Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces.

The putative vector of trachoma, Musca sorbens, prefers to lay its eggs on human faeces on the ground. This study sought to determine whether M. sorbens females were attracted to volatile odours from human faeces in preference to odours from the faeces of other animals, and to determine whether specific volatile semiochemicals mediate selection of the faeces. Traps baited with the faeces of humans and local domestic animals were used to catch flies at two trachoma-endemic locations in The Gambia and one in Ethiopia. At all locations, traps baited with faeces caught more female M. sorbens than control traps baited with soil, and human faeces was the most successful bait compared with soil (mean rate ratios 44.40, 61.40, 10.50 [P<0.001]; 8.17 for child faeces [P = 0.004]). Odours from human faeces were sampled by air entrainment, then extracts of the volatiles were tested by coupled gas chromatography-electroantennography with laboratory-reared female M. sorbens. Twelve compounds were electrophysiologically active and tentatively identified by coupled mass spectrometry-gas chromatography, these included cresol, indole, 2-methylpropanoic acid, butanoic acid, pentanoic acid and hexanoic acid. It is possible that some of these volatiles govern the strong attraction of M. sorbens flies to human faeces. If so, a synthetic blend of these chemicals, at the correct ratios, may prove to be a highly attractive lure. This could be used in odour-baited traps for monitoring or control of this species in trachoma-endemic regions.

Introduction

The Bazaar Fly, Musca sorbens, is the putative vector of the blinding eye disease trachoma [1]. Adult M. sorbens feed on ocular and nasal secretions to obtain nutrition and liquid [2], and in doing so can transmit Chlamydia trachomatis, the bacterium that causes trachoma, from person to person. Chlamydia trachomatis DNA has been found on wild caught M. sorbens [3-5], and a laboratory study demonstrated mechanical transmission of Chlamydia psittaci between the eyes of guinea pigs by the closely related Musca domestica [6]. Strong evidence for the role of M. sorbens as a vector of trachoma comes from a cluster-randomised controlled trial that examined the impact of fly control interventions on trachoma prevalence [7]. Insecticide spraying significantly reduced the number of M. sorbens flies caught from children’s faces by 88%, and a 56% reduction in trachoma prevalence in children was observed. The provision of pit latrines, which by removing sources of open defecation controls M. sorbens juvenile stages, resulted in a 30% decrease in flies on faces and a 30% reduction in trachoma prevalence (non-significant).

These findings demonstrate that controlling the population density of M. sorbens may contribute to a decline in trachoma by reducing the number of fly-eye contacts, highlighting the disease-control potential of effective fly control tools. Odour-baited traps are receiving increased attention with regards to disease vectors, as the knowledge base around insect olfaction and attractive volatile chemicals expands [8-12]. Recent studies demonstrating the epidemiological significance of implementing chemical-based lures for vector-borne disease control [13] bolster the more widely accepted, and longstanding, tsetse fly example [14].

Mass deployment of an odour-baited trap for M. sorbens, based on the attractive volatiles in faeces, may suppress populations sufficiently to decrease the prevalence of trachoma. Alternatively, such traps could be used for entomological surveillance, and for monitoring and evaluating M. sorbens control programmes.

Female M. sorbens deposit their eggs on faeces, in which the larvae develop [15]. Previous studies have shown that M. sorbens preferentially oviposit in human faeces [2,16], and that adults emerging from human faeces are on average larger than those emerging from non-human faeces. This suggests that human faeces may be an optimal larval development medium [16]. More prolific emergence from human faeces could be a result of better larval survival within human faeces, and/or more oviposition in human faeces caused by its relatively greater attraction to female flies, or other unknown factors. It is common for insects to use semiochemicals, volatile airborne chemical signals, to locate resources such as oviposition sites, and to discriminate between resources of varying quality. It is therefore plausible that female M. sorbens visit human faeces more frequently relative to the faeces of other animals because of favourable semiochemical cues.

The aim of this study was to investigate the relative attractiveness of faeces from different mammalian species, including humans and local domesticated animals, to the Bazaar fly M. sorbens at two locations in The Gambia and one in Ethiopia. Using chemical ecology techniques, we then sought to identify putative M. sorben attractants in human faecal odours.

Methods

Study areas and study periods

The study was carried out in The Gambia and Ethiopia. In The Gambia, there were two sites, the village of Boiram, Fulado West, Central River Division, and the rural town of Farafenni. Studies were conducted in Boiram during the June-August 2009 rainy season, and in Farafenni in November-December 2009, immediately after the rainy season. The third site was Bofa Kebele (Oromia, central Ethiopia), identified as having highly prevalent active trachoma by the Global Trachoma Mapping Project. There, the study was conducted in February 2017 [17].

Ethics

The Gambian study was approved by the Joint Gambian Government/Medical Research Council Laboratories Joint Ethics Committee (protocol number L2010.90, re: L2009.67, 01/12/09), and the Ethiopian study by the LSHTM ethics committee (reference number 11979/RR/5821) and the Oromia Regional Health Bureau Ethics Committee. In the Gambian study, written informed consent was provided by the heads of all compounds in which traps were sited or from which faeces were collected. In the Ethiopian study, written informed consent was provided by all participants including guardians of children.

