ORIGINAL PAPER

Fluazuron-induced morphophysiological changesin the cuticle formation and midgut of Rhipicephalussanguineus Latreille, 1806 (Acari: Ixodidae) nymphs

Patrícia Rosa de Oliveira & Izabela Braggião Calligaris &

Gislaine Cristina Roma & Gervásio Henrique Bechara &

Maria Izabel Camargo-Mathias

Received: 1 May 2012 /Accepted: 24 August 2012# Springer-Verlag 2012

Abstract The present study demonstrated the effects of thearthropod growth regulator, fluazuron (Acatak®), in the for-mation of the integument and digestive processes of Rhipice-phalus sanguineus nymphs fed on rabbits treated withdifferent doses of this chemical acaricide. For this, threedifferent doses of fluazuron (20, 40, or 80 mg/kg) wereapplied “pour on” to the hosts (groups II, III, and IV), as wellas distilled water to the control group. On the first day aftertreatment (24 h), the hosts were artificially infested with R.sanguineus nymphs. After full engorgement (7 days), thenymphs were removed, placed on labeled Petri dishes, andkept in biochemical oxygen demand incubator for 7 days. Theengorged nymphs were then taken for morphological, histo-chemical, and histological analyses. The results showed theoccurrence of cytological, morphohistological, and histo-chemical alterations in the integument and midgut of nymphsfrom all the different treated groups. These alterations oc-curred at cuticular level in the subdivisions of the cuticle,related to the size of the digestive cells, amount of accumu-lated blood elements, and digestive residues, as well as thepresence of vacuoles in the cytoplasm of the digestive cells.Thus, this study demonstrated that fluazuron acts on theintegument and midgut cells of R. sanguineus nymphs fedon treated rabbits and pointed out the possibility of the use ofthis chemical—which is more specific, less toxic, and less

harmful to the environment and nontarget organisms—in thecontrol of R. sanguineus, at least in the nymphal stage of itsbiological cycle.

Introduction

The brown dog tick Rhipicephalus sanguineus is undoubtedlythe most important tick species in the veterinary field (Labrunaand Pereira 2001; Soares et al. 2006; Dantas-Torres 2008) as itis the vector and reservoir of several pathogens and producesdirect and indirect damage to the hosts, such as blood spolia-tion and cutaneous lesions (Sonenshine 1991). Over the lastyears, due to globalization, climatic changes, and also forhaving occupied the urban areas, it has become an importanturban plague and thus of great interest for public health (Paz etal. 2008; Dantas-Torres 2010; Roma et al. 2012). The chemicalcontrol via synthetic acaricides has been the main tool to fightthe ticks, despite the problems related to its application, theaccumulation of residues on food, its broad spectrum, and thedevelopment of resistance by the target species (Crampton etal. 1999; Pruett 1999; Mencke et al. 2003; Oliveira et al. 2008;Oliveira et al. 2009).

Although literature has shown that most acaricides act onthe tick’s nervous system, a new category of substances isemerging in the market, the arthropod growth regulators(AGR) (Coop et al. 2002), which act in the process ofecdysis, either interfering in the metabolism of chitin or inthe production of hormones which are involved in thisprocess. This leads to morphophysiological changes duringthe arthropod development and metamorphosis, resulting inthe deposition of nonviable eggs and in the complete inhibi-tion of the emergence of new individuals (Graf 1993; Fournetet al. 1995; Pawar et al. 1995; Hoffmann and Lorenz 1998;Taylor 2001).

