Infection, Genetics and Evolution xxx (2014) xxx–xxx
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges Christopher J. Sanders ⇑, Simon Carpenter Vector-borne Viral Disease Programme, The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF, UK
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Article history: Available online xxxx Keywords: Immunomarking Capture–mark–recapture Vector Dispersal Bluetongue virus Schmallenberg virus
a b s t r a c t Capture–mark–recapture techniques are used to determine the dispersal and survival of arthropods, including vector groups such as Culicoides. An assumption of these studies is that capture and the subsequent marking process does not impact of the survival and behaviour of the marked individual. The small size of Culicoides means that a significant mortality and disruption of normal behaviour such as host-location can be caused by the process of collection. Here we evaluate a technique, novel to the study of dispersal in vectors, to mark Culicoides directly and indirectly without prior capture. The acquisition and subsequent detection of marker protein by Culicoides exposed to a treated substrate was investigated in the laboratory. The technique was then assessed in a small-scale field trial where a defined section of resting habitat was sprayed with an egg white solution and Culicoides caught within the vicinity were tested for the presence of egg protein. It was found that up to 100% of Culicoides acquired the protein marker in the laboratory with no apparent impact on survival. In the field, pools of Culicoides obsoletus collected next to the treated area were found to be positive for the protein, suggesting that the technique could be used in larger-scale studies. The definition of a behaviourally non-invasive technique for marking Culicoides will greatly increase our understanding of the natural dispersal behaviour of Culicoides and other vectors. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The use of capture, re-capture techniques to provide estimates of population size, birth, death and emigration rates is a fundamental technique in ecological research (Southwood and Henderson, 2000). Key assumptions in such studies are that the behaviour or life expectancy of marked individuals is not altered by the marking process, the marks are not lost over time and that marked individuals become entirely mixed in the population (a factor determined by the impact of the marking technique on dispersal ability and the proportion of the total population marked). Methods for this purpose have been reviewed in detail both for insects (Hagler and Jackson, 2001) and specifically for Dipteran vectors of pathogens such as mosquitoes (Silver, 2008; Verhulst et al., 2013). A significant determinant in the degree to which these marking techniques fulfil the pre-requisites for use in capture, recapture experimentation lies in the size and robustness of the target vector species. Hence, whilst large, mobile flies existing at low population density (e.g. Tsetse) provide close to ideal targets for such studies, other haematophagous genera such as ⇑ Corresponding author. Tel.: +44(0)1483231206. E-mail address: [email protected] (C.J. Sanders).
Phlebotominae (sandflies) or Ceratopogonidae (biting midges) provide far more challenging subjects in being small, vulnerable to physical damage and locally abundant. The emergence and spread of bluetongue and Schmallenberg viruses in northern Europe in the last decade has elevated the importance of Palearctic Culicoides (Diptera: Ceratopogonidae) as biological vectors of livestock arboviruses (Carpenter et al., 2009; Hoffmann et al., 2012). This has led to interest in this region in quantifying dispersal of Culicoides over both sea and land as a means of tracing spread patterns of arboviruses and predicting likely routes of incursion (Burgin et al., 2012). The conditions promoting long-distance (>10 km) flights of Culicoides over water bodies can be predicted with a high degree of accuracy, in part due to the availability of disease outbreak data on island habitats, where confounding animal movements can be discounted as a means of introduction. In contrast, understanding dispersal at a local scale over land is more challenging as restrictions on animal movement are more difficult to enforce and dispersal tends to be driven by a wider variety of stochastic and deterministic variables within habitats that are of far greater ecological complexity. Retrospective analysis therefore typically use detection of arbovirus presence, dispersal estimates from studies of behaviour of Culicoides from outside the region of interest or random dispersal to infer
http://dx.doi.org/10.1016/j.meegid.2014.01.020 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
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spread patterns (Ducheyne et al., 2007; Hendrickx et al., 2008; Szmaragd et al., 2009; Pioz et al., 2011; Sedda et al., 2012). Studies quantifying the dispersal behaviour of Culicoides have employed either indirect approaches, where unmarked insects are collected at known distances from their larval development sites or direct approaches based upon capture, recapture. A major criticism of indirect studies is that unless habitats occupied by Culicoides larvae are very accurately defined and/or localised there is a significant risk that overlooked sites will contribute to an overestimation of dispersal ability. Species investigated using this technique include Culicoides impunctatus in the Scottish Highlands (Kettle, 1951a,b, 1960), Culicoides furens in the Caribbean (Williams, 1962) and Culicoides sonorensis on farms in the USA (Zimmerman and Turner, 1984). Direct capture re-capture methods have also been carried out with Culicoides using fluorescent pigments or dusts (Lillie et al., 1981a,b, 1985; Murray and