EFFICACY OF P 547/17 FORMULATION ON DERMANYSSUS GALLINAE EGGS AND LARVAE

High reproductive power and short development cycle, in addition to other factors, enable D.gallinae to produce adverse effects in the poultry industry. Not all development stages have the same significance, nor are they equally sensitive to the methods and formulations used for D.gallinae control. Laboratory tests were conducted by exposing eggs, larvae and protonymphs to P 547/17 formulation of inert oils (Pulcap). The testing was carried out with 20% oil-in-water emulsion with short exposure (1 min), and with  10%, 20%, 50% and 100% oil-in-water emulsion with continuous exposure. In the first control group, water was used (with continuous exposure), and in the other control group, eggs, larvae and nymphs were not treated. We determined that in all cases, eggs were laid in high percentage (89-100%). In addition to this, in tested liquids, larvae were present in high percentage and they change into protonymphs (8-89%). In the conditions of full exposure, where parasitic stages cannot leave the emulsion, P 547/17 in time achieves complete efficacy on development stages. In short exposure of eggs, when dirt is present, or on absorbent surfaces, P 547/17 emulsion cannot control them. However, this flaw is not essential in practical conditions. When applied correctly, P 547/17 formulation is efficient in D.gallinae control.


INTRODUCTION
Poultry red mite Dermanyssus gallinae (De Geer, 1778) is the most significant poultry ectoparasite. Its signifi cance in poultry industry arises from its prevalence, degree of control, overall impact on the human and poultry health and the environment, as well as the level of direct and indirect damage.
High reproductive power and short development cycle result in massive numbers of mites, which, in addition to other factors, have harmful eff ects of D. gallinae in the poultry industry. High intensity D. gallinae infestations can reach the numbers of 25,000 to 50,000, and in exceptional cases even up to 250,000 mites per hen (Van Emous, 2005). Th e development of D. gallinae has fi ve stages: eggs, larvae, protonymphs, deutonymphs, and adults (Wood, 1917) (Figure 1 and 2). D. gallinae reproduction occurs in the gonadotropic cycle, and is conditioned by blood consumption (Pritchard et al., 2015). During 12-24 hours aft er blood consumption, the female will start to lay eggs in hidden places. Th e size of eggs is usually 400x270 μm. Females usually lay up to 9 eggs in one batch, with several batches in their lifetime (Nordenfors, 2000). Temperature conditions required for laying eggs range from 5-45 °C, while the maximum numbers of eggs are recorded at 25 °C and 70% humidity (Nordenfors et al., 1999). Aft er one to three days, at the temperature of 20-45 ºC, larvae are laid from the eggs (Maurer and Baumgartner, 1992;Nordenfors et al.1999). Larvae do not feed. Th ey have 3 pairs of legs. Th eir colour is seashell white. In the next 1-2 days they change into the protonymph. Protonymphs and deutonymphs have 4 pairs of legs, and in order to transform into the next form, they need to consume blood. In optimal environmental conditions, the development cycle from an egg to an adult can be fi nished in seven days (Nordenfors, 2000). In practical conditions, this cycle takes longer. For example, at 25 °C, the development of one generation of D. gallinae fi nishes in 16.8 days (Maurer and Baumgartner, 1992). Th e tests conducted in Sao Paolo, Brazil, showed that 15 to 42 generations of D. gallinae can develop in one year (Tucci et al., 2008).
Th e control of diff erent development stages can have diff erent impact depending on the species of arthropods.
Th e aim of our examination was to determine the effi cacy of P 547/17 formulation (Pulcap) on the eggs and larvae, and the relevance of the fi ndings for D. gallinae control.

MATERIAL AND METHODS
Female D. gallinae fed in isolation chambers in the laboratory, were moved to plastic Petri dishes, in order to lay eggs. Th e lids of Petri dishes were paired up according to the width and sticked with a duct tape in order to prevent mites from coming out. Aft er the third day, adult mites were removed from the Petri dishes, and the laid eggs were counted and included in the testing.
Th e fi rst testing group was exposed for 1 minute to the 20% water emulsion of P 547/17 formulation (hereinaft er w.e.f.). Th is was done by fi rst covering the eggs with w.e.f. P 547/17, and aft er 1 minute, the emulsion was poured out and drained for the next hour. Despite the draining, the Petri dishes still contained a certain amount of left over water emulsion, i.e. the tested formulation. Th e testing groups were constantly exposed to 10%, 20%, 50% water emulsion, and to the concentrated P 547/17 (100%) formulation. Th e testing procedure was also conducted with two control groups: 1. water, with constant exposure and 2. negative control without exposure. Th e development of eggs, larvae and protonymphs was monitored until the 10 th day of exposure (0-10). Th e temperature in the environment ranged from 19 to 23 ºC, and humidity from 49 to 61%.
Th e results obtained by laboratory testing were interpreted by comparison with the clinical trials of the effi cacy of P 547/17 water emulsion.

RESULTS
Our results are presented in tables 1 to 8, and fi gures 2 and 3.

