Octanal 1014 2-Ethylhexanol or 2-octene 1056 Shoulder para cresol (4-methylphenol) and 3-octen-2-ol, peak octen-1-ol 1084 Nonanal 1118 Unknown 1139 2-Ethyl benzaldehyde 1150 4-Ethyl benzaldehyde 1186 Decanal 1199 Dodecane 1239 Ethylacetonphenone 1389 Unknown 1428 Geranylacetone .sup.(a)Analytes identified by retention index (RI) were selected if eliciting >3 responses in one of the treatment groups (Plasmodium infection, no gametocytes, n = 6; Plasmodium infections, sub-microscopic P. falciparum gametocytes, n = 7; high-density P. falciparum gametocytes, n = 7; parasite-free individuals, n = 7; control, n = 7)
[0135] No EAG-active analytes were specific to any of the infection profiles (1-4), indicating that any Plasmodium-induced change in the compounds used by host-seeking Anopheles must manifest through variation in the relative amounts of compounds that are present in parasite-free individuals. This is in accordance with the ‘deceptive signalling’ hypothesis, whereby host cues already used by host-seeking insects are exaggerated, increasing the attractiveness of that vertebrate to biting insects, but when the blood-meal is in fact unfavourable to the insect.sup.31. If the disadvantages (e.g. reductions in fecundity.sup.24-26, shortened lifespan.sup.27-29) of taking an infected blood meal outweigh any advantages (e.g. reduced host defences.sup.2, faster engorgement.sup.30), the evolution of an infected-host avoidance phenotype might be expected. Anopheles may less easily select against an infected-host phenotype comprising ‘normal’ stimuli.
[0136] Plasmodium Infection-Associated Compounds (IAC)
[0137] To investigate whether Plasmodium infection indeed results in quantitative changes in the production of volatile compounds, we compared the profiles of 117 foot, and 59 control (empty entrainment bag), odour samples. A total of 56 individuals participated in air entrainment odour sampling (
[0138] For all IAC, we found a quantitative relationship: the majority of individuals produced these compounds, but the quantity produced increased with Plasmodium infection. An average of 177 (standard error 5.23) analytes were captured per sample, and the IACs were disproportionately abundantly produced (
[0139] Mosquito Response to IAC
[0140] To determine whether the IAC were attractive to mosquitoes, and, therefore, likely to be responsible for the increased attractiveness observed in infected individuals, we tested all six IACs in behavioural bioassays with An. coluzzii. First, the odour of parasite-free children (worn socks) was supplemented with the IAC individually, and tested at a minimum of two concentrations each. Of these, adding 10 μL of heptanal at 10.sup.−8 g/mL to parasite-free odour significantly increased attractiveness, relative to parasite-free odour alone (GLM, 95 CI: 0.60-0.84,
[0141] Aldehydes are found in the skin odour of various mammalian species.sup.37, and have previously been determined to be among the chemicals used by haematophagous insects for host location.sup.38. These oxygenated compounds can be synthesised when reactive oxygen species attack a lipid-dense membrane structure.sup.39, i.e. lipid peroxidation, caused by oxidative stress. Oxidative stress is known to characterise malaria infection.sup.40, occurring in the erythrocytes and liver. The probable presence of other infections in this cohort of children, including schistosomiasis, specifically associates the observed effect with Plasmodium infection itself, as a more general ‘scent of infection’ would likely be still present in the malaria-free individuals. Alternatively, or additionally, the aldehydes found here may have been produced directly by Plasmodium parasites: a recent publication found the aldehydes octanal, nonanal and decanal to be among volatile compounds emitted by red blood cell (RBC) cultures that had been supplemented by (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP).sup.41. HMBPP is a precursor in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, apparently used by Plasmodium for isoprenoid production, and it was suggested that HMBPP triggered enhanced release of these compounds from infected RBC, with a subsequent impact on mosquito attraction. Additionally, terpenes were isolated from HMBPP RBC, and another study also isolated terpenes above Plasmodium infected RBC cultures.sup.42. Although the MEP pathway is a possible source of terpenes via isoprenoid production in infected RBC.sup.42, the source of terpenes in HMBPP RBC remains unknown.sup.41. We did not find an association between Plasmodium infection and the emission of terpenes from the skin, corroborating earlier findings in Plasmodium-infected mice.sup.5. It should be emphasised that laboratory-based studies of the volatile compounds isolated above iRBC cultures do not characterise the human body odour used by mosquitoes during host location. As such, they do not fully capture the complex biological and biochemical host-parasite interactions that occur in natural Plasmodium infections. In our study, the production of aldehydes was increased in individuals with Plasmodium infection. The extent to which parasite-specific release of aldehydes from iRBC would contribute to a profound and systemic increase in aldehyde production, caused by malaria-induced oxidative stress, remains unexplored. Finally, it is important to note that while the lipid peroxidation pathway for aldehyde production is well-established, the skin microbiota are also known to produce aldehydes. This is particularly relevant to our study, as odour samples were taken from the feet. Feet harbour skin microflora that produce volatiles that are attractive to mosquitoes.sup.43, and differences in microflora have been associated with differences in attractiveness.sup.16.