Boorman, 1981; Brenner et al., 1984; Linhares and Anderson, 1989; Carpenter, 2001; Kirkeby et al., 2013), radioactive isotopes or rare elements (Davies, 1965; Holbrook et al., 1991) and ingestion of food dye in 10% sucrose solutions (Campbell and Kettle, 1976) to mark individuals. In their entirety, these studies indicate that mean dispersal from the site of release in direct studies is generally limited to a few hundred metres, but that maximum range can reach several kilometres with the maximum recorded recapture occurring at 6 km from the release site for Culicoides mohave (Brenner et al., 1984). Whilst crude laboratory-based estimates of survival in marked Culicoides have occasionally been made for these techniques (Lillie et al., 1981a; Holbrook et al., 1991), none have been assessed in their impact on flight activity or other behaviours. In the vast majority of Culicoides species it is also not possible to rear adults under laboratory conditions in numbers sufficient for release, with the notable exception of C. sonorensis in the USA, which can be collected as pupae from waste water ponds (Holbrook et al., 1991). Studies therefore utilise Culicoides that have been collected using light- or semiochemical-baited suction traps, a process that is known to inflict mortality and is also likely to influence dispersal ability. It has also been documented that certain species of Culicoides are often reluctant to feed in the laboratory after field capture in light suction traps (Venter et al., 2005). This calls into question whether host-seeking behaviour would be accurately reproduced in marked populations and raises a significant concern for studies that infer likely patterns of arbovirus spread. In this study we examine immunomarking of haematophagous Diptera for the first time and assess the potential for the use of this technique to address the limitations of current marking techniques. In these systems insects are exposed to a protein marker and enzyme-linked immunosorbent (ELISA) based assays are then used to detect exposure. The use of ELISA-based detection allows the rapid screening of thousands of individuals or pools of insects to identify marked individuals, in a cost effective manner with non-specialist equipment used by many laboratories for other applications (Jones et al., 2006). In contrast to fluorescent making, the sensitivity of these assays allows detection of extremely small quantities of antigen, meaning that insects can commonly be marked by walking over coated surfaces or by flying through very fine particulate dusts (Hagler and Jackson, 2001). These techniques were originally developed to mark plant pests (Hagler et al., 1992) and later applied to minute parasitoid wasps in greenhouses (Hagler and Jackson, 1998). The use of readily available, low cost proteins allows the marking of a wide area of habitat such as crop refugia, enabling the marking of thousands of insects within it with limited environmental impact (Horton et al., 2009). In order to establish the utility of immunomarking Culicoides, we first investigated the efficacy of passive marking by exposure to treated surfaces and subsequent recovery of protein from
washes of individuals. Secondly, we investigate whether Culicoides can be marked in areas of potential emergence and resting by wide-scale application of marker protein and captured close to the treated area. This approach would inform of the sensitivity of the ELISA in determining sufficient ovalbumin can be recovered from the bodies of marked Culicoides and the efficacy of the habitat marking technique to mark Culicoides and other small haematophagous flies (e.g. sand flies). 2. Methods 2.1. Passive marking trial, colony insects Colony individuals of Culicoides nubeculosus, a species that is routinely maintained at The Pirbright Institute (Boorman, 1974), were exposed to egg treated paper dipped to simulate the uptake of marker protein on the bodies of Culicoides from a treated substrate. The treatment paper (40 80 mm, 80 gsm copier paper, Banner value paper, white, 70,234) was dipped in 20% egg white solution in distilled water (Two Chicks™ Liquid Egg White, Two Chicks, Kettering, UK) and allowed to dry at 25 °C for 30 min until dry to the touch, before being placed in a 50 mm cardboard pillbox (Watkins and Doncaster, UK) with fine mesh covering. Two hundred C. nubeculosus adults, at 2 days post emergence were introduced into the egg exposure pot for a period of one hour. During this time, all C. nubeculosus had free access to land and walk on the egg treated paper. Following the exposure period, seven groups of 20 C. nubeculosus were removed from the exposure pot and placed in each of seven clean pots to test the recovery of ovalbumin at intervals of 0, 1, 3, 5, 24, 48 and 72 h post exposure. An additional group of 20 C. nubeculosus of the same age were placed in a clean pot and were not exposed to the treatment as a control to assess mortality. The pots for each group were placed at 20 °C for 15 min to kill the insects. Individual insects were placed in 2 ml cryotubes with 500 ll of sample diluent (Chicken Egg Ovalbumin ELISA kit cat. no. 6050, Alpha Diagnostics International, San Antonio, USA). Prior to testing, samples were vortexed for 5 s and spun down in a microcentrifuge to release protein into the sample diluent. Small pieces of the treated paper (3 3 mm) were treated in the same way to provide a positive control for the ELISA assay. The trial was repeated with a second group of 200 C. nubeculosus and control. 2.2. Passive marking trial, wild insects The paper exposure technique was additionally used to mark live Culicoides within a CDC 4 W UV light trap (John Hock Ltd., USA) to investigate the potential to mass mark Culicoides within the trap. A piece of paper (150 150 mm) was dipped in 20% egg white solution and placed in the live insect collection pot of a CDC trap. The trap was hung at 1.7 m in a tree adjacent to a horse paddock (51° 170 25.400 N, 0° 390 11.300 W) where Culicoides had previously been shown to be abundant and operated overnight. The collection pot was then returned to the laboratory and placed at 20 °C to kill collected arthropods. Culicoides were then identified to species group level using wing characteristics placed in pools of either 10 or 20 individuals and transferred to 2 ml cryotubes containing 500 ll of sample diluent. The diluent was then tested using the ELISA technique. In a subsequent repeat of this trial the Culicoides collected were placed individually in tubes and tested in an identical fashion. 2.3. Habitat marking trial Habitat marking trials were conducted on a small dung heap next to a horse stable on a sheep farm in Oxfordshire (51° 420