Trapping

Trap design

Fly traps were placed on the ground. The basic design of the ‘Bristow Trap’ was a lidded white plastic pot (Vegware, Edinburgh, UK) containing bait, with bait volatiles being released through a hole in the lid covered with mesh (Fig 1). Baits were 50 g of faeces, soil, or none (empty). The pot lid was made sticky to catch flies, by attaching circles cut from ‘sticky paper’ (Gambian study, “Yellow Sticky Trap” [Agrisense BCS Ltd, Pontypridd, UK]; Ethiopian study, “Yellow Sticky Trap” [Agralan, Wiltshire, UK]. A nylon (Gambian study, 0.4 mm gauge [Lockertex, Warrington]) or polyester (Ethiopian study, unknown gauge size [The Textile House, Huddersfield]) mesh was used to cover the hole to prevent fly entry. Traps were replaced daily, and a barrier of thorny acacia branches was placed in front of all traps to stop animals interfering with them.

Fig 1

‘Bristow Trap’—an odour-baited trap for Musca sorbens.

(A) Trap design and dimensions (hole diameter used in the two studies was different; Gambian study 3.2 cm, Ethiopian study 4 cm) (B) A fly trap ‘in situ’ in Ethiopia; (insert) typical fly catch on the yellow sticky discs on the trap.

In the Gambian studies only, the following modifications were used. A layer of glue (“Rat Stop”, 92% polybutene, 8% hexane [Taizhou Aiyon Adhesive Co., China]) was added to the top of the yellow disc to increase trap catch. A thin band of the same glue was applied to the side of each pot to prevent ants from gaining access to trap catch. To protect from rain, traps were placed inside wire frame boxes (15 cm x 15 cm x 15 cm [locally sourced]) with blue plastic sheeting (locally sourced) on the uppermost side and above the trap.

Collection of faeces bait

In the Gambian study, faeces (50 g; calf, cow, dog, donkey, horse, human and sheep) were collected for trap bait from open defecation in the compounds between 07:00 h and 11:30 h and weighed on a balance (Salter ARC 1066, accurate to ± 1 g). In Boiram, human faeces were obtained from two adjacent compounds, the children of which defaecated on the ground. In Farafenni, human faeces were obtained from a compound with five children between the ages of eight and 15 years, who defaecated in a plastic potty in the morning. Only fresh faeces (< 1 day old) were used in traps. Cow and calf faeces were collected from areas where cattle were confined at night. Cattle grazed in the bush, calves were under one-year-old and fed on milk. Dog faeces were unavailable in Boiram. Donkeys and horses were fed cous (millet), supplemented in the village by grazing. Their faeces were collected from family compounds. Sheep faeces were collected from shelters where the animals were kept overnight. Sheep in the village grazed in the bush, whilst those in town scavenged in the streets. Tobaski, a religious festival during which rams or sheep are slaughtered, occurred between the first and second experiments. Before Tobaski, sheep had their diets supplemented with milk, millet porridge or Senegalese feed blocks known as repas of unknown composition. After Tobaski the sheep had a less nutritious diet. Soil samples for control bait were taken from areas close to the trap site, but ensuring that no faeces were in the immediate vicinity (within 5 m). In the Ethiopian study, human adult and human child, cow and donkey faeces (50 g) were collected from the compounds where open defecation is commonplace, and weighed (Ascher Portable Digital Scale, accurate to 0.01 g).

Experimental design and data analysis

Traps containing faeces or soil bait were set daily, 50 cm apart, along a transect. Latin Square (LS) designs were used so that baits were rotated between trap positions daily, to prevent trapsite variation and bias, or adjacent trap bait, have any effect on trap catch. In the Gambian study, eight traps were set daily (seven faeces baits and a soil, with an empty pot instead of dog faeces in Boiram). The eight by eight LS was repeated twice at each site, giving 16 trap days per location. In the Ethiopian study, a transect of five traps (four faeces and one soil bait) was set on day one outside one household, and thereafter five-trap transects were deployed outside two (new) households daily until day six, giving a total of 11 trapping days per treatment (bait type). The position of the traps within each transect was rotated according to a LS design. Environmental variables were recorded as follows: Bioram, max/min ambient temperature, presence/absence of rain (thermometers hung nearby and daily observation of rain); Farafenni max/min ambient temperature and humidity (TinytagPlus datalogger, Gemini dataloggers); Oromia, start/finish ambient temperature, start/finish ambient humidity (Colemeter thermometer hygrometer humidity meter).

After 24 h, M. sorbens adhering to the sticky trap lids were identified according to taxonomic keys [18,19] and counted. In The Gambian study, M. sorbens were counted, females dissected for gravidity (carrying fully developed eggs), and Musca domestica were counted. In the Ethiopian study, M. sorbens were counted and sexed. Negative binomial regression, which accounted for the over-dispersed nature of trap catch data, was used to model the relationship between trap bait and the number of flies caught. The effect of trap bait on the likelihood that a trapped M. sorbens was female was analysed using logistic regression (analyses performed in Stata [v. 15, StatCorp], datasets available in S1 Data).

