G Model ACTROP-3354; No. of Pages 7
ARTICLE IN PRESS Acta Tropica xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia E.C.J. Sulston a , A.H. Wardhana b , M.J.R. Hall c , J.G. Logan a , S.A. Gezan d , M.M. Cameron a,∗ a
Department of Disease Control, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK Parasitology Department, Indonesian Research Centre for Veterinary Science (Balai Besar Penelitian Veteriner), Bogor, Indonesia Life Sciences Department, Natural History Museum, London SW7 5BD, UK d School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611, USA b c
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Chrysomya bezziana Old World screwworm fly POC Bezzilure Attractant Indonesia
a b s t r a c t The myiasis fly Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) is an obligate parasite of body orifices and unnatural openings or wounds of vertebrates. It is a major pest of livestock and responsible for economic losses throughout the Old World. Two chemical lures were tested in the present study to assess their effectiveness at trapping Ch. bezziana. Bezzilure B contains a synthetic lure of 2-mercaptoethanol originally identified from cattle wound volatiles. POC is a synthetic lure comprising 4-methyl phenol, 3-propyl phenol and racemic 1-octen-3-ol, originally identified from volatiles of ox odour and now used to control tsetse flies (Diptera: Glossina). Bezzilure B and POC lures were tested alone, and in combination, in a laboratory free flight room bioassay and in a field trial. Sticky traps were used to capture males and females of Ch. bezziana and to determine the efficacy of the lures. In the laboratory, Bezzilure B caught significantly more male and female flies than Bezzilure B + POC (p < 0.001). Similarly, in the field, treatment significantly affected the total number of Ch. bezziana caught (p < 0.001) but, contrastingly, Bezzilure B + POC caught significantly more flies than Bezzilure B (p < 0.05). The differing results between the laboratory and field may simply reflect the different assays and could suggest different roles of semiochemicals at different operational distances. Although ineffective on its own, POC has a synergistic effect with Bezzilure B to increase fly catches in the field: by proportion, Bezzilure B + POC was 2.17 times as efficient as Bezzilure B for Ch. bezziana, and 1.16–2.68 times as efficient for other flies depending on the species collected. © 2014 M.M. Cameron. Published by Elsevier B.V. All rights reserved.
1. Introduction Old World screwworm fly (OWSF), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), causes traumatic myiasis in subcutaneous tissue of body orifices and wounds of live mammals (Hall, 1995). Ch. bezziana is widespread throughout regions of Africa and Asia, including Indonesia (Wardhana et al., 2012). Although OWSF can affect humans (Ahmadi et al., 2009; Chan et al., 2005; Romero-Cabello et al., 2010; Sharma and Hedge, 2010), particularly the young and elderly (Stevens et al., 2006), human infections are rare. Arguably of more significant importance are infections of livestock due to the significant animal welfare issues and economic losses (Spradbery, 1994) and the potential for invasions and
∗ Corresponding author. Tel.: +44 0 20 7927 2337; fax: +44 0 20 7927 2918. E-mail address: [email protected] (M.M. Cameron).
consequent losses in new areas, for example if OWSF invades Australia (Anaman et al., 1994; Atzeni et al., 1997; Robinson et al., 2009; Sutherst et al., 1989). Trapping screwworm flies plays a valuable role in surveillance of invasions, and for monitoring of eradication programmes, such as the Sterile Insect Technique (SIT) (Spradbery et al., 1989; VargasTeran et al., 2005), particularly in the New World for Cochliomyia hominovorax (Krafsur et al., 1987). In the past, traps used liver as bait (Coppedge et al., 1977; Laake, 1936), but great effort has since been made to develop synthetic lures for operational use to ease handling, maintain attractiveness over time, increase the sensitivity of catches and to maximise the numbers of males collected. The first synthetic lure, Swormlure, developed to catch Co. hominovorax, contained 10 volatile compounds derived from degradation products of animal proteins and fatty acids that are likely to be found in wounds (Jones et al., 1976). This was later modified by
http://dx.doi.org/10.1016/j.actatropica.2014.04.017 0001-706X/© 2014 M.M. Cameron. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model ACTROP-3354; No. of Pages 7 2
ARTICLE IN PRESS E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