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
C.J. Sanders, S. Carpenter / Infection, Genetics and Evolution xxx (2014) xxx–xxx
56.200 N, 1° 220 51.500 W). This represented a small, discrete breeding site where previous trapping had consistently collected between 100 and 250 Culicoides per night. As a precaution against possible contamination of personnel and equipment, one operator sprayed the habitat, whilst another set up traps, retrieved collections and subsequently ran the ELISA assay. All equipment was transported to the site sealed in plastic bags to separate the spray treatment from traps and collection jars. Three spray applications were made on 3 nights in September 2012. On the first occasion, 3 L of a 10% egg white solution (Two Chicks Liquid Egg White, UK) was sprayed to run off using a 7 L garden sprayer (Hozelock, UK) over an area of the dung heap of approximately 9 m2 (egg white dose = 0.03 l/m2) (Fig. 1). In the following two trials, 5 L of 20% egg white solution was sprayed to run off over the same area (egg white dose = 0.11 L/m2). Application of the egg spray took place 3 h prior to sunset. Ultraviolet light traps (Miniature Downdraft Blacklight (UV) Trap Model 912, John W. Hock Company, Florida USA) were set at 0, 10 and 20 m from the centre of the treated area and were run from 1 h to +2 h from sunset (Fig. 2). At the end of this period, trap catches were collected and taken back to the laboratory and placed at 20 °C overnight. Captured Culicoides were identified and tested in pools of 10 or 20 individuals according to the methods for the passive marking trial. 2.4. ELISA protocol A commercial ELISA kit for the quantification of egg white albumin was used during all studies (Chicken Egg Ovalbumin ELISA kit cat. no. 6050, Alpha Diagnostics International, San Antonio, USA), which utilised antibodies monospecific for ovalbumin. The kit included all plates, antibodies and washes used in the ELISA and had a minimum detection threshold of 0.1 ng ml1 ovalbumin. Ovalbumin concentration in samples was visually assessed against the protein standards within the kit, ranging from 0.2 to 4 ng ml1, from which ovalbumin concentrations in samples were estimated. 3. Results 3.1. Passive marking trial, colony insects Ovalbumin was successfully recovered from the C. nubeculosus exposed to egg-treated paper at up to 72 hours post exposure. Of 20 individual C. nubeculosus tested immediately after exposure over the two assays 19 were positive for egg (95%). Individuals in the second assay demonstrated a stronger response, with concentrations of greater than 4 ng ml1 in three out of the ten individuals
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Fig. 2. Percentage of Culicoides nubeculosus individuals exposed to a treated paper substrate which were positive for egg white albumin with time from exposure.
tested, compared to concentrations of 0.5–2 ng ml1. Positive responses were recorded from treated C. nubeculosus at all time periods post-exposure, although the number of individuals from which a positive response was recorded declined with the time post exposure, most noticeably in the first assay (Fig. 2.). Pools of 10 individuals at all periods post-exposure (n = 2/period post-exposure) were strongly positive for ovalbumin (>4 ng ml1). Culicoides not exposed to the treatment did not give positive results in the ELISA. Mortality of Culicoides was not significantly affected by the treatment as 100% survival was recorded over the 72 h period. 3.2. Passive marking trial, wild insects All Culicoides obsoletus complex (C. obsoletus/Culicoides scoticus) pools of either 10 (n = 9) or 20 (n = 10) individuals taken from the treatment light trap were positive for ovalbumin when tested following collection (0.5–2 ng ml1). A single pool of 10 Culicoides pulicaris also gave a strong positive response (>4 ng ml1). Similarly, individual C. obsoletus complex from the trap gave a 100% positive response in the assay (0.5–>4 ng ml1, n = 30). Samples from the treated paper gave a very strong positive result (>4 ng ml1). 3.3. Habitat marking trial In the first habitat marking trial, 5 of 10 pools of 20 C. obsoletus complex caught directly above the marked area and 2 of 10 pools from the trap at 10 m were found to be positive for the egg marker (0.2–0.5 ng ml1). In addition, 1 of 3 pools of 20 C. obsoletus complex collected from the trap at 20 m was positive. In the two subsequent trials with increased concentration and volume of ovalbumin sprayed over the same area, a greater recovery of marked insects was achieved. In pools of 10 C. obsoletus group from directly above the marked area, 100% were positive (2–3 ng ml1, n = 20), only one pool of Culicoides from 20 m was positive, with no positive pools from 10 m in the two trials (n = 20 pools). One of two specimens of Culex pipiens (Diptera: Culicidae) collected at 10 m from the marked habitat was positive for ovalbumin (1 ng ml1). 4. Discussion
Fig. 1. Immunomarking site and CDC traps.