Example 2
[0142] Table 3 below shows the results of analysis of sampled foot odour from Plasmodium-infected and non-infected individuals. The percentage composition of foot odours for the various volatile compounds are shown, as well as the mean amounts, in ng, relative to nonanal. The amounts of compounds were collected in 100 minutes sampling from the foot only.
TABLE-US-00010 Percentage of Mean amount found odour sample relative to nonanal, ng* Individual: Negative Infected Negative Infected Hexanal 1.699 2.786 64.234 70.326 Heptanal 0.610 1.077 23.050 29.750 1-Octen-3-one 0.402 0.753 15.216 19.014 2-Octanone 0.032 0.051 1.229 1.410 Octanal 3.157 5.224 119.392 144.275 (E )-2-Octenal 0.455 0.805 17.208 20.348 Nonanal 9.837 17.237 371.977 476.035 (E )-2-Decenal 1.176 2.275 44.484 57.509
[0143] Discussion
[0144] We demonstrated that elevated production of specific aldehydes in skin odour is associated with increased attractiveness to mosquitoes in Plasmodium-infected people. We found that odour from all P. falciparum-infected individuals was more attractive than that of parasite-free individuals, and the increased production of IACs was correlated with total parasite density. The association between P. falciparum asexual parasite biomass and gametocyte density is generally positive.sup.44-46, and we also observed this in our study. The application of identifying such infection-associated compounds, with their demonstrated impact on mosquito behaviour, are far-reaching: we better understand parasite-vector-host transmission events, and their over-dispersed nature in human populations. These compounds are expected to permit further improvement of already highly functional lures for trapping malaria mosquitoes, or to serve as biomarkers for malaria, providing a basis for novel and non-invasive diagnostic tools.
[0145] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0146] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0147] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
Example 3
[0148] A synthetic lure composed of the Mbita blend (MB5) and the Plas 5 blend has been found to attract Anopheles mosquitoes in a field trial undertaken in Bubaque, Bijagos archipelago. The synthetic lure was used to bait Centre of Disease Control (CDC) Light traps at three concentrations (0.1% (v/v), 1% (v/v) and 10% (v/v) Plas 5 with MB5. As well as successfully attracting Anopheles mosquitoes, mosquitoes of the genera Aedes and Culex were also attracted to the lures. A total of 2134 mosquitoes were caught over 25 nights across the five treatments. Of this, 206 (9.7%) consisted of female anophelines. Traps baited with MB5+1% Plas 5 had the highest Anopheles females capture rate (n=13.4) and this treatment caught the most substantial number consistently. The combination of MB5+10% Plas 5 had the highest absolute mosquito capture rate (n=106).