Chemistry of bait attraction

Collection of volatiles

Air entrainment of faeces samples was performed only during the Gambian study. Five human faeces samples were collected (50 g) from Farafenni and Boiram into individual sterile polyethyleneterephthalate (PET) cooking bags (Sainsbury’s Ltd, UK). Volatiles from the faeces were collected using a portable air entrainment kit (Barry Pye, Rothamsted Research, Harpenden). The bag containing the sample was sealed to an aluminium disc with air inlet and outlet holes using bulldog clips. The air entrainment kit, comprising an inflow and outflow pump and charcoal filters (VWR Chemicals BDH, 10–14 mesh, 50 g, preconditioned under a stream of nitrogen at 150 °C for a minimum of two hours), was connected to the bag and air inflow (16 L/min) set higher than outflow (2 x 7 L/min), creating positive pressure to prevent entrance of environmental volatiles. PTFE tubing and rubber ferrules were used for all connections. The apparatus was cleaned before and after use with ethanol (100%, Sigma-Aldrich, Gillingham, UK). Volatiles were collected in the outflow onto Porapak Q polymer (50 mg, mesh size 50/80, Supelco), contained inside a glass tube and held with two plugs of sterile silicanised glass wool (‘Porapak tubes’). These had been conditioned prior to use by repeated washing with redistilled diethyl ether and heating to 132 °C for 2 h under a stream of constant (charcoal-filtered) nitrogen. After 12 h of volatile collection, Porapak tubes were sealed in ampoules under filtered nitrogen for transport and storage. Ampoules were initially stored at the study site at 4 °C (for a maximum of six weeks), then in the UK volatiles were eluted from the Porapak tubes using freshly-distilled diethyl ether (‘extract’) and stored in vials at -20 °C until analysis.

Fly rearing

After counting and identification, live female M. sorbens were collected from the sticky traps with blunt forceps and placed into insect rearing cages (BugDorm, 32.5 x 32.5 x 32.5 cm) containing water (soaked into blue paper towelling) and white sugar cubes. Either human faeces (20 g presented on a small pot of damp soil to replicate the ‘naturalistic’ setting), or full fat Ultra-high temperature processing (UHT) milk (soaked into cotton wool) were given as an oviposition medium and protein source [20]. Every third day larvae were transferred to a larval diet medium: molasses sugar (8 g), dried yeast (7 g), full fat UHT milk (100 mL), water (200 mL) and wheat bran (added until the correct consistency was achieved). Cages containing artificial diet were kept indoors in The Gambia at 25–28 oC and 25–50% relative humidity (RH), while cages containing faeces were kept in natural daylight in a ventilated outbuilding, at 17–44 oC and 8–65% RH, with a roughly 12:12 light/dark photo-period. Six days after they were placed in the cages, two samples of artificial larval diet and one sample of faeces were removed from oviposition cages and the pupariae carefully exposed. Emerging adults were used for GC-EAG; rearing flies on artificial media was not sustainable over more than three generations.

Coupled gas chromatography-electroantennography (GC-EAG)

Musca sorbens females from the laboratory-reared colony were chilled on ice until movement ceased. The heads severed to kill the fly, and abdomens dissected to determine gravidity. One antenna was removed to reduce noise in the recording. The tip of the other antenna was cut off before inserting the antenna into a glass electrode containing a silver/silver chloride wire and filled with Ringer’s solution (7.55 g NaCl, 0.64 g KCl, 0.22 g CaCL2, 1.73 g MgCl2, 0.86 g Na2HCO3, 0.61 g Na3PO4/L distilled water). Another electrode was inserted into the back of the flies’ head to complete the circuit. This assemblage was held in a constant stream (1 L/min) of humidified, charcoal-filtered air. Air entrainment extracts were concentrated (from 750 to 50 μL) under a stream of charcoal-filtered nitrogen. A sample of extract (1 μL of a representative 50 μL concentrated 12-h air entrainment extract) was injected onto a 30 m non-polar polydimethylsiloxane (HP1) column (internal diameter 0.32 mm, solid phase thickness 0.52 μm) in a gas chromatograph (Hewlett Packard HP6890 with a cool-on-column injector, hydrogen carrier gas and flame ionisation detector). The following GC program was used: oven temperature maintained at 40 °C for 2 min, increased by 5 °C per min to 100 °C, then raised by 10 °C per min to 250 °C. Emerging compounds were delivered simultaneously to the flame ionisation detector and the airstream blowing over the antenna. The signal was amplified 10,000 times by the Intelligent Data Acquisition Controller-4, and signals were analysed by using EAD 2000 software (both Syntech, The Netherlands). Antennal responses were correlated visually to compound peaks by overlaying traces on a light box, and the procedure repeated for four female M. sorbens flies.

