Comparative Results of Action of Natural and Synthetic Acaricides in Reproductive and Salivar Systems of Rhipicephalus sanguineus - Searching by a Sustainable Ticks Control

Ticks have been well studied and are one of the most important groups amongst the Arthropod, showing that there is of great interest in the medical and veterinarian aspects, mainly because of the fact that they appear to be carriers of many illnesses that attack domestic and wild animals, as well as humans (Sonenshine, 1991). The ticks in rural or urban environment represent a big problem for society. For dairy producers and especially for meat and leather producer these ectoparasites cause huge financial losses due to their fixation in its hosts. Already in urban environments infestations in domestic dogs, as well as common contaminate the home environment, through the transmission of serious diseases to man.


Introduction
Ticks have been well studied and are one of the most important groups amongst the Arthropod, showing that there is of great interest in the medical and veterinarian aspects, mainly because of the fact that they appear to be carriers of many illnesses that attack domestic and wild animals, as well as humans (Sonenshine, 1991).The ticks in rural or urban environment represent a big problem for society.For dairy producers and especially for meat and leather producer these ectoparasites cause huge financial losses due to their fixation in its hosts.Already in urban environments infestations in domestic dogs, as well as common contaminate the home environment, through the transmission of serious diseases to man.

Rhipicephalus sanguineus ticks
The importance of the ticks meanly Rhipicephalus sanguineus in the domestic world can be explained by the introduction of dogs in houses, for example, as companion animals consequently its ectoparasites facilitates the propagation of biopathogens those cause diseases both to the dogs and the human being; therefore these ticks are nowadays considered "urban plagues" Numerous studies are currently under way to find an effective control strategy that would minimize the damage caused by these ectoparasites.A new tick control approach is an immunological one consisting in the identification, isolation and synthesis of proteins those protect tissues and organs of the tick, mainly those of the reproductive and glandular systems, with the aim of developing a vaccine (Tellam et al, 1992;Willadsen, 1997).However, nowadays the most efficient method to control tick populations is by suing chemical compounds, such as permethrin, fipronil and selamectin (active ingredients of different acaricides) which frequently act in the nervous system of the ticks (Mencke et al., 2003).

Ticks control
The challenge of controlling these arthropods has long been subject of several studies and the results obtained have been the formulation of new acaricides (synthetic chemicals) that are now widely used.However, collateral damages has been reported, including the emergence of individuals resistant to the drugs applied, which raises the source of new generations of ticks those are little affected by application of the acaricides.Also in this demand, the pharmaceutical industry (meanly of veterinary products) frequently releasing acaricides in an attempt to circumvent this problem.However, specific studies on the tick's cell biology are still scarce and little is known about the consequences of its utilization in non-target organisms.

Permethrin acaricide
The toxicity of an acaricide is defined as extent or degree to which a chemical substance to kill or injure the target organism.In this way, the toxicity of a drug is determined by running laboratorial tests on animals and it is expressed as LD 50 (lethal dose fifty) and LC 50 (lethal concentration fifty) values and are the amount or concentration, respectively, of the pesticide's active ingredient that is required to kill 50% of the tested animals under standardized tests conditions (Garcia-Garcia et al., 2005).The first study to described a detailed protocol of laboratorial procedures to determinate de LC 50 of permethrin using semi-engorged females of R. sanguineus ticks was performed by Roma et al. (2010) who established the LC 50 =2062 ppm (1.549-2.675).Acaricides currently available in the market have permethrin (chemical substance that cause a nervous impulses disorders and the ticks suffer excitement, indicated by tremors and spasms followed by paralysis and death) as active ingredient in concentration higher than 300.000 ppm and according Roma et al. (2010) permethrin in lower concentrations (approximately 100 times less) would be enough to kill R. sanguineus ticks, although this process would be slower.