adding methyl disulfide in an improved formulation, Swormlure-2, that required two rather than three dispensers, and was adopted as the synthetic lure for use in the Screwworm Eradication Program for Co. hominovorax in the USA (Coppedge et al., 1977). Further modifications, regarding the relative concentrations of the 10 compounds in different blend formulations, led to the development of Swormlure-4, which improved catches of Co. hominovorax (Mackley and Brown, 1984). Subsequent research has identified 25 electophysiologically-active compounds found in wounds infected with Co. hominovorax (Cork, 1994), which were shown to significantly attract females but not males in the field (Cork and Hall, 2007). However, less attention has been focussed on the development of synthetic lures specifically for OWSF, although LuciLure, a synthetic blend derived from wound odours and containing 2mercaptoethanol, developed to attract Lucilia sericata, caught Ch. bezziana at levels comparable to a liver standard (Urech et al., 2004). The LuciTrap (Bioglobal Pty Ltd., Australia) baited with Lucilure for the control of L. sericata in Australia, included a small proportion (9.3%) of Chrysomya sp. and Calliphora sp. in collections (Urech et al., 2009). More recently, attractant formulations based on Swormlure and Lucilure, Bezzilure and/or Bezzilure-2, have been tested against field populations of Ch. bezziana using either sticky traps and/or LuciTraps in Indonesia and Malaysia (Urech et al., 2012; Wardhana, 2010). In Indonesia, where only baited sticky traps with Bezzilure were used, low catches (0.32% of the total catch) of Ch. bezziana were obtained compared with other myiasis species (Wardhana, 2010). In Malaysia, where the relative efficacies of different combinations of lure (Bezzilure or Bezzilure-2) and trap (sticky or LuciTraps) were tested, the optimal combination for selectively collecting higher numbers of Ch. bezziana, in relation to nontarget flies, was the LuciTrap/Bezzilure-2 system (Urech et al., 2012). Although mean numbers of Ch. bezziana in Bezzilure-2baited LuciTraps were relatively high compared with other field studies (8.0 flies/trap) (Urech et al., 2012), there is scope for further improvements to synthetic lures for the detection of Ch. bezziana at sites with low densities of OWSF. Although LuciTraps allow for specialised attraction of Ch. bezziana, they require an additional exploratory behaviour to catch flies. Sticky traps represent an efficient means of examining off target effects of the chemical lures against other myiasis causing agents which may not exhibit the behaviour necessary to be caught by LuciTraps. In a widespread survey looking for active cases of Ch. bezziana in Indonesia, 65% (167/255) of hosts infested by larvae were cattle, followed by goats (22%), and, based on livestock census data, cattle were more likely to be infested by OWSF than goats (Wardhana, 2010). If Ch. bezziana prefer cattle hosts, it is possible that they may respond to synthetic cattle-derived attractants. During the 1980’s several volatile chemicals from ox odour were identified as attractants to tsetse flies such as carbon dioxide, 1-octen-3-ol and acetone (Hall et al., 1984; Vale and Hall, 1985). Since then, many laboratory and field studies have been performed to isolate volatile components of ox odour and optimise lures for attracting tsetse flies. Of these, two refined blends, POC (P = 3-n-propylphenol, O = racemic1octen-3-ol, C = 4-methylphenol) and POCA (POC + A = acetone), significantly increased catches of Glossina palpalis gambiensis without using carbon dioxide, which can be expensive for large-scale use in surveillance programmes (Rayaisse et al., 2010). The aim of the present study was to compare the efficacy of two different types of lure, a wound lure (Bezzilure B) and a cattlederived lure (POC), alone and in combination, for capturing Ch. bezziana.
2. Materials and methods 2.1. Study area: Biogeographical regions, climate and land cover The laboratory based bioassays were conducted at the Indonesian Research Centre for Veterinary Science (IVETRI), Bogor, Java. The field study took place in two study sites in the province of Kediri in East Java (−07◦ 54.640 , 112◦ 01.411 and −07◦ 56.105 , 112◦ 03.605 ) between July and August 2011. 2.2. Fly colony for laboratory bioassays The colony of Ch. bezziana originated from wild samples collected from Bogor in 1994, which was refreshed with introductions from populations collected from Sumba and Sulawesi in 2002. Although the laboratory flies were originally collected from different field sites and populations from Bogor in West Java to the islands of Sumba and Sulawesi to the East of Java, and the flies sampled in the field tests were from East Java, all flies belonged to the same cyt b sub-lineage (2.2) (Wardhana et al., 2012). Flies were kept at 28 ◦ C and RH:75% in a 12:12 h photocycle. Approximately 10,000 flies were reared each week using a standard protocol (Sukarsih et al., 2000). 