Coupled gas chromatography-mass spectrometry (GC-MS)

Compounds found to be electrophysiologically-active were identified by GC-MS only. One concentrated extract was diluted tenfold prior to injecting (1 μL) onto a HP1 column (dimensions as for GC-EAG; Hewlett HP 5890 GC fitted with a cool-on-column injector, helium carrier gas and FID, with a deactivated HP1 pre-column [0.53 mm ID]). The following program was used: oven temperature maintained at 30 °C for 5 min, increased by 5 °C per min to 250 °C. A VG Autospec double-focusing magnetic sector mass spectrometer (MS) using electron impact ionisation (70 eV, 250 °C) was coupled to the GC and data analysed using an integrated data system (Fisons Instruments, Manchester, UK). Compounds were identified using the NIST 2005 database of standards (NIST/EPA/NIH mass spectral library version 2.0, Office of the Standard Reference Data Base, National Institute of Standards and Technology, Gaithersburg, Maryland).

Results

In Boiram, a total of 1734 muscid flies were caught across all traps between 17th July 2009 and 7th August 2009. Of these, 382 were M. sorbens, 1046 M. domestica and 306 were unidentified Musca spp. All had a female majority (M. sorbens, 82.9%; M. domestica, 69.7%; unidentified Musca spp., 80.3%). In Farafenni, a total of 1899 flies were caught between 18th November 2009 and 10th December 2009, of which 1754 were M. sorbens and 145 M. domestica. Again, the majority of caught flies were female (M. sorbens, 96.5%; M. domestica, 86.2%). In Boiram and Farafenni, across all faeces bait types other than horse and sheep, more than 60% of females caught were gravid. In Oromia, a total of 152 M. sorbens were caught between 19th and 27th February 2017. Of those that could be sexed (96.1%), most were female (90.4%). Gravidity was not measured, and other flies/arthropods were not counted. The distribution of M. sorbens trap catches per day (24-hour period) by bait type are shown in Fig 2, S1 Table, S2 Table.

Fig 2

Male and female Musca sorbens caught by different faeces bait, in the three studies (boxes, median and interquartile range; points, outliers).

Trap days per bait are Boiram, n = 16; Farafenni, n = 16 (sheep n = 15); Oromia, n = 11.

In all three studies, the number of female M. sorbens caught was strongly associated with the type of trap bait used (P<0.001, Table 1), and human faeces was consistently and strongly the most attractive bait overall. When comparing female M. sorbens trap catch to the soil bait (as the baseline), however, the relative performance of different bait types (estimated rate ratios) varied substantially between studies. In Boiram, 44.4 times more female M. sorbens were caught in human faeces-baited traps (mean rate ratio [RR]; 95% confidence intervals [CI] 15.5–127.3, P<0.001), in Farafenni 61.4 times more (95% CI 32.3–117.0, P<0.001), and in Oromia 10.5 times more in traps baited with adult faeces (95% CI 2.5–43.5, P = 0.001) and 8.2 times more in traps baited with child faeces (95% CI 2.0–34.0, P = 0.004) (Table 1). Where measured, the strong preference for human faeces was not observed in M. domestica (S1 Fig).

Table 1

Female and male Musca sorbens caught by different baits at three study sites in The Gambia (Boiram and Farafenni) and Ethiopia (Oromia).

Soil and/or empty pots were used as negative controls, and the impact of climate (ambient temperature, rainfall/relative humidity) on trap catch assessed.

		Female Musca sorbens			Male Musca sorbens
BOIRAM		Rate ratio (95% CI)	P-valueA	P-valueB	Rate ratio (95% CI)	P-valueA	P-valueB
Bait	Calf faeces	4.60 (1.50–14.12)	0.008		0.80 (0.61–4.13)	0.790
	Cow faeces	2.20 (0.66–7.31)	0.198		1.00 (0.21–4.86)	1.000
	Donkey faeces	1.40 (0.39–5.04)	0.607		C
	Empty pot	1.40 (0.39–5.04)	0.607	<0.001	1.20 (0.26–5.60)	0.817	0.086
	Horse faeces	3.20 (1.01–10.15)	0.048		1.80 (0.41–7.82)	0.433
	Human faeces	44.40 (15.49–127.30)	<0.001		5.60 (1.43–21.96)	0.014
	Sheep faeces	5.20 (1.71–15.83)	0.004		1.60 (0.36–7.08)	0.536
	Soil	1.0			1.0
Climate	Av. tempD,E, continuous	1.15 (0.92–1.45)		0.225	1.45 (1.08–1.94)		0.014
	RainfallF,E, Yes	1.21 (0.40–3.62)		0.737	0.82 (0.21–3.21)		0.779
FARAFENNI
Bait	Calf faeces	2.00 (0.97–4.11)	0.059		0.33 (0.06–1.78)	0.198
	Cow faeces	1.70 (0.83–3.58)	0.145		0.50 (0.12–2.17)	0.355
	Dog faeces	20.70 (10.80–39.57)	<0.001		1.17 (0.35–3.85)	0.800
	Donkey faeces	1.20 (0.57–2.63)	0.607	<0.001	0.83 (0.23–3.00)	0.781	<0.001
	Horse faeces	3.70 (1.85–7.28)	<0.001		1.17 (0.02–1.46)	0.106
	Human faeces	61.40 (32.26–117.02)	<0.001		5.33 (1.97–14.47)	0.001
	Sheep faeces	2.50 (1.22–5.08)	0.012		0.89 (0.25–3.21)	0.857
	Soil	1.0			1.0
Climate	Av. tempG, continuous	1.11 (0.96–1.27)		0.150	1.26 (1.05–1.52)		0.015
	Av. RHF, continuous	1.02 (0.98–1.06)		0.294	1.02 (0.97–1.07)		0.412
OROMIA
Bait	Cow faeces	1.33 (0.20–6.35)	0.718		0.17 (0.02–1.42)	0.102
	Donkey faeces	1.00 (0.21–5.01)	1.000		0.33 (0.06–1.71)	0.189
	Adult human faeces	10.50 (2.54–43.48)	0.001		0.50 (0.12–2.09)	0.342
	Child faeces	8.17 (1.96–34.03)	0.004	<0.001	0.33 (0.06–1.71)	0.189	0.359
	Soil	1.0			1.0
Climate	Av. tempG, continuous	0.89 (0.70–1.14)		0.366	1.48 (1.08–2.04)		0.015
	Av. RHF, continuous	1.01 (0.96–1.07)		0.721	1.09 (1.02–1.16)		0.007