Permethrin x Rhipicephalus sanguineus salivary gland
The feeding success of the ticks is the result of the action of the metabolism of the salivary glands, organs that are responsible for the fixation of the tick in the host (Moorhouse and Tatchell, 1966), by osmoregulation (Sonenshine, 1991), the inhibition of the host defense mechanisms such as coagulation and inflammation (Nuttal andStrickland, 1908: Paesen et al., 1999) and by the digestion of the tissues (Walker et al., 1985).Therefore, salivary glands are organs indispensable for the feeding process and therefore make ticks very biological successful organisms.The tick's salivary glands are paired organs found in its celomatic cavity and contain approximately 1400 acini each (Walker et al., 1985).Many histological descriptions of salivary glands have been made in different species of ixodides ticks including by Furquim et al (2007Furquim et al ( , 2008)).In summary, there are three different types of acini in females (I, II, III) and four in males (I, II, III and IV) and type I acini cells are agranular and have the osmoregulatory process as the main function, while types II, III and IV acini cells are granular and responsible for the other previously mentioned functions.According to Nodari et al. (2011) the permethrin chemical induced morphophysiological changes in salivary glands of R. sanguineus semi-engorged females, even at lower concentrations, causing tissue changes compromising the organ metabolism, essential to the 393 feed process of the tick.The salivary glands in individuals subjected to 206 ppm of permetrhrin presented acini I morphologically altered (irregular shape and dilated lumen) corroborating Pereira et al. (2011), who reported that these acini in the same tick species were also affected by fipronil acaricide.These authors suggested that the acini I would be ormoregulators and through the saliva, they could remove the toxic compound from the hemolimph, since the lumen diameter of the acini suffer a significant increase.This authors showed that females subjected to permethrin at 2062 ppm presented only few acini I, with the other ones being classified as indeterminate, since they have lost their morphological and histological characteristics due the degeneration process by the toxic agent.The Nodari et al. (2011) results confirmed that permethrin, besides the proven neurotoxic action, also accelerates glandular tissue degeneration, an event that will occur naturally and with greater intensity only after full female engorgement (Fig. 1,2).

Fipronil x Rhipicephalus sanguineus salivary glands
In this sense, Oliveira et al. (2011) performed another study bringing information about the action of fipronil acaricide, using R. sanguineus as a biological model.Fipronil is a broadspectrum chemical agent available in the market in a range of formulations from attractive baits for ant and cockroach control to sprays used in veterinary products for the control of ticks, fleas and mites (Penaliggon, 1997;Hugnef et al,. 1999;Higgins et al. 2001) and his effect are not fully known, as well as, the direct and indirect consequences of its use.The fipronil act mainly in the ticks GABA system, consisting of inhibitory neurotransmitters of neuromuscular junctions and central nervous system synapses (Denny, 2001).The authors developing an appropriate protocol for an in vitro bioassay (AIT) monitored daily.The LC 50 and the 95% confidence interval were also determined.The increase in the concentration of fipronil (1ppm-100 ppm) caused a progressive increase in the mortality of semi-engorged R. sanguineus females.Pereira et al. (2011) in a study performed with the same chemical substance showed the action of fipronil in salivary glands of the same species females subjected to different concentrations.The acaricide accelerated the natural degeneration process of these glands, as well as reduced the number of the acini present in the gland as the concentration of the chemical compound is increased, making the organ unable to secrete its final saliva.From this process only the glandular ducts are preserved probably for being resistant to fipronil and having all the ducts (acinar, intermediate and common excretory ones) with lumen covered by cuticle, which prevented both the diffusion and the action of the acaricide on the epithelial cells.In the acini with intact cells the organelles were intact too (Fig. 3).In the altered cells there was evidence of apoptotic cell death which characteristic is to affect the cells individually and as synchronically.One common morphological characteristic of the cytoplasm of salivary gland of the R. sanguineus is the spherocrystal presence, constituted by phosphate and calcium carbonate and originated from the cisternae of the rough endoplasmic reticulum, however despite their presence in various systems of different species, no author stated what would be their real function (reserve of calcium, detoxication, ionic balance, etc.).In females subjected to 10 ppm of fipronil the glandular damages were extensive, considering that even the most resistant structure-the spherocrystals-became disorganized, resulting in a structure that went from perfectly round to completely irregular (Fig. 4, 5).