This study has demonstrated the utility of an immunomarking assay for marking Culicoides both directly through contact exposure and indirectly by spraying of a selected adult emergence and resting site. Whilst durability of detection in directly marked individuals was limited, the fact that ovalbumin could be found on Culicoides that only walked over a treated surface demonstrates a significant advance on techniques relying on invasive and potentially behaviour modifying marking procedures (e.g. the use of
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
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fluorescent dusts). The detection of ovalbumin on up to 100% of Culicoides caught directly above marked adult emergence and resting habitat and recovery of marked individuals from 20 m distance from this site also suggests that the use of this technique on a large scale will be successful in studying Culicoides dispersal over greater distances. The technique should provide better estimates of the natural dispersal behaviour of Culicoides, essential for describing and predicting of the movement of the viruses that they transmit. The trial has also alluded to the potential to use the immunomarking technique in the investigation of other aspects of vector biology, where adults exhibiting specific behaviours could be marked, such as the selection of breeding and resting sites by adults. Whilst the detection threshold of the ELISA kit used was sufficient to detect ovalbumin on individual marked Culicoides recovered up to 72 h post-exposure, it was noticeable that detection rates fell off rapidly during this period. Egg albumin has generally been found to be a highly effective marking protein in immunomarking studies on pear psylla (Cacopsylla pyricola; Homoptera: Psyllidae) (Hagler and Jones, 2010), and was the most durable marker for lady beetles (Hippodamia convergens; Coleoptera: Coccinellidae), outperforming rabbit IgG, milk and soy milk treatments (Slosky et al., 2012). The minute parasitoids, Gonatocerus ashmeadi (Hymenoptera: Mymaridae), were unable to self-mark when exposed to dry protein residues of egg albumin, but were marked efficiently in exposure to dried casein residues (Irvin et al., 2012). The difference in marking may be due to the difference in proportion of protein within the milk (80%) used which was much higher than that present in the egg white solution (1%), although both proteins were effective markers when applied topically and were retained on the parastioids for the entire 11-day period of the study (Irvin et al., 2012). It is therefore unlikely that a different marker would improve durability and the presence of cattle would preclude the use of milk as a marker as it may be present on hosts. The assay could be potentially be more effectively optimised through the use of a higher concentration of marker and/or by the selection of wetting agent as an adjuvant during application (Jones et al., 2011). In terms of interpretation of wide-scale trials, several further areas need to be addressed including understanding the potential for contamination between captured Culicoides. Due to the sensitivity of assays involved this is a significant issue and has led in some cases to the use of sticky traps that ensure a lack of contact (Jones et al., 2011). In the case of Culicoides, sticky inserts could be placed within modified light- or semiochemical-baited suction traps to ensure that catch rates remain sufficient. A second key consideration is the extreme sensitivity of the ELISA assay, which has required the development of procedures to reduce risk of contamination and generation of false positive results. The use of two operators to separate treatment from collection of insects was extremely useful in the current study and minimised this issue. Following the success of the habitat-marking trial, this work will be extended to cover a much larger area and examine dispersal over greater distances. The increased recovery of marked Culicoides recorded when an increased volume and concentration of egg white solution was sprayed over the treatment area suggests this would be most appropriate for future studies. A high-throughput form of the ovalbumin ELISA (Hagler and Jones, 2010) will be used due to the greater number of insects that require testing. An ELISA plate reader will additionally be used to assess the optical density of samples relative to negative controls to refine the resolution of the assay and prevent the identification of false positives (Sivakoff et al., 2011). In addition to the ovalbumin ELISA described here, there is the potential to use two or more different protein sources, for example egg white and soya, and test insects in serial ELISAs for the different markers used. In this scenario, two or more habitats
could be treated at the same time with different marker proteins, and the insects collected tested for both proteins, allowing dispersal from two or more habitats to be investigated simultaneously. The immunomarking proteins could also be utilised in self-marking emergence traps such as those used with fluorescent dusts (Linhares and Anderson, 1989) allowing marking of Culicoides emerging from breeding habitats over a period of time or for the marking of insects visiting specific hosts in mixed herds. Such studies will greatly increase our understanding of the natural dispersal of Culicoides vectors and the diseases they transmit at an intra- and inter-farm scale.