2.3. Dispensing mechanisms and chemical lures A stock solution of 600 ml of Bezzilure B was produced as reported previously (Urech et al., 2012) and was distributed between 18 white 30 ml HDP plastic bottles with a cotton wick 0.5 cm in diameter and 10 cm long. Lures were stored at 5 ◦ C with a securely fastened cap and placed in a plastic zip lock bag to minimise loss between experiments. Similarly, POC was produced as previously described (FAO/IAEA, 2003), and 1 ml was dispensed from a cotton wick contained within a polyethylene sachet (5 cm2 ) of 0.15 mm thickness. POC sachets were heat sealed inside individual aluminium plastic bags to minimise loss between experiments. Release rates of lures were determined from the room bioassay experiments by weighing the lures at the start and end of each experimental day to calculate the change in weight (release rate) per hour. 2.4. Experimental procedure and set up for laboratory bioassays Modified pot traps, as developed by Julie Bristow, LSHTM (pers. comm) were used. These consisted of a high density plastic paint pot (Leyland® SDM, UK), top diameter 15 cm, bottom diameter 10 cm, height 7 cm, with a lid (diameter 15 cm). A hole was cut out of the lid (3 cm diameter) and a circle of nylon mesh (3 mm gauge) was attached to the inside of the lid to prevent flies from accessing the bait. Yellow sticky card (Agrisense BCS Ltd, UK) was then cut to the size and shape of the lid. The card was sticky on both sides; one used to attach to the lid and one used to trap flies. Lures were placed inside the pots, and the baited raps were placed on two stands at 1.2 m above the ground in a sealed experimental room measuring 5.35 m × 4.4 m × 2.85 m. They were positioned at two points to ensure equal distances to the air inlet pipes, light levels and distance from the fly release position. An extraction fan opposite the entrance allowed for the air to be refreshed between replicates. Effectiveness of the lures was determined by offering flies a choice of attractant in the free flight room. Flies were exposed to pairs of traps containing either: (A) Control or Control; (B) Control or POC; (C) Control or Bezzilure B; (D) Control or Bezzilure B + POC; and (E) Bezzilure B or POC. The pairings of exposure were tested using randomised 5 × 5 Latin square designs (5 treatment pairs, 5 periods of exposure, 5 days) repeated three times to account for
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model ACTROP-3354; No. of Pages 7
ARTICLE IN PRESS E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
site, period and day effects. For each replicate, three hundred flies (150 female, 150 male) were released from a plastic tray at a fixed point in the room and exposed to the two treatments for 30 min. The traps were then removed and male and female flies caught on each trap were recorded. Flies remaining in the room were collected using a vacuum cleaner and removed. The process was repeated for the next random exposure pairing with a new set of previously unexposed flies. 2.5. Experimental procedure for field trials Two transects were constructed (07◦ 56.105 S 112◦ 03.605 E to 07◦ 56.173 S 112◦ 03.593 E and 07◦ 54.640 S, 112◦ 01.411 E, to 07◦ 54.641 S, 112◦ 01.484 E) with locations for four traps marked 50 m apart, a distance used in previous studies testing baited traps and targets (Coppedge et al., 1977; Cork and Hall, 2007). Sites for transects were selected on the requirements of being close to farms ( 0.05) and therefore were dropped from the models. To calculate the comparative potency of Bezzilure B versus Bezzilure B + POC for collecting different species of field-caught flies, mean catches using Bezzilure B + POC were divided by mean catches using Bezzilure B alone and expressed as a proportion. Similar comparisons for control and POC treatments could not be performed, as there were too many zero values (non-trapped insects) for most treatments, providing limited information.
3. Results 3.1. Laboratory bioassay The mixed linear model showed that treatment was a significant factor influencing the total catch of Ch. bezziana (Wald = 747.42, F = 248.86, p < 0.001). Bezzilure B + POC caught significantly more flies than POC alone (p < 0.001); and POC caught significantly more flies than the control (p < 0.001) (Fig. 1). The data were also used to examine the effect of sex on attractiveness to the lures by fitting the same model to each sex separately. Treatment was found to be significantly different for both females (Wald = 440.79, F = 146.76, p < 0.001) and males (Wald = 212.54, F = 70.76, p < 0.001), with Bezzilure B catching significantly more flies than Bezzilure B + POC (p < 0.001 and p < 0.05, respectively). Release rates were calculated during the first room bioassay, using the average change in mass for each lure over each day. The release rates of Bezzilure B were, on average, 8 times larger than that of POC. The mean release rates for all lures are displayed in Table 1.
3.2. Field study catches of Ch. bezziana The mixed linear model showed that treatment was a significant factor affecting the total catch of Ch. bezziana (Wald = 23.34, F = 7.78, p < 0.001). Furthermore, Bezzilure B + POC caught significantly more Ch. bezziana than Bezzilure B (p < 0.05; Fig. 2). However, this effect was only significant for female flies (Wald = 31.78, F = 10.59, p < 0.001) and not for males (Wald = 5.19, F = 1.73, p > 0.1). Neither POC nor the control group caught any Ch. bezziana. When comparing the potency of Bezzilure B + POC versus Bezzilure B for collecting Ch. bezziana, Bezzilure B + POC was 2.17 times as efficient as Bezzilure B.