A P-value comparing this category with baseline (soil)

B P-value testing hypothesis that bait is associated with number of flies trapped

C Value could not be estimated as no male Musca sorbens caught in traps baited with donkey faeces

D Adjusted for rainfall

E Based on 13 days data (two days missing)

F Adjusted for average temperature

G Adjusted for average RH

For non-human faeces baits, in Farafenni, only dog, horse and sheep faeces caught more female M. sorbens than soil-baited traps (dog, RR 20.7, 95% CI 10.8–39.6, P<0.001; horse, RR 3.7, 95% CI 1.9–7.3, P<0.001; sheep, RR 2.5, 95% CI 1.2–5.1, P = 0.012 Table 1). In Boiram, only traps baited with horse, sheep and calf faeces were more attractive than soil-baited traps (RR 3.2, 95% CI 1.0–10.2, P = 0.048; RR 5.2, 95% CI 1.7–15.8, P = 0.004; RR 4.6, 95% CI 1.5–14.1, P = 0.008 respectively, Table 1). Dog faeces were not tested in Boiram nor Oromia. In Oromia no evidence was found of a difference in catch rates between the non-human faeces baits (cow or donkey) and the soil bait (P>0.05).

Human faeces were not only more attractive than soil bait, but were found to be more attractive than the second most attractive bait at each site (P<0.001). In Boiram, human faeces-baited traps caught 8.5 times as many female M. sorbens (RR; 95% CI 4.2–17.2) than sheep faeces-baited traps. In Farafenni, human faeces-baited traps caught 3.0 times more female M. sorbens (RR; 95% CI 1.9–4.7) compared to dog faeces-baited traps, and in Oromia, 7.9 (RR; 95% CI 2.0–30.8) and 6.1 (RR; 95% CI 1.6–24.1) times more were caught by human and child faeces-baited traps than cow faeces-baited traps.

In all studies, there were increased odds that M. sorbens caught by human faeces bait relative to those caught by soil bait would be female (Boiram, odds ratio [OR] 7.93 [95% CI 2.16–29.1] P = 0.002; Farafenni, OR 11.52 [95% CI 4.29–30.96] P<0.001; Oromia adult, OR 14 [95% CI 3.63–53.99) P<0.001; Oromia child, OR 32.67 [95% CI 5.59–191.06] P<0.001; S3 Table). Similarly, increased odds for female M. sorbens being caught were observed for dog and horse faeces baits in Farafenni, and cow faeces bait in Oromia (S3 Table). Generally, fewer male M. sorbens were trapped than females. In Boiram and Farafenni, only human faeces attracted more males than the soil control (P<0.05); this was not observed in Oromia (P>0.05, Table 1). Although it appears that males were possibly more affected by climatic variables (average ambient temperature affecting trap catch at each study site, P<0.05; ambient relative humidity affecting male catch at Oromia, P<0.05 [not measured at other sites], Table 1), low male catch rates may have affected these estimates (total males caught: Bioram, 65; Farafenni, 61; Oromia, 14).

Coupled gas chromatography-electroantennography

Twelve compounds from the headspace of the human faecal sample elicited an antennal response from two or more female M. sorbens. These compounds were subsequently tentatively identified by GC-MS as 3-ethylpentane, 2-methylpropanoic acid, butanoic acid, pentanoic acid, hexanoic acid, cresol, 2-phenylethanol, valerolactam, dimethyl tetrasulphide, indole, 2-dodecanone and an unidentified cholesterol derivative (Table 2).

Table 2

Twelve compounds in the odour of a human faeces sample elicited an electrophysiological response from female Musca sorbens.

These were tentatively identified by coupled gas chromatography-mass spectrometry.