Acknowledgements This study was funded by Defra grant number SE: 4211. The authors would like to acknowledge the assistance of Giles Weaver in conducting field experiments and the owners of the two farms involved in the study for their support.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges Christopher J. Sanders ⇑, Simon Carpenter Vector-borne Viral Disease Programme, The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF, UK
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Article history: Available online xxxx Keywords: Immunomarking Capture–mark–recapture Vector Dispersal Bluetongue virus Schmallenberg virus
a b s t r a c t Capture–mark–recapture techniques are used to determine the dispersal and survival of arthropods, including vector groups such as Culicoides. An assumption of these studies is that capture and the subsequent marking process does not impact of the survival and behaviour of the marked individual. The small size of Culicoides means that a significant mortality and disruption of normal behaviour such as host-location can be caused by the process of collection. Here we evaluate a technique, novel to the study of dispersal in vectors, to mark Culicoides directly and indirectly without prior capture. The acquisition and subsequent detection of marker protein by Culicoides exposed to a treated substrate was investigated in the laboratory. The technique was then assessed in a small-scale field trial where a defined section of resting habitat was sprayed with an egg white solution and Culicoides caught within the vicinity were tested for the presence of egg protein. It was found that up to 100% of Culicoides acquired the protein marker in the laboratory with no apparent impact on survival. In the field, pools of Culicoides obsoletus collected next to the treated area were found to be positive for the protein, suggesting that the technique could be used in larger-scale studies. The definition of a behaviourally non-invasive technique for marking Culicoides will greatly increase our understanding of the natural dispersal behaviour of Culicoides and other vectors. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The use of capture, re-capture techniques to provide estimates of population size, birth, death and emigration rates is a fundamental technique in ecological research (Southwood and Henderson, 2000). Key assumptions in such studies are that the behaviour or life expectancy of marked individuals is not altered by the marking process, the marks are not lost over time and that marked individuals become entirely mixed in the population (a factor determined by the impact of the marking technique on dispersal ability and the proportion of the total population marked). Methods for this purpose have been reviewed in detail both for insects (Hagler and Jackson, 2001) and specifically for Dipteran vectors of pathogens such as mosquitoes (Silver, 2008; Verhulst et al., 2013). A significant determinant in the degree to which these marking techniques fulfil the pre-requisites for use in capture, recapture experimentation lies in the size and robustness of the target vector species. Hence, whilst large, mobile flies existing at low population density (e.g. Tsetse) provide close to ideal targets for such studies, other haematophagous genera such as ⇑ Corresponding author. Tel.: +44(0)1483231206. E-mail address: [email protected] (C.J. Sanders).
Phlebotominae (sandflies) or Ceratopogonidae (biting midges) provide far more challenging subjects in being small, vulnerable to physical damage and locally abundant. The emergence and spread of bluetongue and Schmallenberg viruses in northern Europe in the last decade has elevated the importance of Palearctic Culicoides (Diptera: Ceratopogonidae) as biological vectors of livestock arboviruses (Carpenter et al., 2009; Hoffmann et al., 2012). This has led to interest in this region in quantifying dispersal of Culicoides over both sea and land as a means of tracing spread patterns of arboviruses and predicting likely routes of incursion (Burgin et al., 2012). The conditions promoting long-distance (>10 km) flights of Culicoides over water bodies can be predicted with a high degree of accuracy, in part due to the availability of disease outbreak data on island habitats, where confounding animal movements can be discounted as a means of introduction. In contrast, understanding dispersal at a local scale over land is more challenging as restrictions on animal movement are more difficult to enforce and dispersal tends to be driven by a wider variety of stochastic and deterministic variables within habitats that are of far greater ecological complexity. Retrospective analysis therefore typically use detection of arbovirus presence, dispersal estimates from studies of behaviour of Culicoides from outside the region of interest or random dispersal to infer
http://dx.doi.org/10.1016/j.meegid.2014.01.020 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
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spread patterns (Ducheyne et al., 2007; Hendrickx et al., 2008; Szmaragd et al., 2009; Pioz et al., 2011; Sedda et al., 2012). Studies quantifying the dispersal behaviour of Culicoides have employed either indirect approaches, where unmarked insects are collected at known distances from their larval development sites or direct approaches based upon capture, recapture. A major criticism of indirect studies is that unless habitats occupied by Culicoides larvae are very accurately defined and/or localised there is a significant risk that overlooked sites will contribute to an overestimation of dispersal ability. Species investigated using this technique include Culicoides impunctatus in the Scottish Highlands (Kettle, 1951a,b, 1960), Culicoides furens in the Caribbean (Williams, 1962) and Culicoides sonorensis on farms in the USA (Zimmerman and Turner, 1984). Direct capture