Cytotoxicity of fipronil on mice liver cells.

In the present study, mice livers were examined following exposure to different doses of fipronil (15, 25, and 50 mg/kg). Histological and histochemical techniques were used to determine the cytotoxic potential of this compound and to assess the damage it caused to livers. Mice were divided into four groups: control group and groups I, II, and III were exposed to 15, 25, and 50 mg/kg fipronil, respectively. Our findings revealed cytological, morphohistological, and histochemical alterations in liver cells of animals from groups I, II, and III compared to group control animals. These changes included Kupffer-cell proliferation, hepatocyte hypertrophy, accumulation and distribution of proteins, polysaccharides, lipids, and vacuoles in the cytoplasm of hepatocytes, and congestion of blood vessels. These phenotypes mainly characterize the following: (a) autophagic processes, (b) steatosis, and (c) cell death by necrosis, which demonstrate the damage caused by fipronil on nontarget organisms in artificial conditions.

Ultrastructural analysis of the oocytes of female Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks subjected to the action of Azadirachta indica A. Juss (neem).

The present study provides ultrastructural information about the acaricidal effects of neem (Azadirachta indica A. Juss) on the ovaries of R. sanguineus engorged females. In general, the main damage caused in the oocytes was alteration in the shape of the cell and of the germinal vesicle, ring-shaped nucleolus, cytoplasmic vacuolation, and disorganization of the organelles and of the cell membranes (including the chorion), all of which indicate that these cells could be in the process of death. The results showed that azadirachtin would be an efficient acaricide agent for inhibiting and/or neutralizing the reproduction process of R. sanguineus females, impairing the reproductive ability of this species.

Fipronil (active ingredient of acaricide Frontline®) acting on the mice thyroid.

Fipronil, active ingredient of Frontline® acaricide, is a phenyl-pyrazolic derivative, and its efficacy in the elimination of several plagues, even in lower concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the present study had the objective to evaluate the effects of different doses of fipronil in mice and determine its cytotoxic potential and possible compromises on nontarget organisms in artificial conditions. The results showed that animals exposed to fipronil presented significant alterations in the thyroidean tissue with evident follicular disorganization, decreased in the size of most follicles culminating with the rupture of smaller follicles and still with posterior fusion in a big and irregular "follicle" filled with colloid. The present study also revealed that the action of fipronil not only causes disorganization in the thyroidean tissue but alsoalters the chemical composition of the colloid itself (prehormone), modifying its proteic basis, which was confirmed by histochemical test for protein detection, making it clear that, in thyroids exposed to fipronil, the colloid was weakly positive or even negative for the presence of proteins in its constitution.

Genotoxic and mutagenic effects of fipronil on mice.

The toxic effects of several compounds on ecosystems are not restricted to ecological disturbances, and may also affect long-term human health. Fipronil is highly efficient in the control of pests, including those resistant to pyrethroid, organophosphate, and carbamate insecticides. Relatively little is known about the action of fipronil in vertebrates. This study was aimed to evaluate the genotoxic and mutagenic potential of this compound in mice exposed to different doses and demonstrates the damage caused by fipronil on non-target organisms in artificial conditions. Mice were divided into five groups: group I=30% of DL(50) (15 mg/kg), group II=50% of the DL(50) (25 mg/kg), group III=DL(50) (50 mg/kg), group IV=negative control, and group V=positive control. Peripheral blood was collected for the comet assay (24 h after exposure) and the micronucleus test (24, 48 and 72 h after exposure). Our findings revealed that doses of 15 mg/kg (group I) and 25 mg/kg (group II) of fipronil did not have genotoxic or mutagenic effects. Only the highest dose tested (50 mg/kg) induced DNA damage 24 h after exposure, indicating the mutagenic potential of fipronil. Therefore, this or higher doses are not recommended, as they may be toxic to non-target organisms.

Action of the chemical agent fipronil (active ingredient of acaricide Frontline®) on the liver of mice: an ultrastructural analysis.