DISCUSSION
In untreated, control group, all the eggs transformed into larvae and subsequently into protonymphs. According to the research by Nordenfors et al. (1999), 99% of eggs developed into larvae at 25 °C, and 92% of larvae further transformed into protonymphs.
Mineral oils have been used for the control of harmful insects and mites for more than a century (Agnello, 2002 Pulcap is the fi rst specialised formulation (P 547/17) and technology (project ID 1115) which uses water emulsion of inert oils for D. gallinae control. Th e aim is for the biological effi cacy of P 547/17 to be thoroughly examined and documented. Preliminary observations from previous tests (Pavlićević et al., 2018) showed that P 547/17 water emulsion in working solutions has no signifi cant eff ect on D. gallinae eggs.
Th ere is general knowledge about the eff ect of inert oils. Unlike acaricides and insecticides (in the narrow sense of the word synthetic neurotoxic chemical compounds), oils do not act through specifi c receptors, but rather in multiple ways. Th eir eff ect depends on interaction between physical, chemical, anatomic, developmental and physiological characteristics and behaviour of the targeted mites or insects. Th e following have been described: affi nity to epicuticle, immobilisation (in active stages), preventing respiration, disturbing water balance, penetrating the organism, disturbing cellular functions, etc. (Buteler and Stadler, 2011).
Certain oils are known to have eff ect on some arthropods' eggs. For example, in insects, Al Dabel et al. (2008) found high effi cacy of horticultural oil on Ostrinia nubilalis eggs applied in 3-10% concentration. Buteler and Stadler (2011) determined that 2% horticultural mineral oil causes almost total mortality of Choristoneura rosaceana eggs.
Large numbers, short development cycle, i.e. great reproductive capacity of D. gallinae require high effi cacy of a formulation used for their control. For direct 1 minute exposure, biological effi cacy of formulations for D. gallinae control should be 95-100%. If constant and direct exposures are combined, effi cient formulations should achieve 100% effi cacy in laborato (ideal) conditions. Th e tests were designed to maximise the contact of eggs and larvae with w.e.f. P 547/17 by direct and constant exposure. In spite of this, most eggs underwent their embryonic development smoothly. Th e degree of egg exposure ranged from 89 to 100%. Larvae existed in the tested fl uids in high percentage and changed into protonymphs in 8-89 % of cases. 50% w.e.f. P 547/17 showed the highest degree of effi cacy on larval stage where 8% changed into protonymphs.
Th ere is a signifi cant diff erence in sensitivity between the development stages of eggs and larvae compared to adult D. gallinae. For adult D. gallinae, 1 minute direct exposure to 10% w.e.f. P 547/17 would be lethal (Pavlićević et al., 2017). Eggs and larvae were able to withstand, to a high degree, a constant direct exposure to water emulsion (10%, 20%, 50%), even concentrated (100%) P 547/17 formulation.
Th e results of conducted research indicate that eggs and larvae of D. gallinae can stand anoxia. We are not aware of any tests on the anoxia of D. gallinae eggs and larvae. However, hypoxia and anoxia have been indirectly tested in adult D. gallinae. Mites were put in Petri dishes, directly exposed to CO2 (2,500 ppm), by spraying for 10 seconds. Aft er 120 hours, the eff ect of anoxia was 100% mortality of adult D. gallinae (Kang et al., 2020). Furthermore, we can assume that additional research will fi nd that eggs and larvae of D. gallinae are, to a great extent, not sensitive to controlled CO2 management.
In comparison, controlled CO2 management has been used since the 1980s in plant protection in order to control harmful mites (Aharoni et al., 1981). Exposure to 60% CO 2 at 30 ºC results in up to 100% mortality of adult two-spotted spider mite Tetranychus urticae ( Laboratory tests were done in the conditions which enable the maximum eff ect of w.e.f. P 547/17. However, in practical conditions, it is not possible to expose all eggs and larvae of D.gallinae directly, suffi ciently and constantly. Th e reasons for this are mites' way of life on hidden places, limited distribution (overlapping and covered places) of water emulsion in external application by spraying, as well as the very conditions in the poultry industry (absorbent surfaces or presence of dirt). Th erefore, in practical conditions, we can expect worse results compared to those found in laboratory tests. Even in ideal laboratory conditions, even with high concentrations, the eff ect of P 547/17 formulation on D. gallinae eggs and larvae did not fulfi l the conditions of total effi cacy, and should be taken into account. Th e observed fl aw of w.e.f. P 547/17 needs to be overcome by planning the method of application. Th e choice of method of w.e.f. P 547/17 application starts with the choice of the moment of application. Although w.e.f. P 547/17 can be used in both empty and populated poultry houses, the specifi c conditions and eff ects of control will vary signifi cantly.