re-capture methods have also been carried out with Culicoides using fluorescent pigments or dusts (Lillie et al., 1981a,b, 1985; Murray and Boorman, 1981; Brenner et al., 1984; Linhares and Anderson, 1989; Carpenter, 2001; Kirkeby et al., 2013), radioactive isotopes or rare elements (Davies, 1965; Holbrook et al., 1991) and ingestion of food dye in 10% sucrose solutions (Campbell and Kettle, 1976) to mark individuals. In their entirety, these studies indicate that mean dispersal from the site of release in direct studies is generally limited to a few hundred metres, but that maximum range can reach several kilometres with the maximum recorded recapture occurring at 6 km from the release site for Culicoides mohave (Brenner et al., 1984). Whilst crude laboratory-based estimates of survival in marked Culicoides have occasionally been made for these techniques (Lillie et al., 1981a; Holbrook et al., 1991), none have been assessed in their impact on flight activity or other behaviours. In the vast majority of Culicoides species it is also not possible to rear adults under laboratory conditions in numbers sufficient for release, with the notable exception of C. sonorensis in the USA, which can be collected as pupae from waste water ponds (Holbrook et al., 1991). Studies therefore utilise Culicoides that have been collected using light- or semiochemical-baited suction traps, a process that is known to inflict mortality and is also likely to influence dispersal ability. It has also been documented that certain species of Culicoides are often reluctant to feed in the laboratory after field capture in light suction traps (Venter et al., 2005). This calls into question whether host-seeking behaviour would be accurately reproduced in marked populations and raises a significant concern for studies that infer likely patterns of arbovirus spread. In this study we examine immunomarking of haematophagous Diptera for the first time and assess the potential for the use of this technique to address the limitations of current marking techniques. In these systems insects are exposed to a protein marker and enzyme-linked immunosorbent (ELISA) based assays are then used to detect exposure. The use of ELISA-based detection allows the rapid screening of thousands of individuals or pools of insects to identify marked individuals, in a cost effective manner with non-specialist equipment used by many laboratories for other applications (Jones et al., 2006). In contrast to fluorescent making, the sensitivity of these assays allows detection of extremely small quantities of antigen, meaning that insects can commonly be marked by walking over coated surfaces or by flying through very fine particulate dusts (Hagler and Jackson, 2001). These techniques were originally developed to mark plant pests (Hagler et al., 1992) and later applied to minute parasitoid wasps in greenhouses (Hagler and Jackson, 1998). The use of readily available, low cost proteins allows the marking of a wide area of habitat such as crop refugia, enabling the marking of thousands of insects within it with limited environmental impact (Horton et al., 2009). In order to establish the utility of immunomarking Culicoides, we first investigated the efficacy of passive marking by exposure to treated surfaces and subsequent recovery of protein from
washes of individuals. Secondly, we investigate whether Culicoides can be marked in areas of potential emergence and resting by wide-scale application of marker protein and captured close to the treated area. This approach would inform of the sensitivity of the ELISA in determining sufficient ovalbumin can be recovered from the bodies of marked Culicoides and the efficacy of the habitat marking technique to mark Culicoides and other small haematophagous flies (e.g. sand flies). 2. Methods 2.1. Passive marking trial, colony insects Colony individuals of Culicoides nubeculosus, a species that is routinely maintained at The Pirbright Institute (Boorman, 1974), were exposed to egg treated paper dipped to simulate the uptake of marker protein on the bodies of Culicoides from a treated substrate. The treatment paper (40 80 mm, 80 gsm copier paper, Banner value paper, white, 70,234) was dipped in 20% egg white solution in distilled water (Two Chicks™ Liquid Egg White, Two Chicks, Kettering, UK) and allowed to dry at 25 °C for 30 min until dry to the touch, before being placed in a 50 mm cardboard pillbox (Watkins and Doncaster, UK) with fine mesh covering. Two hundred C. nubeculosus adults, at 2 days post emergence were introduced into the egg exposure pot for a period of one hour. During this time, all C. nubeculosus had free access to land and walk on the egg treated paper. Following the exposure period, seven groups of 20 C. nubeculosus were removed from the exposure pot and placed in each of seven clean pots to test the recovery of ovalbumin at intervals of 0, 1, 3, 5, 24, 48 and 72 h post exposure. An additional group of 20 C. nubeculosus of the same age were placed in a clean pot and were not exposed to the treatment as a control to assess mortality. The pots for each group were placed at 20 °C for 15 min to kill the insects. Individual insects were placed in 2 ml cryotubes with 500 ll of sample diluent (Chicken Egg Ovalbumin ELISA kit cat. no. 6050, Alpha Diagnostics International, San Antonio, USA). Prior to testing, samples were vortexed for 5 s and spun down in a microcentrifuge to release protein into the sample diluent. Small pieces of the treated paper (3 3 mm) were treated in the same way to provide a positive control for the ELISA assay. The trial was repeated with a second group of 200 C. nubeculosus and control. 