Rhipicephalus sanguineus ovaries
The tick ovaries in general are classified like a panoistic type; therefore it lacks nurse and follicular cells, as demonstrated in Amblyomma cajennense (Denardi et al, 2004), R. sanguineus (Oliveira et al, 2005) and A. triste females (Oliveira et al. 2006).It consists of a wall of epithelial cells, the pedicel, cellular structure that, besides supplying elements of the yolk to the oocytes, also connect them to the ovary wall (Till, 1961;Oliveira et al. 2005Oliveira et al. , 2006)).The ovaries are usually filled with many oocytes, which will go through various developmental stages (I to V or VI) until they are ready for oviposition (Said, 1992, Saito el al. 2005).

Fipronil x Rhipicephalus sanguineus ovaries
Fipronil substance that acts inhibiting the development of ticks in pets animals (Taylor, 2001) and as a neurotoxic substance (Cole et al, 1993) also acts in the reproductive process of the ticks by altering both the structure and function of germinative cells of R. sanguineus (Oliveira et al., 2008(Oliveira et al., , 2009)).In these individuals ovaries the same authors detected alterations related with the size and morphology of the oocytes as well as presence of vacuoles in the cytoplasm and oocytes that had problem to succeeded in arriving at stage V (mature one), some of them had ruptured or withered, and had released their contents before the end of maturation.In females subjected to 10 ppm of fipronil (the maximal concentration tested) the oocytes in stage I were no longer observed, showing clearly that as the concentrations of fipronil increase the number and type of morphological changes in oocytes I increase as well (Fig. 6).Other data that would confirm the action of fipronil on the development of the R. sanguineus ovary would change the size of the oocytes, when comparing the control group to the three groups of treatment (1, 5 and 10 ppm of fipronil).The exposure of R. sanguineus feeding females to the fipronil leads to the susceptibility of its oocytes (in various degrees of development) to the chemical agent applied and, consequently, indicate the potential to cause damage in the reproductive process and to reduce the fertility of these ectoparasites (Fig. 7).

Natural acaricides
Thinking in the non-target organisms as well as in the environment preservation found alternative ticks control using biocompounds has an interesting subject with the intent to resolve some problems.In this sense is necessary to prevent the damages those are inflicted on non-target organisms, whereas the addition of financial savings the natural products could bring to producers and to the environment a sustainable management, since the environmental pollution caused by the indiscriminate use of toxic substances selects resistant animals, making the search for alternatives to natural and efficient chemicals, urgent.Plant compounds have been shown to be an important alternative for the control of these ectoparasites (Olivo et al., 2008).

Castor been oil esters x Rhipicephalus sanguineus salivary glands
Of the many plant species that have thus far been studied for future use in effective control of ticks is included the castor bean plant (Ricinus communis) which has recently emerged as a great promise, since their components such as oil extracted from it, is already widely known (biodiesel and dermatologic products).Also this plant has been supplying active principles that have enabled the fabrication of medical protheses, which besides being used in Brazil has been widely exported to the world.Recently a new application for the components extracted of castor plants R. communis has emerged from studies conducted by BCSTM (Brazilian Center of Studies on Ticks Morphology) headquartered at UNESP, Rio Claro, SP, Brazil coordinated by Profa.Dra.Maria Izabel Camargo Mathias in an attempt to offer an alternative way for tick control.Ricinoleic acid esters from castor oil R. communis have been shown to efficiently in control micro and ectoparasites.Recent studies on the use of non-polluting plant the esters have shown that they act in the hydrolysis of saccharides and the dissolution of lipids in different biological systems (Mandelbaum et al., 2003).The consumption of castor oil esters by rabbits hosting the dog tick R. sanguineus significantly affected the vitellogenesis of the female, by making the oocytes non-viable and/or causing their death, and thus preventing new offspring from developing.The action of the ester in salivary glands was also studied by Arnosti et al (2010a), and they showed that these esters, provided through food (feed+esters) for the rabbits hosts, enhancing and accelerated their degenerative process, that would otherwise occur slowly.It was also shown that the damage caused by esters in glandular cells was proportional to the concentration of the product, in other words at higher concentration (5g of ester (stabilized in NaCl)/Kg of commercial food for a period of 10 days.The results showed that, unlike conventional methods of chemical synthetic control, which kills ticks by neurotoxic action, different concentration of ester of R. communis oil did not cause the death of the ticks, but interfered in two interdependent systems, which are of paramount importance to these ectoparasites (by inducing premature degeneration of the salivary glands), and also acting in the reproductive process (preventing the vitellogenesis) (Fig. 8).Thus the action of esters on the salivary glands makes the ticks feeding process deficient causing the absence of some components in the hemolymph and consequently impairing the vitellogenic process of the oocytes (Arnosti et al., 2010a,b).These first results performed in laboratorial assays by Arnosti et al (2010 a, b) were very promising showing the effect of this component not only in salivary glands but on the reproductive system of ticks R. sanguineus (reducing significantly the eggs viability), avoiding the individuals from completing the blood meal process considered necessary for effective ticks success (Fig. 9).