[0149] For each genera-sex group (
[0150] A greater number of Anopheles mosquitoes (
TABLE-US-00011 TABLE 4 Trap catches indexes comparing the absolute trap catches and the total Anopheles mosquitoes across treatments. Absolute Anopheles Absolute Anopheles Absolute Anopheles Absolute Anopheles Hexane — — — — — — — — MB5 1.937759336 1.2 — — — — — — 0.1% Plas 5 2.012448133 2.25 1.038544 1.875 — — — — 1% Plas 5 1.705394191 3.35 0.880086 2.791666667 0.847423 1.48888889 — — 10% Plas 5 2.199170124 2.5 1.134904 2.083333333 1.092784 1.11111111 1.289538 0.746268657
[0151] Treatment 4, MB5+1% Plas 5 was shown to be the concentration that was the most successful in attracting Anopheles mosquitoes. In addition to the highest capture rate as mentioned previously, the trap catch index was also the greatest (3.35). Furthermore, the MB5+1% Plas 5 catch indexes (table 4) were higher than 1 against both the hexane control and MB5, as well as to MB5+0.1% Plas 5 and MB5+10% Plas 5.
[0152] Treatment Effects
[0153] There was no correlation between the environmental variables of temperature, wind speed, humidity or rainfall present with the trap catches. Therefore, the model effects of these parameters were not included as covariates in the models created.
[0154] Anopheles Females
[0155] Tukey post-hoc results (see table 5 below) confirmed the significant effect of treatment on the relative abundance of Anopheles females. There was no significant difference between the control and MB5 treatments, p=0.77. However, there were significant differences between the control and 0.1% Plas 5 (p<0.05) and 10% Plas 5 (p<0.01) with the significant difference between the control and 1% Plas 5 being the greatest (p<0.001). Comparing the combined MB5+Plas 5 baits to MB5 alone, there was only a significant effect between 1% Plas 5 and MB5 (p<0.01). There were no other significant differences between treatments.
TABLE-US-00012 TABLE 5 Multiple comparisons of treatments to determine the direction of the odour treatment effect on Anopheles: Estimate Standard Treatment of error of Z- comparison coefficient coefficient value P-value MB5 − Hexane 0.33647 0.29275 1.149 0.77573 MB5 + 0.1% 0.81093 0.26872 3.018 0.02080 Plas 5 − Hexane MB5 + 1% 1..16315 0.25615 4.541 <0.001 Plas 5 − Hexane MB5 + 10% 0.89609 0.26532 3.377 0.00637 Plas 5 − Hexane MB5 + 0.1% 0.47446 0.24070 1.971 0.27466 Plas 5 − MB5 MB5 + 1% 0.82668 0.22658 3.648 0.00238 ** Plas 5 − MB5 MB5 + 10% 0.55962 0.23690 2.362 0.12251 Plas 5 − MB5 MB5 + 1% 0.35222 0.19454 1.810 0.36123 Plas 5 − MB5 + 0.1% Plas 5 MB5 + 10% 0.08516 0.20647 0.412 0.99377 Plas 5 − MB5 + 0.1% Plas 5 MB5 + 10% −0.26706 0.18982 −1.407 0.61728 Plas 5 − MB5 + 1% Plas 5
[0156] For Culex females (see table 6 below), Poisson regression models revealed that there was a significant difference between all treatments and the control with p<0.001. Treatment 3 and treatment 5 had a significant difference of p<0.005 and p<0.001 respectively. Therefore, treatment 4 had a significantly lower abundance than the other MB5+Plas 5 treatments. None of the treatments was significantly different to MB5 alone.