Table 1 Mean release rates in mg/hr of the lures over all three replicates of the first room bioassay. Treatment
Mean release rate (mg/h)
95% CI
POCa POCb Bezzilure Ba Bezzilure Bb
0.354 0.339 2.15 2.85
0.2672–0.4406 0.1772–0.5008 0.5002–3.7912 0.3560–5.3538
a b
Represents lures used independently. Represents lures that were used together for the POC + Bezzilure B treatment.
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model
ARTICLE IN PRESS
ACTROP-3354; No. of Pages 7
E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
4
10 Arithemec Mean Catch
9 8 7 6 5
Females
4
Males
3
Total
2 1 0
Control
POC
Bezzilure + POC
Bezzilure
Treatment Fig. 1. Mean number of flies caught per treatment (with least significant differences at the 1% significance level for females and total, and 5% significance level for males). A total of 300 flies (150 females and 150 males) were used per replicate in the laboratory bioassay.
3.3. Field study catches of other myiasis causing agents
4. Discussion
Over the 12 days of trapping in the field, a total of five additional non-target genera or species relating to myiasis were caught that were either facultative (Ch. megacephala, Ch. rufifacies, Hemipyrellia, Sarcophaga) or accidental (Musca domestica) agents. The mean daily catch of each genus/species caught by each treatment is shown in Table 2. The treatment that caught the greatest number of flies was Bezzilure B + POC (5229 flies). The dominant genus caught was Hemipyrellia (5901 flies). After fitting a model for total catches to each individual genus/species, treatment was found to be a significant factor, with Bezzilure B + POC catching more flies than Bezzilure B, for Hemipyrellia (Wald = 95.66, F = 31.89, p < 0.001), Ch. megacephala (Wald = 126.55, F = 42.18, p < 0.001), Ch. ruififacies (Wald = 62.73, F = 20.91, p < 0.001), Sarcophaga (Wald = 166.19, F = 55.40, p < 0.001), but the contrary was true for Musca (Wald = 21.03, F = 7.01, p < 0.001). When comparing the potency of Bezzilure B + POC versus Bezzilure B for collecting the different genera/species, Bezzilure B + POC was 2.68, 1.69, 1.47 and 1.16 as efficient as Bezzilure B for catching Ch. ruififacies, Ch. megacephala, Hemipyrellia, and Sarcophaga, respectively. Similar significant trends whereby Bezzilure B + POC caught more flies than Bezzilure B were observed when analysing sexes separately for Hemipyrellia, Ch. megacephala and Ch. ruififacies, (Table 2), but not for Musca females where more flies were collected in traps baited with Bezzilure B than with Bezzilure B + POC (Wald = 3.49, F = 7.83, p < 0.001). It was difficult to differentiate between males and females of Sarcophaga, so this analysis was not undertaken.
4.1. Main findings Catches of Ch. bezziana from the laboratory were low, the best catch being 2.82%, which could result from either low activity of laboratory reared flies in an artificial environment and/or low trap efficiency (the diameter of the target was only 10 cm). Nevertheless, significant differences between lures were detected whereby Bezzilure B caught more flies than Bezzilure B + POC, which in turn caught significantly more flies than POC. One interpretation of the results is that, when exposed to chemical cues in close proximity, Ch. bezziana is more attracted to host wound volatiles than host derived volatiles, or a combination of the two, but that a combination of the two is more attractive than host derived volatiles alone. Evidence to support this hypothesis comes from behavioural observations: at close range the fly will be exhibiting a landing response, for which the wound volatiles are used (Hall, 1995). However, it is worth bearing in mind that the release rates for Bezzilure B as presented in the bioassay were higher than for POC. Likewise in the field trial catches of Ch. bezziana were low, representing only 0.65% of the total field catch, but significant differences between lures were detected. In contrast to the laboratory bioassay, the main finding from the field study was that Bezzilure B + POC caught significantly more flies than Bezzilure B or POC on their own. This could indicate that cattle-derived volatiles are used by Ch. bezziana, either for activation or orientation to the host. Cattle volatiles (such as the components of POC) are presumably present in vast quantities around a herd, whereas wound volatiles, that are
Arithemec Mean Catch
2.5 2 1.5 Females 1
Males Total
0.5 0
Control
POC
Bezzilure + POC
Bezzilure
Treatment Fig. 2. Mean daily catches of C. bezziana (with least significant differences at the 5% level) for each of the four treatments during field collections performed in Kediri, East Java (n = 24).