Peak number	Compound identified
1	3-Ethylpentane
2	2-Methylpropanoic acid
3	Butanoic acid
4	Pentanoic acid
5	Hexanoic acid
6	Isomer of cresol
7	2-Phenylethanol
8	Valerolactam
9	Dimethyl tetrasulphide
10	Indole
11	2-Dodecanone
12	Cholesterol derivative

Discussion

We found evidence that M. sorbens, the putative vector of trachoma, is strongly attracted to odours produced by human faeces, which attracted and caught the greatest number of M. sorbens in all three studies. This was particularly so with female M. sorbens relative to males. In the Gambian studies, the majority of females were found to be gravid. These results corroborate previous studies finding that female M. sorbens are attracted to faeces for oviposition [2,15,21]. They further demonstrate that volatile cues alone are responsible for this attraction, as the faeces baits were only visible to flies if they were very close, directly above, and could see down through the mesh. A previous study conducted in The Gambia [16] demonstrated attraction to human faeces, but because the faeces were not hidden from the flies, visual cues could not be ruled out as a stimulus. Traps with no faeces bait (soil control and empty pot) consistently caught very few flies, suggesting that the volatiles alone were responsible for (faeces-baited) trap attractiveness, and that neither adjacent traps, nor nearby faeces sources, influenced trap capture. In the current study, there was also evidence that some of the non-human faeces baits were relatively more attractive than others (i.e. calf and sheep in Boiram attracted more female M. sorbens than the soil control, as did dog and horse in Farafenni). The high rate ratio for female M. sorbens caught using dog faeces in Farafenni (the only site with dogs) may indicate a preference for the faeces of non-herbivores. The mean rate ratio of M. sorbens females caught using human faeces relative to the soil control varied between the three sites, but in all cases a large effect was seen. The Gambian studies were conducted in two different ecological settings and at different times of the year, making it impossible to account for the differences in fly abundance between sites. At the Oromia site, trap catch may have been removed by birds or other insectivores as no protective wire frames were used. Even more so, the environment (including local abiotic and biotic factors) in Ethiopia is so different to that in The Gambia that comparison of population density between studies, and based on a small sampling window, would not be appropriate. As well as differences between local conditions and sampling timeframes precluding meaningful comparisons of trap catch between study sites, further limitations of this trapping work are as follows. The same faeces types were not available at each study site, therefore the considerable attractiveness of dog faeces observed in Farafenni was not tested at any other site. Faeces freshness was not taken into consideration; although all faeces baits were collected as fresh as possible, freshness would have varied between samples and may account for, or contribute to, differences in attractiveness seen. The volatile compounds (odour) released by the faeces bait would constitute a stronger signal the fresher the faeces sample, with fresh samples most likely eliciting a stronger attraction response from flies. Future studies could estimate the longevity of attractiveness by measuring faecal moisture content as a proxy for freshness.

Differential attraction to different types of faeces could be due to the presence or absence of certain semiochemicals that attract or repel the flies. Electroantennography was used to determine whether there are chemicals in the odour of human faeces that the antennae of M. sorbens detect and respond to. Importantly, the reported antennal responses do not inform of the nature of the response, only indicating that the fly detects the chemical. For this reason, EAG studies should be followed by robustly designed behavioural bioassays, able to differentiate between attractive and repellent compounds. Given the significant numbers of female M. sorbens caught in the three trapping studies, and the high proportion of gravidity observed where measured, we speculate that some or all of the compounds identified here as EAG-active serve as oviposition attractants to female flies. Of the M. sorbens EAG-active compounds tentatively identified here, short chain fatty acids (SCFA, including 2-methyl propanoic acid, butanoic acid, pentanoic acid and hexanoic acid), indoles, cresols and sulphur compounds are known volatile organic compounds in faeces [22-25]. SCFA are produced in the gut by bacterial fermentation of carbohydrates and proteins [26], and are commonly found in vertebrate-associated volatiles, including urine and faeces [22,27-30]. Their detection by host-seeking arthropods is well documented [31-35]. The aromatic compounds identified (cresol, 2-phenylethanol, indole) are likely to be fermentation products of the aromatic amino acids tyrosine, phenylalanine and tryptophan [24]. All GC-EAG replicates were conducted using the same human faeces odour extract, chosen as a ‘representative’ sample. This does not negate the importance of the compounds identified as eliciting a response. However, it could be that the sample omitted other compounds that are important in the detection of human faeces by M. sorbens, or indeed contained compounds that are not commonly present in faecal samples. This could be resolved in future studies by creating an odour ‘blend’ of extract for testing, combining odour from several human faeces samples.