2.2. Passive marking trial, wild insects The paper exposure technique was additionally used to mark live Culicoides within a CDC 4 W UV light trap (John Hock Ltd., USA) to investigate the potential to mass mark Culicoides within the trap. A piece of paper (150 150 mm) was dipped in 20% egg white solution and placed in the live insect collection pot of a CDC trap. The trap was hung at 1.7 m in a tree adjacent to a horse paddock (51° 170 25.400 N, 0° 390 11.300 W) where Culicoides had previously been shown to be abundant and operated overnight. The collection pot was then returned to the laboratory and placed at 20 °C to kill collected arthropods. Culicoides were then identified to species group level using wing characteristics placed in pools of either 10 or 20 individuals and transferred to 2 ml cryotubes containing 500 ll of sample diluent. The diluent was then tested using the ELISA technique. In a subsequent repeat of this trial the Culicoides collected were placed individually in tubes and tested in an identical fashion. 2.3. Habitat marking trial Habitat marking trials were conducted on a small dung heap next to a horse stable on a sheep farm in Oxfordshire (51° 420
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
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56.200 N, 1° 220 51.500 W). This represented a small, discrete breeding site where previous trapping had consistently collected between 100 and 250 Culicoides per night. As a precaution against possible contamination of personnel and equipment, one operator sprayed the habitat, whilst another set up traps, retrieved collections and subsequently ran the ELISA assay. All equipment was transported to the site sealed in plastic bags to separate the spray treatment from traps and collection jars. Three spray applications were made on 3 nights in September 2012. On the first occasion, 3 L of a 10% egg white solution (Two Chicks Liquid Egg White, UK) was sprayed to run off using a 7 L garden sprayer (Hozelock, UK) over an area of the dung heap of approximately 9 m2 (egg white dose = 0.03 l/m2) (Fig. 1). In the following two trials, 5 L of 20% egg white solution was sprayed to run off over the same area (egg white dose = 0.11 L/m2). Application of the egg spray took place 3 h prior to sunset. Ultraviolet light traps (Miniature Downdraft Blacklight (UV) Trap Model 912, John W. Hock Company, Florida USA) were set at 0, 10 and 20 m from the centre of the treated area and were run from 1 h to +2 h from sunset (Fig. 2). At the end of this period, trap catches were collected and taken back to the laboratory and placed at 20 °C overnight. Captured Culicoides were identified and tested in pools of 10 or 20 individuals according to the methods for the passive marking trial. 2.4. ELISA protocol A commercial ELISA kit for the quantification of egg white albumin was used during all studies (Chicken Egg Ovalbumin ELISA kit cat. no. 6050, Alpha Diagnostics International, San Antonio, USA), which utilised antibodies monospecific for ovalbumin. The kit included all plates, antibodies and washes used in the ELISA and had a minimum detection threshold of 0.1 ng ml1 ovalbumin. Ovalbumin concentration in samples was visually assessed against the protein standards within the kit, ranging from 0.2 to 4 ng ml1, from which ovalbumin concentrations in samples were estimated. 3. Results 3.1. Passive marking trial, colony insects Ovalbumin was successfully recovered from the C. nubeculosus exposed to egg-treated paper at up to 72 hours post exposure. Of 20 individual C. nubeculosus tested immediately after exposure over the two assays 19 were positive for egg (95%). Individuals in the second assay demonstrated a stronger response, with concentrations of greater than 4 ng ml1 in three out of the ten individuals
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Fig. 2. Percentage of Culicoides nubeculosus individuals exposed to a treated paper substrate which were positive for egg white albumin with time from exposure.
tested, compared to concentrations of 0.5–2 ng ml1. Positive responses were recorded from treated C. nubeculosus at all time periods post-exposure, although the number of individuals from which a positive response was recorded declined with the time post exposure, most noticeably in the first assay (Fig. 2.). Pools of 10 individuals at all periods post-exposure (n = 2/period post-exposure) were strongly positive for ovalbumin (>4 ng ml1). Culicoides not exposed to the treatment did not give positive results in the ELISA. Mortality of Culicoides was not significantly affected by the treatment as 100% survival was recorded over the 72 h period. 3.2. Passive marking trial, wild insects All Culicoides obsoletus complex (C. obsoletus/Culicoides scoticus) pools of either 10 (n = 9) or 20 (n = 10) individuals taken from the treatment light trap were positive for ovalbumin when tested following collection (0.5–2 ng ml1). A single pool of 10 Culicoides pulicaris also gave a strong positive response (>4 ng ml1). Similarly, individual C. obsoletus complex from the trap gave a 100% positive response in the assay (0.5–>4 ng ml1, n = 30). Samples from the treated paper gave a very strong positive result (>4 ng ml1). 3.3. Habitat marking trial In the first habitat marking trial, 5 of 10 pools of 20 C. obsoletus complex caught directly above the marked area and 2 of 10 pools from the trap at 10 m were found to be positive for the egg marker (0.2–0.5 ng ml1). In addition, 1 of 3 pools of 20 C. obsoletus complex collected from the trap at 20 m was positive. In the two subsequent trials with increased concentration and volume of ovalbumin sprayed over the same area, a greater recovery of marked insects was achieved. In pools of 10 C. obsoletus group from directly above the marked area, 100% were positive (2–3 ng ml1, n = 20), only one pool of Culicoides from 20 m was positive, with no positive pools from 10 m in the two trials (n = 20 pools). One of two specimens of Culex pipiens (Diptera: Culicidae) collected at 10 m from the marked habitat was positive for ovalbumin (1 ng ml1). 4. Discussion
Fig. 1. Immunomarking site and CDC traps.