Fipronil, active ingredient of the acaricide Frontiline®, is a phenyl-pyrazolic derivative, and its efficacy in the elimination of several plagues, even in low concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the objective of this study was to evaluate the effects of different dosages of fipronil on the liver of mice in artificial conditions. Results showed that the animals exposed to fipronil present significant ultrastrucutural changes in hepatic cells with evident cellular and cytoplasm disorganization in hepatocytes characterized by an increase in the number of organelles, mainly mitochondria and rough endoplasmic reticulum, organelles that, in the case of the exposed animals, were probably responsible for the enzymes' synthesis that have the function of inactivating the toxic metabolites. A fat accumulation in the hepatocytes' cytoplasm (steatosis) was observed, in addition to extended vacuolated areas, mainly in regions next to the cell nucleus. Alterations observed in the nuclei of the hepatocytes pointed out cell death processes. Moreover, Kupffer cells increased in number (hyperplasia) suggesting an increase in the phagocytic activity of the liver in the exposed animals.

Inhibitory action of neem aqueous extract (Azadirachta indica A. Juss) on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks.

The present study revealed unheard of data about the action of aqueous extracts of neem leaves (Azadirachta indica) on the vitellogenesis of Rhipicephalus sanguineus ticks, proving that these extracts in 10 and 20% concentrations do not have the potential to kill the females; however, in lower concentrations (10%) provokes great morphological alterations in germinative cells such as the emergence of extended cytoplasmic vacuolization areas as well as the fragmentation of the germinal vesicle, even in those oocytes which were in initial stages of development (I-III), showing that neem is a potent agent which acts impeding one of the main metabolic stages of the ticks, i.e., the reproduction. In oocytes in final stages of development (IV-V) azadirachtin (neem's active principle) caused significant reduction in the size and quantity of proteic granules of the yolk and the inversion of their localization where the smaller granules before inside the cell (normal oocyte) were posteriorly observed in the periphery, and the bigger ones in the central region. Thus, the study showed that the alterations found both in the oocytes and in the pedicel cells indicated that azadirachtin acts on the process of tick's reproduction and signalizes that this plant can be used in the future to control ticks with the advantage of not being aggressive to nontarget organism or the environment. Furthermore, data here obtained showed that the most significant efficiency of the aqueous extract of neem is related to the concentration of 10%, proving that higher doses would not be so efficient.

Salivary glands of Amblyomma cajennense (Acari: Ixodidae): a histological and an ultrastructural overview.

The present study on the salivary glands of semi-engorged Amblyomma cajennense females has identified the various cell types present in this tissue and allowed its morphohistochemical characterization. Marking techniques were applied to detect polysaccharides (PAS), proteins (bromophenol blue), lipids (Nile blue) and calcium (von Kossa), as well as those of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results obtained by TEM showed and confirmed that these individuals' glands are also formed by round acini that are connected to the common excretory duct through acinar and intermediate ducts. Histological data as well as ultrastructural ones showed that the glands are formed by types I, II and III acini. In this study with salivary glands polysaccharides, proteins, lipids and calcium were observed in the cytoplasm and/or cell secretion granules-both free or forming complexes, as the intensity of the marking varied according to the cell as well as the type of acini analyzed, showing the structural and functional complexity of the tick salivary glands, characteristics that give the multifunctional character to this organ.

Toxicity effect of the acaricide fipronil in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): preliminary determination of the minimum lethal concentration and LC(50).

Chemical acaricides, especially fipronil (active ingredient of Frontline®), are still the most effective method to control tick populations. In this study, the effectiveness of fipronil was assessed in semi-engorged females of the tick Rhipicephalus sanguineus. A protocol for an in vitro bioassay (AIT) was developed, and the LC(50) (lethal concentration 50%) and 95% confidence interval were determined. Ticks were immersed in Petri dishes with different concentrations of fipronil or distilled water for 2 min, dried, and placed in an incubator for 7 days. Dead R. sanguineus females treated with the 14 concentrations of fipronil were counted daily. Mortality results were compared with the Probit analysis, and the LC(50) and 95% confidence interval were calculated, g (95): LC(50) = 9.647 (4.711 to 13.470). This study was aimed at developing a more appropriate and updated protocol for an in vitro bioassay (AIT--adult immersion test), and providing information on the toxic potential of fipronil (elimination of ectoparasites with lower concentrations) and sensitivity of ticks, especially R. sanguineus, a pest of great interest, due to its occurrence in urban environments.

Rhyncophorus palmarum L. (Linnaeus, 1758): a morphological and histological study of the female reproductive system.

Rhyncophorus palmarum (Coleoptera) is a pest of great economic importance because of the damage caused in the plants of the Palmae family. This study showed that the female reproductive system of this beetle is composed of two ovaries of the telotrophic meroistic type invariably containing two ovarioles per ovary. Each ovariole is home to about 50 oocytes at different stages of maturation. The proximal region of the ovary is rather peculiar because it houses inside it a large quantity of bacilliform structures immersed in a glycolipoprotein substance, which has never before been described in the literature. The function of these structures is still unknown. However, it is suggested that they can function as a method of neutralizing the resistance of the plant because these insects are responsible for the transmission of the red ring disease to the palm plants.