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model
ARTICLE IN PRESS
ACTROP-3354; No. of Pages 7
0 0 1.88 1.54 21.03 7.01
ARTICLE IN PRESS Acta Tropica xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia E.C.J. Sulston a , A.H. Wardhana b , M.J.R. Hall c , J.G. Logan a , S.A. Gezan d , M.M. Cameron a,∗ a
Department of Disease Control, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK Parasitology Department, Indonesian Research Centre for Veterinary Science (Balai Besar Penelitian Veteriner), Bogor, Indonesia Life Sciences Department, Natural History Museum, London SW7 5BD, UK d School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611, USA b c
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Chrysomya bezziana Old World screwworm fly POC Bezzilure Attractant Indonesia
a b s t r a c t The myiasis fly Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) is an obligate parasite of body orifices and unnatural openings or wounds of vertebrates. It is a major pest of livestock and responsible for economic losses throughout the Old World. Two chemical lures were tested in the present study to assess their effectiveness at trapping Ch. bezziana. Bezzilure B contains a synthetic lure of 2-mercaptoethanol originally identified from cattle wound volatiles. POC is a synthetic lure comprising 4-methyl phenol, 3-propyl phenol and racemic 1-octen-3-ol, originally identified from volatiles of ox odour and now used to control tsetse flies (Diptera: Glossina). Bezzilure B and POC lures were tested alone, and in combination, in a laboratory free flight room bioassay and in a field trial. Sticky traps were used to capture males and females of Ch. bezziana and to determine the efficacy of the lures. In the laboratory, Bezzilure B caught significantly more male and female flies than Bezzilure B + POC (p < 0.001). Similarly, in the field, treatment significantly affected the total number of Ch. bezziana caught (p < 0.001) but, contrastingly, Bezzilure B + POC caught significantly more flies than Bezzilure B (p < 0.05). The differing results between the laboratory and field may simply reflect the different assays and could suggest different roles of semiochemicals at different operational distances. Although ineffective on its own, POC has a synergistic effect with Bezzilure B to increase fly catches in the field: by proportion, Bezzilure B + POC was 2.17 times as efficient as Bezzilure B for Ch. bezziana, and 1.16–2.68 times as efficient for other flies depending on the species collected. © 2014 M.M. Cameron. Published by Elsevier B.V. All rights reserved.
1. Introduction Old World screwworm fly (OWSF), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), causes traumatic myiasis in subcutaneous tissue of body orifices and wounds of live mammals (Hall, 1995). Ch. bezziana is widespread throughout regions of Africa and Asia, including Indonesia (Wardhana et al., 2012). Although OWSF can affect humans (Ahmadi et al., 2009; Chan et al., 2005; Romero-Cabello et al., 2010; Sharma and Hedge, 2010), particularly the young and elderly (Stevens et al., 2006), human infections are rare. Arguably of more significant importance are infections of livestock due to the significant animal welfare issues and economic losses (Spradbery, 1994) and the potential for invasions and
∗ Corresponding author. Tel.: +44 0 20 7927 2337; fax: +44 0 20 7927 2918. E-mail address: [email protected] (M.M. Cameron).
consequent losses in new areas, for example if OWSF invades Australia (Anaman et al., 1994; Atzeni et al., 1997; Robinson et al., 2009; Sutherst et al., 1989). Trapping screwworm flies plays a valuable role in surveillance of invasions, and for monitoring of eradication programmes, such as the Sterile Insect Technique (SIT) (Spradbery et al., 1989; VargasTeran et al., 2005), particularly in the New World for Cochliomyia hominovorax (Krafsur et al., 1987). In the past, traps used liver as bait (Coppedge et al., 1977; Laake, 1936), but great effort has since been made to develop synthetic lures for operational use to ease handling, maintain attractiveness over time, increase the sensitivity of catches and to maximise the numbers of males collected. The first synthetic lure, Swormlure, developed to catch Co. hominovorax, contained 10 volatile compounds derived from degradation products of animal proteins and fatty acids that are likely to be found in wounds (Jones et al., 1976). This was later modified by
http://dx.doi.org/10.1016/j.actatropica.2014.04.017 0001-706X/© 2014 M.M. Cameron. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model ACTROP-3354; No. of Pages 7 2
ARTICLE IN PRESS E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