Several of these faeces-associated volatiles have been described in similar entomological studies involving other higher Dipterans. Nine compounds in pig faeces volatiles elicited antennal response in M. domestica; butanoic acid and indole both eliciting strong responses in subsequent dose-response EAG [36]. Both m- and p-cresol in the headspace of canine faeces elicited antennal response in the green bottle fly, Lucilia sericata, and a chemical blend including these compounds was as attractive to flies as the faeces [37]. Cresols (isomer not identified) in volatiles from rat carrion also elicited antennal response from L. sericata [38], and female and male M. domestica responded (by EAG) to volatiles including butanoic acid, hexanoic acid, 2-phenylethanol and p-cresol in the headspace of vinegar [39]. Similarly, butanoic acid and p-cresol were among chemostimulants of Stomoxys calcitrans detected in the headspace of rumen volatiles, and were also found to elicit activation and attraction in a wind tunnel [40]. Microbial degradation is thought to lead to the production of m- and p-cresol in cattle urine, again found to elicit an EAG response in S. calcitrans [41]. Stomxys calcitrans is a muscid fly with coprophagous larvae, known to be attracted to faecal odours. This fly has been shown to select faeces by their odour [42], as demonstrated here with M. sorbens, and the chemostimulant compounds thought to be responsible for that attraction included butanoic acid, indoles, p-cresol and sulphides.

Taken together, the faecal semiochemicals described here are commonly isolated and/or detected because they are products of bacterial decomposition. As such, they are frequently detected by filth flies, most likely as cues for oviposition sites. To underpin the specific attractiveness of human faeces to M. sorbens therefore, either variation in amounts emitted, in the ratios of compounds present, or the presence of further compounds not identified in this study, must distinguish human faeces as a preferable oviposition medium.

Conclusion

Our study demonstrates that female M. sorbens at three different study locations, in both West Africa and East Africa, are preferentially attracted to the volatiles of human faeces, as evidenced by attraction in the absence of visual cues. We provide evidence that twelve compounds are putative attractants that may play a role in this response, by identifying, for the first time, compounds including short chain fatty acids and aromatic compounds that are detected by the antennae of M. sorbens. Further work is required to optimise chemical blends and release rates, to produce a synthetic lure to which the behavioural responses of M. sorbens can be investigated. Establishing those with attractive properties may lead to the design of baits for odour-baited traps, which could be used for M. sorbens surveillance or even population suppression or control.

Median female Musca sorbens/trap/24-hours (IQR).

(DOCX)

Click here for additional data file.

Median male Musca sorbens/trap/24-hours (IQR).

(DOCX)

Click here for additional data file.

Odds of Musca sorbens caught by different bait types being female.

(DOCX)

Click here for additional data file.

Musca sorbens trapping dataset (raw).

(XLS)

Click here for additional data file.

Male and female Musca domestica caught by different faeces bait, in the two studies in The Gambia (boxes, median and interquartile range; points, outliers).

Trap days per bait are Boiram, n = 16; Farafenni, n = 16 (sheep n = 15).

(TIF)

Click here for additional data file.

23 Nov 2019

Dear Dr Robinson:

Thank you very much for submitting your manuscript "Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces" (PNTD-D-19-01361) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file ); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) Appropriate Figure Files

Please remove all name and figure # text from your figure files upon submitting your revision. Please also take this time to check that your figures are of high resolution, which will improve both the editorial review process and help expedite your manuscript's publication should it be accepted. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plosntds/s/figures

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We hope to receive your revised manuscript by Jan 22 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit your revised files, please log in to https://www.editorialmanager.com/pntd/

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Jeremiah M. Ngondi, MB.ChB, MPhil, MFPH, Ph.D

Associate Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Objectives could have been more strongly stated. A trapping system was developed to capture adult flies and flies were reared to produce adults for the GC-EAG studies. Description of traps is confusing. One complete trap design should be described, then the modifications made to arrive at the next design and so on. Line 165 - sounds like the yellow sticky trap is really a yellow disc coated with adhesive, but do not know. Lines 166-167 - Nylon and polyester mesh used to prevent entry of flies into the traps, correct? Is there a mesh size to be used? Line 170 - which company manufacturers Rat Stop? Lines 172-173 - Wire frames. Made from individual wires, pieces of wire fence or what? Coated in black electrical tape? Size of frames given in cubic cm. This can be many shapes. Need length, width and height. Discussion of feces collections do not strongly state how fresh the feces must be to qualify for the studies. Line 196 - soil from uncontaminated areas. Contaminated with what? Collected near trap sites. How near or far? Moisture content of feces would be interesting to know. Line 201 - traps were separated by 50 cm. This seems a bit close. Line 227 - was the portable air entrainment kit made by the authors, or a commercial model? Fly rearing paragraph could be more clearly stated. Line 252 - need dimensions of BugDorms, not cubic cm. How many BugDorms and how many flies in each? Line 253 - what is blue roll? Lines 253-254 - on a small pot of damp soil? Lines 264-266 - run on sentence. Actual photos of the traps would be better than the casual drawings. Was GC-EAG done on only 4 flies?

Reviewer #2: (No Response)

Reviewer #3: Well done authors!

This is an important piece of work which is well over-due. The objectives and methods are clearly stated and described and the interpretation appropriate. This represents a HUGE amount of work!!

I have a couple of minor comments and noticed a few typos

Major comments:

None

Minor comments:

1) In the title and throughout the ms the authors use the term "putative trachoma vector". I suggest removing the word 'putative' as there is no longer any reasonable question as to whether M. sorbens is a trachoma vector. M. sorbens is a mechanical vector of trachoma, it does not however, have an obligatory role in the transmission of trachoma as Aedes mosquitoes do in the transmission of human Yellow Fever and Dengue. It would be worth mentioning this in the introduction. The relative importance of eye-seeking flies in trachoma transmission will likely vary in time and space. Controlling eye-seeking flies will not block trachoma transmission. Stopping fly-eye contact will not block trachoma transmission.