TABLE-US-00013 TABLE 6 Multiple comparisons of treatments to determine the direction of the odour treatment effect on Culex females: Standard Treatment Estimate error of comparison coefficient coefficient Z-value P-value MB5 − Hexane 0.75803 0.09058 8.369 <0.001 MB5 + 0.1% 0.83606 0.08948 9.343 <0.001 Plas 5 − Hexane MB5 + 1% 0 0.57781 0.09339 6.187 <0.001 Plas 5 − Hexane MB5 + 10% 0. 87872 0.08892 9.882 <0.001 Plas 5 − Hexane MB5 + 0.1% 0.07803 0.07099 1.099 0.80463 Plas 5 − MB5 MB5 + 1% −0.18023 0.07585 −2.376 0.11983 Plas 5 − MB5 MB5 + 10% 0.12069 0.07027 1.717 0.41917 Plas 5 − MB5 MB5 + 1% −0.25826 0.07454 −3.465 0.00471 Plas 5 − MB5 + 0.1% Plas 5 MB5 + 10% 0.04266 0.06886 0.620 0.97158 Plas 5 − MB5 + 0.1% Plas 5 MB5 + 10% 0.30092 0.07386 4.074 <0.001 Plas 5 − MB5 + 1% Plas 5
[0157] In Culex males (see table 7 below), there were less significant differences in abundances between treatments. Only treatment 5 was significantly different, to the control with p<0.05 and to MB5, p<0.05. there was no significant difference between MB5+Plas 5 treatments or for the treatments against the control.
TABLE-US-00014 TABLE 7 Multiple comparisons of treatments to determine the direction of the odour treatment effect on Culex males. Standard Estimate error of Treatment comparison coefficient coefficient Z-value P-value MB5 −Hexane 0.07696 0.27754 0.277 0.9987 MB5 + 0.1% Plas 5 − Hexane 0.27763 0.26513 1.047 0.8318 MB5 + 1% Plas 5 − Hexane 0.39204 0.25887 1.514 0.5505 MB5 + 10% Plas 5 − Hexane 0.73237 0.24335 3.010 0.0217 * MB5 + 0.1% Plas 5 − MB5 0.20067 0.25950 0.773 0.9377 MB5 + 1% Plas 5 − MB5 0.31508 0.25311 1.245 0.7229 MB5 + 10% Plas 5 − MB5 0.65541 0.23721 2.763 0.0450 * MB5 + 1% Plas 5 − MB5 + 0.11441 0.23944 0.478 0.9893 0.1% Plas 5 MB5 + 10% Plas 5 − MB5 + 0.45474 0.22256 2.043 0.2433 0.1% Plas 5 MB5 + 10% Plas 5 − MB5 + 0.34033 0.21508 1.582 0.5062 1% Plas 5
[0158] Molecular Identification
[0159] Anopheles:
[0160] Overall 206 female Anopheles were caught across traps of the five treatments. PCR results of the trap catches (
TABLE-US-00015 TABLE 8 The abundance of each species Anopheles gambiae s.l complex with treatment. Treatment Hexane MB5 0.1% Plas 5 1% Plas 5 10% Plas 5 Anopheles n % n % n % n % n % An. melas 9 4.37 18 8.74 26 12.62 34 16.50 21 10.19 An. Coluzzii (M) 2 0.97 1 0.49 2 0.97 5 2.43 6 2.91 An. gambiae (S) 5 2.43 4 1.94 3 1.46 11 5.34 8 3.88 An. Gambiae 2 0.97 2 0.97 6 2.91 10 4.85 11 5.34 hybrid (M/S) Total 12 25 37 60 46
[0161] PCR determined that there was no significant effect of treatment on the species of Anopheles caught, p=0.1718. This is illustrated in table 7.
[0162] Following the CSP ELISA, the sporozoite rate of infective anophelines was calculated as 0.97%. Of the Anopheles females with sporozoites detected, one was An. gambiae (S) under 1% Plas 5 treatment. The other was Anopheles melas and was under the hexane control treatment. The sample size of Anopheles was not large enough to be the basis for an accurate depiction of local sporozoite rates.
[0163] Culex:
[0164] Morphologically, only two of the ten morphological groups were able to be identified to species. These were Culex quinquefasciatus and Culex tritaenoiorhynchus. The remaining species are possible of non-medical importance as they could not be identified with the medical mosquito keys.
[0165] The results of the trapping experiment indicate an optimisation of attractant of Plas5 at about 1% (v/v) in MB5 in order to trap proportionally more Anopheles female mosquitoes than the MB5 blend alone. Plas5 may be used, for example in the range 0.5%-5% (v/v) in order to potentiate existing attractant blends to increase their attractiveness for Anopheles females.
REFERENCES
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