adding methyl disulfide in an improved formulation, Swormlure-2, that required two rather than three dispensers, and was adopted as the synthetic lure for use in the Screwworm Eradication Program for Co. hominovorax in the USA (Coppedge et al., 1977). Further modifications, regarding the relative concentrations of the 10 compounds in different blend formulations, led to the development of Swormlure-4, which improved catches of Co. hominovorax (Mackley and Brown, 1984). Subsequent research has identified 25 electophysiologically-active compounds found in wounds infected with Co. hominovorax (Cork, 1994), which were shown to significantly attract females but not males in the field (Cork and Hall, 2007). However, less attention has been focussed on the development of synthetic lures specifically for OWSF, although LuciLure, a synthetic blend derived from wound odours and containing 2mercaptoethanol, developed to attract Lucilia sericata, caught Ch. bezziana at levels comparable to a liver standard (Urech et al., 2004). The LuciTrap (Bioglobal Pty Ltd., Australia) baited with Lucilure for the control of L. sericata in Australia, included a small proportion (9.3%) of Chrysomya sp. and Calliphora sp. in collections (Urech et al., 2009). More recently, attractant formulations based on Swormlure and Lucilure, Bezzilure and/or Bezzilure-2, have been tested against field populations of Ch. bezziana using either sticky traps and/or LuciTraps in Indonesia and Malaysia (Urech et al., 2012; Wardhana, 2010). In Indonesia, where only baited sticky traps with Bezzilure were used, low catches (0.32% of the total catch) of Ch. bezziana were obtained compared with other myiasis species (Wardhana, 2010). In Malaysia, where the relative efficacies of different combinations of lure (Bezzilure or Bezzilure-2) and trap (sticky or LuciTraps) were tested, the optimal combination for selectively collecting higher numbers of Ch. bezziana, in relation to nontarget flies, was the LuciTrap/Bezzilure-2 system (Urech et al., 2012). Although mean numbers of Ch. bezziana in Bezzilure-2baited LuciTraps were relatively high compared with other field studies (8.0 flies/trap) (Urech et al., 2012), there is scope for further improvements to synthetic lures for the detection of Ch. bezziana at sites with low densities of OWSF. Although LuciTraps allow for specialised attraction of Ch. bezziana, they require an additional exploratory behaviour to catch flies. Sticky traps represent an efficient means of examining off target effects of the chemical lures against other myiasis causing agents which may not exhibit the behaviour necessary to be caught by LuciTraps. In a widespread survey looking for active cases of Ch. bezziana in Indonesia, 65% (167/255) of hosts infested by larvae were cattle, followed by goats (22%), and, based on livestock census data, cattle were more likely to be infested by OWSF than goats (Wardhana, 2010). If Ch. bezziana prefer cattle hosts, it is possible that they may respond to synthetic cattle-derived attractants. During the 1980’s several volatile chemicals from ox odour were identified as attractants to tsetse flies such as carbon dioxide, 1-octen-3-ol and acetone (Hall et al., 1984; Vale and Hall, 1985). Since then, many laboratory and field studies have been performed to isolate volatile components of ox odour and optimise lures for attracting tsetse flies. Of these, two refined blends, POC (P = 3-n-propylphenol, O = racemic1octen-3-ol, C = 4-methylphenol) and POCA (POC + A = acetone), significantly increased catches of Glossina palpalis gambiensis without using carbon dioxide, which can be expensive for large-scale use in surveillance programmes (Rayaisse et al., 2010). The aim of the present study was to compare the efficacy of two different types of lure, a wound lure (Bezzilure B) and a cattlederived lure (POC), alone and in combination, for capturing Ch. bezziana.
2. Materials and methods 2.1. Study area: Biogeographical regions, climate and land cover The laboratory based bioassays were conducted at the Indonesian Research Centre for Veterinary Science (IVETRI), Bogor, Java. The field study took place in two study sites in the province of Kediri in East Java (−07◦ 54.640 , 112◦ 01.411 and −07◦ 56.105 , 112◦ 03.605 ) between July and August 2011. 2.2. Fly colony for laboratory bioassays The colony of Ch. bezziana originated from wild samples collected from Bogor in 1994, which was refreshed with introductions from populations collected from Sumba and Sulawesi in 2002. Although the laboratory flies were originally collected from different field sites and populations from Bogor in West Java to the islands of Sumba and Sulawesi to the East of Java, and the flies sampled in the field tests were from East Java, all flies belonged to the same cyt b sub-lineage (2.2) (Wardhana et al., 2012). Flies were kept at 28 ◦ C and RH:75% in a 12:12 h photocycle. Approximately 10,000 flies were reared each week using a standard protocol (Sukarsih et al., 2000). 