2) The findings of the work do not identify the role of the electrophysiologically active semio-chemicals identified. It is reasonable to assume that they are attractants for oviposition, since their presence was associated with a convincingly high proportion of gravid female sorbens. However, the presence of an electrophysiological response does not in itself predict behaviour. The compounds may act individually as attractants or repellents, or in combination elicit a different response. This should be the focus of future work

Typos and other nit-picking:

Lines 127-130: Starting 'It is unknown however, ..." the sentence is presented as an 'either/or' construction, when both are reasonable suggestions. I would suggest constructing the sentence to be 'The more prolific emergence could be a result of better larval survival, more eggs being laid as a result of its greater relative attractiveness to ovipositing flies or other factors'. For me I'd continue to consider it as the preferred and 'better' medium for larval development due to the size of the flies emerging after pupation.

Line 145: Repetition of The Gambia

Line 217: Suggest defining gravidity for the lay-audience and the specific context. It is typically used to mean 'mated with developed eggs', was the stage of gravidity estimated?

Line 300: Any idea what the 'unidentified Musca spp' were in Oromia? That's intriguing. Any idea if these unidentified species are caught from human eyes too?

Line 306: Do you mean 'per night' or per 24hr period?

Line 379: "...a preference for non-herbivore faeces." made me chuckle. Surely the preference is for "the faeces of non-herbivores"?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results are weakly stated with relation to the stats. Lines 302-303 - For feces baits other than horse and sheep, > 50% of flies collected were gravid. Lines 303-305 - run on sentence. Too long. Figure 2 might be more effective in tabular form with means and standard deviation show along with letters indicating statistical separation of means. Tables need more complete titles.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Data basically support conclusions. Limitations of analysis not mentioned. Authors discuss how data might be helpful. Authors do not mention the possibility of feces management. This might be difficult to impossible, but people need to know that these are sources of the flies that are transmitting disease to them and their children. Lines 384-387 and lines 405-408 - run on sentences. Too long.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The paper could use a thorough revision for clarity and organization. I think the science is basically ok, but the paper can be difficult to read in some places. I have tried to attach a scan of the paper so more of my comments can be seen. I would call this revision minor.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study is a very simple one, but very important for determining the breeding materials used by this fly. Also the evaluation of chemical compounds found in the feces might be developed into efficacious baits. The sentence structure needs to be tightened up and things more clearly stated.

Reviewer #2: This is an excellent paper overall. The writing is impeccable. With the exception of a single typographical error on line 202 (bais for bias) I did not find any errors throughout the manuscript.

The work is important, and the degree of replication in different study sites sufficient to make the results quite convincing.

A few minor comments for the authors consideration:

1) A photograph of the assembled trap would be helpful, either instead of or supplemental to the schematic drawing.

2) Most of the flies collected from Boiram were M. domestica. Although the focus of the paper was on M. sorbens, it would be interesting to see the collection results for domestica as well. How were the house fly collections distributed? Was the pattern substantially different from that of M. sorbens?

3) The climate information is mentioned in the Methods but never mentioned again except for some entries in Table that I was unable to decode. Suggest removing the climate data or discussing it in the context of the collection data.

4) The number of flies examined by EAG seems rather small. Although I have no expertise with this type of work I wonder whether an EAG response by two flies is sufficient to make conclusions about the attractiveness of the chemical constituents.

Reviewer #3: (No Response)

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: Reviewed paper.pdf

Click here for additional data file.

7 Jan 2020

Submitted filename: robinson_rebuttal_20191213.docx

Click here for additional data file.

13 Jan 2020

Dear Dr Robinson,

We are pleased to inform you that your manuscript, "Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please note: your manuscript will not be scheduled for publication until you have made the required changes.

IMPORTANT NOTES

* Copyediting and Author Proofs: To ensure prompt publication, your manuscript will NOT be subject to detailed copyediting and you will NOT receive a typeset proof for review. The corresponding author will have one final opportunity to correct any errors when sent the requests mentioned above. Please review this version of your manuscript for any errors.

* If you or your institution will be preparing press materials for this manuscript, please inform our press team in advance at plosntds@plos.org. If you need to know your paper's publication date for media purposes, you must coordinate with our press team, and your manuscript will remain under a strict press embargo until the publication date and time. PLOS NTDs may choose to issue a press release for your article. If there is anything that the journal should know, please get in touch.

*Now that your manuscript has been provisionally accepted, please log into EM and update your profile. Go to http://www.editorialmanager.com/pntd, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

*Note to LaTeX users only - Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines [http://www.plosntds.org/static/latexGuidelines.action] in the meantime.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Jeremiah M. Ngondi, MB.ChB, MPhil, MFPH, Ph.D

Associate Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

27 Feb 2020

Dear Dr Robinson,

We are delighted to inform you that your manuscript, "Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases