Control of Host-seeking Adults of Hyalomma lusitanicum with Oxalic Acid under Field Conditions

For many years, tick control was based on topical acaricide administration, using similar methodology. Hyalomma lusitanicum is one of the most abundant tick species in the Mediterranean area but no control measures have been specifically developed against it. A previous study has shown the in vitro activity of Oxalic acid (OA) against H. lusitanicum. In this study OA was evaluated to control host seeking adults of H. lusitanicum under field conditions. Two different OA application systems were used. The first system, long hoses attached to a tractor that were used to treat non accessible areas, and the second system was an ultra low volume (ULV) device for applying test materials in tracks, paths and other accessible areas. Different concentrations of OA ranging from 6.3 to 0.5% by hose and 10.0% by ULV applications were tested in this study. Hose application of OA 6.3% was found to be the most effective leading to 77.9 to 100% reduction of the tick population. However, OA 3% achieved 80.9% reduction in tick population. The ULV application of 10.0% OA led a 75.9% reduction in tick population. The ULV method was easy to use and can cover large areas more efficiently.


Introduction
Ticks are blood seeking ectoparasites adapted to very different habitats and hosts.Although the direct impact in animal health is important, the greatest significance of tick infestation is transmission of pathogens to animal and human beings.In fact, ticks are, after mosquitoes, the second most important group of vectors.For many years, tick control has been based on similar methodologies as topical acaricide administration in many parts of the world regardless of the tick species and its chronobiology.
In consequence development of acaricide resistances appeared and resulted on a low efficiency of tick control strategies (Kunz and Kemp 1994).In order to be more efficient, specific control measures should be based on local tick phenology and different modes of acaricide application.
Treating domestic animals alone may not be sufficient to control ticks as the developmental stages could be maintained on wild animals.A perfect example is the tick species Hyalomma lusitanicum which is the most abundant triphasic, exophilic tick in central Spain, and a recognized vector of ruminant Mediterranean theileriosis and potential vector of several zoonotic bacterial agents (Toledo et al. 2008(Toledo et al. , 2009)).Even without paying attention to its vectorial capacity or the direct effect of hyper-infestation on wild and domestic animals, the extreme abundance of host seeking stages in spring and early summer make impossible for people to enjoy open field activities in some areas.
In that context, measures directed to control free living stages of the tick species should be developed.However, the use of synthetic acaricides has a negative environmental impact (Kunz and Kemp 1994).Biopesticides are commonly used as an alternative in organic farming.Some of them, such as thymol, oxalic acid, formic acid and lactic acid, have been reported as organic alternatives for chemical acaricides (Espinosa et al., 2007;George et al., 2008).In the same sense, some authors are successfully studying the in vitro activity of many extracts of several plants and insect growth regulators against ticks (de Oliveira et al. 2011, 2012, Arnosti et al. 2010a, b, Politi et al. 2012, Ramalho et al. 2012, Calligaris et al. 2013, Roma et al. 2013) even entomopathogenic fungi in vitro (Sun et al. 2013) and in field (Valcárcel et al., 2013).
Oxalic acid, a metabolite of ethylene glycol, an ubiquitous molecule in the environment (TOXNET 2007), is one of the most common organic treatment of varroosis in honey bees worldwide (Gregorc and Planinc 2005, Gregorc and Poklukar 2003, Aliano et al. 2006, Pérez 2006) and it has been described as the virulence factor of Beauveria bassiana fungus against ticks (Kirland et al. 2005).Moreover, its effectiveness against H. lusitanicum has been established in vitro (Olmeda et al. 2008).The goal of this study was to evaluate if Oxalic acid was effective in controlling adults of H. Lusitanicum under field conditions.

Study Site
The study was conducted at a natural reserve of plant and animal species representative of the Meso-Mediterranean bioclimatic environment (Central Spain,37º24'78"N;42º59'101"E;HT669m).Accordingly to Köppen classification the climate is Csa (temperate with dry or hot summer) ( AEMET 2011).The study site covered an area of 13,000 ha, at an altitude 500-1,266 meters above sea level.The annual average rainfall was 650 mm and temperatures ranged from -4 to 43 ºC.The main trees include oaks (Quercus ilex), cork (Quercus suber), mastic (Pistacia lentiscus), eucalyptus (Eucalyptus globulus) and pines (Pinus pinea and P.pinaster).There was also a reduced agricultural activity with 265 ha of organically grown olive (Olea europea) where no herbicides or insecticides were used, 1,300 ha of cereals and aromatic and native shrub species that provided food and shelter for wildlife.

Study Design
Experiments were carried out from June 2008 to October 2011.Trials were only performed from April to July because the higher host seeking ticks abundance in the area (Olmeda et al 2005(Olmeda et al , 2007;;Basco-Basco et al. 2008;Cota et al 2009;2010;Barandika et al 2011).
Eleven trials were performed using different doses of OA and two different application methods: hose (four trials, 71 replicates of 1,500 m 2 ) and Ultra Low Volumen (ULV) device (seven trials of 4,000 m 2 ) (Tables 1 to 2).