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females.

Many studies have been conducted with plants whose extracts have the potential to be used for pest control. One of these plants is Azadirachta indica (neem), whose main active ingredient is azadirachtin, a compound shown to have acaricide and insecticide activity. Rhipicephalus sanguineus (brown dog tick) is currently considered to be an "urban pest," because of its high levels of infestation and its ability to attack humans. In the present study partially and fully engorged R. sanguineus females were exposed to aqueous extracts of neem at concentrations of 10% and 20%, and to a control treatment. The results showed that differently from what was observed in the control, the pedicel cells of females exposed to neem at both concentrations lost their original shape. In the latter cases, the cytoplasm of the cells became fully vacuolated, especially near the germinal vesicle (oocyte nuclei) and in the oocyte pole, which is in contact with the cells of the pedicel. Oocytes in early stages of development (I and II) of ticks treated with both concentrations had irregular germinal vesicle, including the presence of two nucleoli as well as fragments of these. Oocytes in stages IV and V of exposed individuals showed full granular cytoplasm with bigger yolk granules when compared to the early stages. Chorion of mature oocytes was also altered, showing folds and deformations along their entire extension. The observed changes in cells of the reproductive system of R. sanguineus, especially in the oocytes, indicated the potential of neem as a new alternative method to control these ectoparasites.

The dynamics of RNA participation in the vitellogenesis of Rhipicephalus sanguineus ticks Latreille 1806 (Acari:Ixodidae). I. Nucleoli or Cajal bodies?

To understand the morphological and histological aspects of internal systems of ticks has become important matter since these arthropods have an impact in the areas of the economy and public health. In this context, this study has provided morphological data on female germinative cells of Rhipicephalus sanguineus ticks, ectoparasites of dogs that maintain a close relationship with human on a daily basis. Oocytes of engorged females were analyzed, through the PAS reaction (detection of polysaccharides) counterstained by methyl green (detection of RNA) revealing information that allowed to infer for the first time the presence of Cajal bodies, in the germinal vesicles (nuclei) of developing oocytes, as well as showing how the RNA and the polysaccharides are involved in the dynamics of the vitellogenesis in this species.

Fat body cells of Amblyomma cajennense partially engorged females (Acari: Ixodidae) and their role on vitellogenesis process.

During the process of Arthropoda reproduction, the synthesis and uptake of proteins, carbohydrates, and lipids by oocytes is termed vitellogenesis. These compounds that will make up the yolk may be in ticks endogenously synthesized by the oocytes and/or exogenously produced by the fat body and pedicel cells. This study examined the fat body of Amblyomma cajennense ticks at the cytochemical ultrastructural level to demonstrate the presence of lipids, proteins and carbohydrates in trophocytes. The lipids were detected in higher quantity than proteins and carbohydrates in the fat body cells, suggesting that the role of the fat body in tick is stored lipids and carbohydrates to convert them in energy, or still they could be used with cell structural purpose. The electrophoresis technique applied at A. cajennense fat body demonstrated specifically the molecular mass of proteins: about 98kDa. By the other hands, the fat body is not the organ responsible for the synthesis of the yolk protein, role probably performed by the pedicel cells.

New morphological data on fat bodies of semi-engorged females of Amblyomma cajennense (Acari: Ixodidae).

The present ultramorphological, histological and ultrastructural study on the fat body of semi-engorged females of Amblyomma cajennense revealed that this tissue is diffuse and consists of strands of cells surrounding the tracheal trunks. Morphometric analysis showed that the cellular and nuclear areas of round-shaped trophocytes are larger than those of cuboidal trophocytes, indicating that the arrangement of the former provides more contact area with the haemolymph. In this species, the fat body is found right underneath the integument and around organs. It consists of two cell types that despite distinct morphological characteristics and locations in the tissue, present the same histological features. In this study, these cells were termed cuboidal trophocytes when arranged as strands of cells and present in larger numbers, and round-shaped trophocytes when lying on these strands and observed in fewer numbers. Histological observations revealed that both types of trophocytes have one nucleus in their cytoplasm and also exhibit numerous vacuoles of different sizes and contents. Ultrastructural examination revealed that the organelles more frequently observed were the vesicular and lamellar rough endoplasmic reticulum, and mitochondria with tubular crests, indicating that they might be involved in lipid synthesis. Smooth endoplasmic reticulum was not observed. Cuboidal trophocytes arranged in strands, despite being closely associated, do not exhibit fused plasma membranes. Rather, the fusion of basal lamina of two neighboring cells is occasionally observed, acting as a selective permeability barrier. Here, a new terminology for tick fat body is proposed. It is based on fat body location (parietal, when located right underneath the integument instead of peripheral; and perivisceral, when located around organs instead of central) terminologies previously suggest by Obenchain and Oliver and for the cells constituting them, cuboidal trophocytes when arranged as strands, and round-shaped trophocytes when lying on these strands. Nephrocytes were not observed in semi-engorged females of A. cajennense.