Azadirachta indica (neem plant) x Rhipicephalus sanguineus ovaries
In this sense, another plant of the Meliacea family (Azadirachta indica) or "neem" plant, have supplied actives products (extracted from leafs and seeds), which are effective to cause the reproduction damage in the ticks, by avoiding the maturation of oocytes causing consequently inhibition of the proliferation of ticks new generations.According by Denardi et al (2010) aqueous extracts obtained from leafs of neem, caused serious damages in the ovary cells of R. sanguineus engorged females, including morphological irregularities in the oocytes shape, changes in the yolk granulation and modifications in the germinal vesicle (oocytes nucleus) which contain the genetic material and consequently inhibing the progress of vitellogenesis, important process to reproductive success of this species (Fig. 10, 11).Azadirachta indica would be an interesting alternative way to control ticks, since the azadiractin the active compound, can be easily obtained by management of the leafs to produce the extracts (aqueous and alcoholic), as well as saving non targets organisms once this plant is also consumed by humans in several products (like tea) in several countries in the world, meanly by Indian people.Besides these alternative option to control the ticks the BCSTM researcher in Brazil showed the effects of synthetic chemicals acaricides (permethrin and fipronil) in reproductive and salivary systems of R. sanguineus and demonstrate that lower doses and concentrations of acaricides would be used to prevent the ticks reproduction and feed, minimizing the damages to non-target individuals as well as to the environment (Roma et al., 2010, Oliveira et al. 2010).The results presented here have also shown that doses of synthetic acaricides would be still efficient and much smaller and much less harmful to non-target organisms and to the environment being enough to reduce the harm caused by these ticks.This study confirm that chemical agents act by reducing and/or preventing the reproduction of females of R. sanguineus ticks, since many of the oocytes from individuals subjected to different acaricides exhibited major changes in the germ cells in relation to the control group.Besides this, the natural compounds can be used in the management and combating of ticks within a context of sustainability of the systems, resulting in reduction of damage both to the hosts and environment (alternative tick control).

Fig. 5 .Fig. 6 .
Fig. 5. Transmission Electron Micrographs (TEM) of semi-engorged females salivary glands of the tick Rhipicephalus sanguineus subjected to the concentration of 10 ppm of fipronil.A. Detail of completely disorganized cell where the irregular nuclei (n) with invaginations in the envelope nuclear (ne).B. Detail of the cytoplasm showing disarrange of the cytoskeleton (ck).C. Detail of the spherocrystals (ca) with completely irregular morphology.D-H.Detail of nuclei in process of cell death with invaginations (arrow) in the nuclear envelope (ne) and cytoplasmic vacuolation (v).Note in G, nuclei (n) with ring-shaped nucleolus (nu), typical characteristic of cells in cell death process.H. Apoptotic cell with irregular (arrow) nuclei (n).I. Detail of apoptotic body (ab) presenting extense process of vacuolation (v) and secretion granules (sg) (Pereira et al, 2011).Bars: B, F-G=1 µm; C-E, H = 2 µm; A, I = 5 µm.