2.3. Dispensing mechanisms and chemical lures A stock solution of 600 ml of Bezzilure B was produced as reported previously (Urech et al., 2012) and was distributed between 18 white 30 ml HDP plastic bottles with a cotton wick 0.5 cm in diameter and 10 cm long. Lures were stored at 5 ◦ C with a securely fastened cap and placed in a plastic zip lock bag to minimise loss between experiments. Similarly, POC was produced as previously described (FAO/IAEA, 2003), and 1 ml was dispensed from a cotton wick contained within a polyethylene sachet (5 cm2 ) of 0.15 mm thickness. POC sachets were heat sealed inside individual aluminium plastic bags to minimise loss between experiments. Release rates of lures were determined from the room bioassay experiments by weighing the lures at the start and end of each experimental day to calculate the change in weight (release rate) per hour. 2.4. Experimental procedure and set up for laboratory bioassays Modified pot traps, as developed by Julie Bristow, LSHTM (pers. comm) were used. These consisted of a high density plastic paint pot (Leyland® SDM, UK), top diameter 15 cm, bottom diameter 10 cm, height 7 cm, with a lid (diameter 15 cm). A hole was cut out of the lid (3 cm diameter) and a circle of nylon mesh (3 mm gauge) was attached to the inside of the lid to prevent flies from accessing the bait. Yellow sticky card (Agrisense BCS Ltd, UK) was then cut to the size and shape of the lid. The card was sticky on both sides; one used to attach to the lid and one used to trap flies. Lures were placed inside the pots, and the baited raps were placed on two stands at 1.2 m above the ground in a sealed experimental room measuring 5.35 m × 4.4 m × 2.85 m. They were positioned at two points to ensure equal distances to the air inlet pipes, light levels and distance from the fly release position. An extraction fan opposite the entrance allowed for the air to be refreshed between replicates. Effectiveness of the lures was determined by offering flies a choice of attractant in the free flight room. Flies were exposed to pairs of traps containing either: (A) Control or Control; (B) Control or POC; (C) Control or Bezzilure B; (D) Control or Bezzilure B + POC; and (E) Bezzilure B or POC. The pairings of exposure were tested using randomised 5 × 5 Latin square designs (5 treatment pairs, 5 periods of exposure, 5 days) repeated three times to account for
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model ACTROP-3354; No. of Pages 7
ARTICLE IN PRESS E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
site, period and day effects. For each replicate, three hundred flies (150 female, 150 male) were released from a plastic tray at a fixed point in the room and exposed to the two treatments for 30 min. The traps were then removed and male and female flies caught on each trap were recorded. Flies remaining in the room were collected using a vacuum cleaner and removed. The process was repeated for the next random exposure pairing with a new set of previously unexposed flies. 2.5. Experimental procedure for field trials Two transects were constructed (07◦ 56.105 S 112◦ 03.605 E to 07◦ 56.173 S 112◦ 03.593 E and 07◦ 54.640 S, 112◦ 01.411 E, to 07◦ 54.641 S, 112◦ 01.484 E) with locations for four traps marked 50 m apart, a distance used in previous studies testing baited traps and targets (Coppedge et al., 1977; Cork and Hall, 2007). Sites for transects were selected on the requirements of being close to farms ( 0.05) and therefore were dropped from the models. To calculate the comparative potency of Bezzilure B versus Bezzilure B + POC for collecting different species of field-caught flies, mean catches using Bezzilure B + POC were divided by mean catches using Bezzilure B alone and expressed as a proportion. Similar comparisons for control and POC treatments could not be performed, as there were too many zero values (non-trapped insects) for most treatments, providing limited information.
3. Results 3.1. Laboratory bioassay The mixed linear model showed that treatment was a significant factor influencing the total catch of Ch. bezziana (Wald = 747.42, F = 248.86, p < 0.001). Bezzilure B + POC caught significantly more flies than POC alone (p < 0.001); and POC caught significantly more flies than the control (p < 0.001) (Fig. 1). The data were also used to examine the effect of sex on attractiveness to the lures by fitting the same model to each sex separately. Treatment was found to be significantly different for both females (Wald = 440.79, F = 146.76, p < 0.001) and males (Wald = 212.54, F = 70.76, p < 0.001), with Bezzilure B catching significantly more flies than Bezzilure B + POC (p < 0.001 and p < 0.05, respectively). Release rates were calculated during the first room bioassay, using the average change in mass for each lure over each day. The release rates of Bezzilure B were, on average, 8 times larger than that of POC. The mean release rates for all lures are displayed in Table 1.
3.2. Field study catches of Ch. bezziana The mixed linear model showed that treatment was a significant factor affecting the total catch of Ch. bezziana (Wald = 23.34, F = 7.78, p < 0.001). Furthermore, Bezzilure B + POC caught significantly more Ch. bezziana than Bezzilure B (p < 0.05; Fig. 2). However, this effect was only significant for female flies (Wald = 31.78, F = 10.59, p < 0.001) and not for males (Wald = 5.19, F = 1.73, p > 0.1). Neither POC nor the control group caught any Ch. bezziana. When comparing the potency of Bezzilure B + POC versus Bezzilure B for collecting Ch. bezziana, Bezzilure B + POC was 2.17 times as efficient as Bezzilure B.