This study has demonstrated the utility of an immunomarking assay for marking Culicoides both directly through contact exposure and indirectly by spraying of a selected adult emergence and resting site. Whilst durability of detection in directly marked individuals was limited, the fact that ovalbumin could be found on Culicoides that only walked over a treated surface demonstrates a significant advance on techniques relying on invasive and potentially behaviour modifying marking procedures (e.g. the use of
Please cite this article in press as: Sanders, C.J., Carpenter, S. Assessment of an immunomarking technique for the study of dispersal of Culicoides biting midges. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.020
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fluorescent dusts). The detection of ovalbumin on up to 100% of Culicoides caught directly above marked adult emergence and resting habitat and recovery of marked individuals from 20 m distance from this site also suggests that the use of this technique on a large scale will be successful in studying Culicoides dispersal over greater distances. The technique should provide better estimates of the natural dispersal behaviour of Culicoides, essential for describing and predicting of the movement of the viruses that they transmit. The trial has also alluded to the potential to use the immunomarking technique in the investigation of other aspects of vector biology, where adults exhibiting specific behaviours could be marked, such as the selection of breeding and resting sites by adults. Whilst the detection threshold of the ELISA kit used was sufficient to detect ovalbumin on individual marked Culicoides recovered up to 72 h post-exposure, it was noticeable that detection rates fell off rapidly during this period. Egg albumin has generally been found to be a highly effective marking protein in immunomarking studies on pear psylla (Cacopsylla pyricola; Homoptera: Psyllidae) (Hagler and Jones, 2010), and was the most durable marker for lady beetles (Hippodamia convergens; Coleoptera: Coccinellidae), outperforming rabbit IgG, milk and soy milk treatments (Slosky et al., 2012). The minute parasitoids, Gonatocerus ashmeadi (Hymenoptera: Mymaridae), were unable to self-mark when exposed to dry protein residues of egg albumin, but were marked efficiently in exposure to dried casein residues (Irvin et al., 2012). The difference in marking may be due to the difference in proportion of protein within the milk (80%) used which was much higher than that present in the egg white solution (1%), although both proteins were effective markers when applied topically and were retained on the parastioids for the entire 11-day period of the study (Irvin et al., 2012). It is therefore unlikely that a different marker would improve durability and the presence of cattle would preclude the use of milk as a marker as it may be present on hosts. The assay could be potentially be more effectively optimised through the use of a higher concentration of marker and/or by the selection of wetting agent as an adjuvant during application (Jones et al., 2011). In terms of interpretation of wide-scale trials, several further areas need to be addressed including understanding the potential for contamination between captured Culicoides. Due to the sensitivity of assays involved this is a significant issue and has led in some cases to the use of sticky traps that ensure a lack of contact (Jones et al., 2011). In the case of Culicoides, sticky inserts could be placed within modified light- or semiochemical-baited suction traps to ensure that catch rates remain sufficient. A second key consideration is the extreme sensitivity of the ELISA assay, which has required the development of procedures to reduce risk of contamination and generation of false positive results. The use of two operators to separate treatment from collection of insects was extremely useful in the current study and minimised this issue. Following the success of the habitat-marking trial, this work will be extended to cover a much larger area and examine dispersal over greater distances. The increased recovery of marked Culicoides recorded when an increased volume and concentration of egg white solution was sprayed over the treatment area suggests this would be most appropriate for future studies. A high-throughput form of the ovalbumin ELISA (Hagler and Jones, 2010) will be used due to the greater number of insects that require testing. An ELISA plate reader will additionally be used to assess the optical density of samples relative to negative controls to refine the resolution of the assay and prevent the identification of false positives (Sivakoff et al., 2011). In addition to the ovalbumin ELISA described here, there is the potential to use two or more different protein sources, for example egg white and soya, and test insects in serial ELISAs for the different markers used. In this scenario, two or more habitats
could be treated at the same time with different marker proteins, and the insects collected tested for both proteins, allowing dispersal from two or more habitats to be investigated simultaneously. The immunomarking proteins could also be utilised in self-marking emergence traps such as those used with fluorescent dusts (Linhares and Anderson, 1989) allowing marking of Culicoides emerging from breeding habitats over a period of time or for the marking of insects visiting specific hosts in mixed herds. Such studies will greatly increase our understanding of the natural dispersal of Culicoides vectors and the diseases they transmit at an intra- and inter-farm scale.
Acknowledgements This study was funded by Defra grant number SE: 4211. The authors would like to acknowledge the assistance of Giles Weaver in conducting field experiments and the owners of the two farms involved in the study for their support.
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