Ultra Low Volume (ULV) Application
Seven trials were performed using an Ultra-Low Volume (ULV) vat tractor device covering a total treated surface of 28,000 m 2 .Other non treated plots covering 28,000 m 2 were used as control.For each trial, a 410 m long path was selected divided in two plots (treated and untreated) of 200 m long x 10 m and separated by a corridor of 10 m.In treated plots a total of 250 l of 10% OA was applied for a period of 10 minutes.The speed of the tractor during the application was 1.2 Km/h.

Host Seeking Ticks
To determine tick abundance before treatments, corridors closed to the study plots were sampled using the blanket dragging technique for 30 minutes 24 hours before treatments (Toledo et al 2008(Toledo et al , 2009)).Similarly, 24 hours after treatments, ticks were sampled from each of the treated, control plots and not treated plots.
Specific flags were specifically used in different treatments (products and solutions).Samplings of host seeking ticks were carried out by five two-people.Each team represented one replicate of each treatment in order to avoid individual bias.

Complementary Data
Data of temperature and relative humidity at ground level and air, vegetation length and wind were taken on the day of treatments.All the experiments were assumed to have been performed under similar climate conditions (Table 2).No experiments were performed under strong wind or raining conditions.A visual inspection of the flora was performed during and after treatments in order to detect any side effect of the products.
Berlesse technique was used to determine the effect of treatments on non-target arthropods from second to fifth hose application replications and, similarly Pitfall technique was also performed in the ULV treatments (Sabu and Shilu 2010).

Efficiency Evaluation
The Tick Abundance Rate (TAR) was estimated as the number of ticks captured during 100 minutes using the blanket dragging techniques: TAR= (tick number/ time capture)*100.As Hyalomma lusitanicum is the most abundant exofilic tick in the central Iberian Peninsula (Basco-Basco et al 2008) this report included the number of only this tick species.
To compare the efficiency of treatments, data of day +1 were used.The percentage of tick reduction comparing different treatments to negative control was calculated as: Tick reduction (%) = ((TAR in -C plots -TAR in treated plots)/ TAR in -C plots) *100.
Sign and Mann-Whitney tests were performed in order to compare differences between treatments.The significant level used was p= 0.05 to 0.01.

Results
As it was expected, non treated and negative control plots showed the highest TAR (9.4-32.6 3).When oxalic acid was applied by a hose, tick reduction was variable but sometimes the efficacy was even higher than positive control.The first trial using hose application (6.3% OA) performed in an evergreen oak area reached a 77.9% of reduction of H. lusitanicum.The second treatment with the same OA concentration (6.3%) resulted in 100% reduction in TAR.Even a lower OA concentration (3.0%) resulted in a 95.9% reduction in TAR in the second trial.In the third trial, OA concentrations (3.0%, 1.0% and 0.5%) also reduced tick population (from 21.3% to 65.9%), although only the 3.0% OA had a statistically lower TAR compared to the negative control.The fourth hose trial achieved again significant tick reductions (from 50.3% to 85.3%).
Using the ULV applicator it also reached a global significantly tick reduction of 75.86% respect the non treated plots ranging from 62.5% to 100% (Table 4).

Discussion
When there is a supper population of ticks in an area, different strategies must be applied for tick control, especially in large areas where the management of animals is difficult as it happens in domestic extensive systems or in game reserves.In these situations, targeting the treatments to host seeking ticks could be very helpful to design a suitable integrated control that allows the reduction of direct/indirect tick effects, transmitted pathogens, and human labour lost, drugs residues in animal products and others.From this point of view, these results seem to indicate that oxalic acid is an alternative useful for tick control.
Oxalic acid is a metabolite of ethylene glycol ubiquitous in the natural medium, both in the smoke of snuff, vegetables and soil or atmosphere (TOXNET 2007).It also forms part, as the potassium salt or calcium, plants and mammals (urine, bones and others).It is one of the most common organic treatments in the varroosis of bees and it seems to be compatible with organic farming and would be relatively safe for other arthropods.Under these conditions there were not severe side effects on plants and non targeted arthropods but further specific studies should be done to confirm that.
Both application methods were effective in the control of host seeking ticks.The hose treatment application needs just a worker that spent about 45 minutes to treat a 1,500 m 2 plot so the labor cost of application is affordable in small areas.Using this system, OA always reduced the level of H. lusitanicum.Although administering the higher dose (OA 6.3%) the best performances were obtained, the difficulty of this dose preparation and the product consumption recommend us to use fewer active substances to optimize the results.In consequence, the intermediate OA concentration (3.0%) always showed significant reductions and quite similar to 6.3%, avoiding preparation troubles and reducing product consumption.
The ULV application was also effective against host seeking ticks allowing to treat large surfaces very quickly reducing time consumption (one worker can treat a 4,000 m 2 plot in just ten minutes) and so the human cost.Once ULV applicator is disposable it seems to be faster and cheaper than hose system and it spends less product (25.00kg and 66.67 kg per 4,000 m 2 ; ULV and hose, respectively).As it is usual when a treatment is performed on field it is important to note that personal protection measures should be taken and no animals or human presence must be allowed when product is applied until fiveten minutes post ULV application.
In conclusion, application of oxalic acid by hose or ULV device methods can be used to control of host seeking adults of Hyalomma lusitanicum tick.So it could be a useful and realistic alternative to traditional acaricides under field conditions, especially when resistance to commercial drugs is developed.