Table 1 Mean release rates in mg/hr of the lures over all three replicates of the first room bioassay. Treatment
Mean release rate (mg/h)
95% CI
POCa POCb Bezzilure Ba Bezzilure Bb
0.354 0.339 2.15 2.85
0.2672–0.4406 0.1772–0.5008 0.5002–3.7912 0.3560–5.3538
a b
Represents lures used independently. Represents lures that were used together for the POC + Bezzilure B treatment.
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model
ARTICLE IN PRESS
ACTROP-3354; No. of Pages 7
E.C.J. Sulston et al. / Acta Tropica xxx (2014) xxx–xxx
4
10 Arithemec Mean Catch
9 8 7 6 5
Females
4
Males
3
Total
2 1 0
Control
POC
Bezzilure + POC
Bezzilure
Treatment Fig. 1. Mean number of flies caught per treatment (with least significant differences at the 1% significance level for females and total, and 5% significance level for males). A total of 300 flies (150 females and 150 males) were used per replicate in the laboratory bioassay.
3.3. Field study catches of other myiasis causing agents
4. Discussion
Over the 12 days of trapping in the field, a total of five additional non-target genera or species relating to myiasis were caught that were either facultative (Ch. megacephala, Ch. rufifacies, Hemipyrellia, Sarcophaga) or accidental (Musca domestica) agents. The mean daily catch of each genus/species caught by each treatment is shown in Table 2. The treatment that caught the greatest number of flies was Bezzilure B + POC (5229 flies). The dominant genus caught was Hemipyrellia (5901 flies). After fitting a model for total catches to each individual genus/species, treatment was found to be a significant factor, with Bezzilure B + POC catching more flies than Bezzilure B, for Hemipyrellia (Wald = 95.66, F = 31.89, p < 0.001), Ch. megacephala (Wald = 126.55, F = 42.18, p < 0.001), Ch. ruififacies (Wald = 62.73, F = 20.91, p < 0.001), Sarcophaga (Wald = 166.19, F = 55.40, p < 0.001), but the contrary was true for Musca (Wald = 21.03, F = 7.01, p < 0.001). When comparing the potency of Bezzilure B + POC versus Bezzilure B for collecting the different genera/species, Bezzilure B + POC was 2.68, 1.69, 1.47 and 1.16 as efficient as Bezzilure B for catching Ch. ruififacies, Ch. megacephala, Hemipyrellia, and Sarcophaga, respectively. Similar significant trends whereby Bezzilure B + POC caught more flies than Bezzilure B were observed when analysing sexes separately for Hemipyrellia, Ch. megacephala and Ch. ruififacies, (Table 2), but not for Musca females where more flies were collected in traps baited with Bezzilure B than with Bezzilure B + POC (Wald = 3.49, F = 7.83, p < 0.001). It was difficult to differentiate between males and females of Sarcophaga, so this analysis was not undertaken.
4.1. Main findings Catches of Ch. bezziana from the laboratory were low, the best catch being 2.82%, which could result from either low activity of laboratory reared flies in an artificial environment and/or low trap efficiency (the diameter of the target was only 10 cm). Nevertheless, significant differences between lures were detected whereby Bezzilure B caught more flies than Bezzilure B + POC, which in turn caught significantly more flies than POC. One interpretation of the results is that, when exposed to chemical cues in close proximity, Ch. bezziana is more attracted to host wound volatiles than host derived volatiles, or a combination of the two, but that a combination of the two is more attractive than host derived volatiles alone. Evidence to support this hypothesis comes from behavioural observations: at close range the fly will be exhibiting a landing response, for which the wound volatiles are used (Hall, 1995). However, it is worth bearing in mind that the release rates for Bezzilure B as presented in the bioassay were higher than for POC. Likewise in the field trial catches of Ch. bezziana were low, representing only 0.65% of the total field catch, but significant differences between lures were detected. In contrast to the laboratory bioassay, the main finding from the field study was that Bezzilure B + POC caught significantly more flies than Bezzilure B or POC on their own. This could indicate that cattle-derived volatiles are used by Ch. bezziana, either for activation or orientation to the host. Cattle volatiles (such as the components of POC) are presumably present in vast quantities around a herd, whereas wound volatiles, that are
Arithemec Mean Catch
2.5 2 1.5 Females 1
Males Total
0.5 0
Control
POC
Bezzilure + POC
Bezzilure
Treatment Fig. 2. Mean daily catches of C. bezziana (with least significant differences at the 5% level) for each of the four treatments during field collections performed in Kediri, East Java (n = 24).
Please cite this article in press as: Sulston, E.C.J., et al., Combining cattle and wound-derived synthetic attractants, POC and Bezzilure B, for sampling Chrysomya bezziana in Indonesia. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.017
G Model
ARTICLE IN PRESS
ACTROP-3354; No. of Pages 7
0 0 1.88 1.54 21.03 7.01