Amblyomma cajennense (Acari: Ixodidae): salivary gland cells of partially engorged females ticks and the production of lipid by their mitochondria.

Morphologically, the salivary glands of ticks are paired structures consisting of a secretory and an excretory portion, lacking a reservoir for the storage of the secretion. The secretory portion is composed in females by cells that form acini classified into the types I, II, and III. The excretory possess a major duct, from which arise several intermediate ducts that then subdivide to form the canaliculi or acinal tubules, which end at the acini from where they collect the secretion. The present study describes the ultrastructural changes that occur in the mitochondria of cells of the acini I, II, and III in the salivary glands of partially engorged females of the Cayenne tick Amblyomma cajennense. The results show that this organelle exhibits completely disarrayed crests due to the presence of lipidic material inside the matrix and between the crests, thus demonstrating their participation in the production of the lipids that would be used structurally by the cells. These organelles with ultrastructural changes were denominated derived mitochondria.

Morphological, histological, and ultrastructural characterization of degenerating salivary glands in females of the cattle-tick Rhipicephalus (Boophilus) microplus (CANESTRINI, 1887) (Acari: Ixodidae).

The present study describes the morphology and ultrastructure of the salivary glands of semi-engorged females of the southern cattle-tick Rhipicephalus (Boophilus) microplus. The acini that compose these glands, at that specific feeding stage, show cells featuring degenerative process of the salivary glands, such as: vacuolated cytoplasm, condensed chromatin, fragmented nuclei, and presence of apoptotic bodies. In addition, the presence of microorganisms was detected, with morphology typical of protozoa, inside these organs.

Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

This study presents the morphology of the ovary, as well as the process of the vitellogenesis in oocytes of the tick Rhipicephalus sanguineus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a wall formed by small epithelial cells with rounded nuclei which delimit the lumen. The oocytes in the different developmental stages in this tick species were classified into five stages (I-V). They remain attached to the ovary during vitellogenesis by a cellular pedicel and afterwards the mature oocytes (stage V) are released into the ovary lumen.

Comparison of the external morphology of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks from Brazil and Argentina.

In the present study the external morphology of semi-engorged Rhipicephalus sanguineus females ticks from Brazil and Argentina were compared by scanning electron microscopy. Many differences were detected among the R. sanguineus collected at the two localities, such as body size, shape of the genital pore, and morphology of the sensory structures. All these characteristics are fundamental for the diagnosis of species of the genus Rhipicephalus and thus indicate the need for further comparisons and, the taxonomical revision of this species of tick in the Neotropics.

Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae).

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-tick Boophilus microplus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a lumen delimitated by a wall of small epithelial cells with rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of the cellular components would be undergoing a degenerative process and/or reabsorption.

Morphological characterization of the ovary and vitellogenesis dynamics in the tick Amblyomma cajennense (Acari: Ixodidae).

In this work we describe the internal morphology of the female reproductive system of the cayenne tick Amblyomma cajennense. This system is represented by a panoistic ovary, which lacks nurse cells in the germarium. This ovary consists of a single tube, in which a large number of oocytes develop asynchronously, thus accompanying the processes of yolk deposition in the oocytes. The oocytes were classified into stages that varied from I to V, according to: cytoplasm appearance, presence of the germ vesicle, presence of yolk granules, and presence of chorion. The study of vitellogenesis dynamics suggest that the yolk elements are deposited in the oocyte following a preferencial sequence, in which the lipids are the first to appear, followed by proteins an finally by the carbohydrates. In this way the yolk of A. cajennense ticks have these three elements that may be free in the cytoplasm or chemically bounded forming glycoprotein or lipoprotein complexes.