Th e development structure of D. gallinae invasion in hosts is reported as 35% eggs, 10% larvae, 33% nymphs and 22% adults (Van Emous, 2005). In a populated house, w.e.f. P 547/17 is used when necessary. Th e conditions in a populated poultry house are characterised by lack of hygiene, worse distribution of the emulsion due to the presence of poultry (it covers the surfaces) and uninterrupted reproduction of D. gallinae (with all development stages present). Th e effi cacy depends on hygienic situation (limited contact and reduced residual effect on surfaces), complexity of environmental conditions, quality of application and intensity and extensity of D. gallinae infestation. In the case of general low intensity infestation, one treatment is implemented, while in the case of general high intensity infestation, there are two treatments. Th e eff ects of suppression with w.e.f. P 547/17 in a populated house usually last for 4-5 months.
Formulation P 547/17 is primarily designed for prevention, since this is the primary and essential task of human and veterinary medicine. Treating an empty poultry house with w.e.f. P 547/17 prevents the infestation of a new, young fl ock of laying hens with D. gallinae invasion (present in the facility).
In an empty facility there is no host (poultry) and therefore no blood which is necessary for laying D. gallinae eggs. Adults will keep the ability to lay eggs for several days aft er emptying the poultry house, based on the previously consumed blood. At the temperature of 27 °C and humidity of 80%, egg laying will last for up to 11 days (Tucci et al., 2009). Th en it will stop. Th e development of eggs and protonymphs will be limited to the phase of deutonymph, because from this moment, further development will depend on blood consumption. Th e absence of blood, i.e. the host (poultry) will prevent further development of protonymphs and deutonymphs. D. gallinae adults remain in an empty poultry house. In some cases, they can endure starvation for more than a year (Pavlićević et al., 2007). Th erefore, the success of controlling adults is crucial for the eff ects which will be achieved in D. gallinae control.
Th e tests performed in laboratory conditions (Maurer and Baumgartner, 1992) indicate that development of eggs into larvae of D. gallinae at 10 °C lasts for 12 days, while at the temperature of 5 °C it lasts for up to 50 days or 28.2 days (Nordenfors et al. 1999). In practical conditions, we have not yet found a problem which could arise from a slower development of D. gallinae eggs and larvae at lower temperatures.
Poultry house preparation includes thorough cleaning, washing, deratisation, disinfection and drying. Aft er that, w.e.f. P 547/17 is applied. On unabsorbent surfaces, it has long-lasting, highly effi cient prolonged eff ect even on subsequently exposed mites (Pavlićević et al., 2017). Th e prolonged eff ect is especially noticeable in empty facilities, since residual layer is not exposed to dirt or removal.
Favourable hygienic situation, good conditions for the distribution of the water emulsion and suffi ciently sensitive development stages of mites, housing down-time (enough time at the temperature conditions in which D. gallinae is active), and correct application optimise the effi cacy of w.e.f. P 547/17. When these conditions are fulfi lled in an empty poultry house, P 547/17 results in D. gallinae eradication from production facilities, i.e. farms. Eradication has so far been proven in fl oor housing of parent fl ocks, cage housing system, conventional and enriched cages for layer housing (Pavlićević et al., 2018;2019a,b, 2020 in the process of preparation). In aviaries and free range housing, the eradication has not yet been tested. D. gallinae eradication in a poultry house is considered successful if during one year of systematic monitoring, not a single mite is detected. If the conditions are only partially met, P 547/17 results in highly effi cient D. gallinae suppression. If no conditions are met, D. gallinae suppression might not be eff ective (Pavlićević et al., 2019a).
Aft er comparing the results obtained from laboratory (unsatisfactory effects) and clinical tests (highly effi cient suppression and eradication), we believe that the development stages of eggs and larvae are not crucial for D. gallinae control. Despite their resistance, the need for feeding and short development period makes them inconsequential for the control procedure. Adult stage is crucial for the control.
In the appendix about the assessment of signifi cance of D. gallinae eggs and larvae for the control, there is the research on the effi cacy of the formulation of active substance fl uralaner (isoxazoline). Fluralaner is administered to poultry per os, in drinking water and it works only on adults and development phases that feed on blood (protonymphs and deutonymphs). Th erefore, fl uralaner has no eff ect on eggs and larvae. Nevertheless, the eff ects of D. gallinae control can last for up to 238 days (Th omas et al., 2017).Th e development of D. gallinae eggs and larvae continues in aquatic environment as well. When washing the poultry house, the water itself (especially on fl oors) will not significantly aff ect the vitality of eggs and larvae (apart from mechanical removal). Th erefore, they are expected to complete their development aft er the surfaces dry off (Th omas et al. 2017). However, a detailed washing is a prerequisite for creating the hygienic conditions which enable a quality contact with the water emulsion and prolonged eff ect on unabsorbent surfaces.

CONCLUSION
P 547/17 formulation does not have a signifi cant impact on D. gallinae eggs and larvae. Although they have a higher resistance level, eggs and larvae are not very important for D. gallinae control. Adult control is crucial. Intensive and long-lasting prolonged eff ect of w.e.f. P 547/17 successfully eliminates adults, thereby including all developmental stages. Th erefore, P 547/17 achieves a highly eff ective suppression, and with an optimal application in an