P. R. de Oliveira : I. B. Calligaris :G. C. Roma :M. I. Camargo-Mathias (*)Institute of Biosciences, Sao Paulo State University-UNESP,Av. 24 A, no 1515, P.O. Box 199, 13506-900, Rio Claro, SP, Brazile-mail: micm@rc.unesp.br

G. H. BecharaFaculty of Agronomic and Veterinary Sciences, UNESP,Via de Acesso Prof. Paulo D. Castellane, s/n,14884-900, Jaboticabal, SP, Brazil

Parasitol ResDOI 10.1007/s00436-012-3103-7

Fluazuron (active ingredient of Acatak®) is an importantarthropod growth regulator (Correia 2003). It inhibits thesynthesis and/or deposition of chitin, one of the main com-ponents of arthropod cuticle, and was the first AGR to beregistered as acaricide. However, its use is still limited tofew tick species (Bull et al. 1996). In fact, there are fewstudies in the literature about its use in the control of R.sanguineus. In the chitin structure, the nitrogenated poly-saccharide serves as a matrix for the deposition of sclero-protein. It should be stressed that the cuticle is responsiblefor the mechanic protection against predator’s attacks, andits permeability prevents desiccation; however, it is also alimiting factor, as it does not allow the growth of theindividuals (Splinder 1990). The actual growth and devel-opment of the arthropods occurs only during the shortperiod of ecdysis as the old chitin cuticle is cast off, anda new one is produced (Chapman 1982). In addition,chitin is also part of the peritrophic membrane found inthe arthropods midgut. This layer surrounds the foodbolus, delimiting the area where the digestive events occurand, consequently, protecting the animal from its own diges-tive enzymes and microorganisms ingested during blood in-gestion (You et al. 2003).

Due to the critical role played by chitin in the survivaland success of the arthropods, the fact that it is not found invertebrate, the scarcity of efficient highly selective controlmethods which are harmless to human beings, to the envi-ronment, and nontarget organisms, research on AGRs hasbeen stimulated, becoming increasingly important in thepresent context (Bowman et al. 1997).

Concerning the information above, this study aimed toverify the effect of different doses of the AGR fluazuron(Acatak®) in the integument and midgut epithelium of R.sanguineus nymphs through morphological, histological,and histochemical studies. This would provide criticalinformation which could help in the development ofnew methods to control R. sanguineus ticks and/or im-prove the existing methods of control, making them morespecific, less toxic, and less harmful to the environmentand nontarget organism and less resistance inducing forthe ticks.

Material and methods

Chemical compound

Fluazuron is a compound of the benzoylphenylurea chemicalclass, chemical name N-[[4-chloro-3-[3-chloro-5-(trifluoro-methyl)pyridin-2-yl]oxyphenyl]carbamoyl]-2,6-difluoroben-zamide and CAS 86811-58-7. For the experiments, it wasobtained from the acaricide Acatak®, a commercial productmanufactured by Novartis.

R. sanguineus ticks (Latreille 1806)

R. sanguineus-fasting nymphs were used and collected froma colony kept in controlled conditions (28 °C, 85 % ofhumidity and photoperiod of 12 h) in a biochemicaloxygen demand (BOD) incubator, in a room of the Biol-ogy Department Biotery (IB), UNESP, campus of RioClaro, SP, Brazil.

Hosts

Twelve naïve New Zealand White rabbits weighing 3–3.5 kg, from the General Biotery, UNESP, Campus of Botu-catu/SP, Brazil, kept in controlled conditions at the Bioteryof UNESP, Campus of Rio Claro, SP, Brazil, were usedthroughout the experiment. During the experiment, the ani-mals were kept in individual cages and received water andcommercial food ad libitum. The Ethics Committee forAnimal Experimentation of UNESP/SP/Brazil, protocolno. 6334/2011, approved the study.

Doses of fluazuron

The initial dose of fluazuron for the treated groups wasdefined based on the recommendations of Acatak® manu-facturer (2.5 mg in 1 mL of fluazuron/kg of body weight).Several doses were evaluated in preliminary tests (pilots) bydiluting fluazuron (Acatak®) in distilled water and a higherdose (over 1 mL/kg of the product) applied “pour on” on thehosts. After this bioassay, the efficacy of fluazuron and thelevel of susceptibility of the nymphs were evaluated for R.sanguineus nymphs, and the lethal doses DL95 and DL50

determined were 100 and 20 mg/kg, respectively. In thisstudy, the doses corresponded to 20, 40, and 80 mg offluazuron/kg of body weight. All the doses of fluazuronwere kept in labeled volumetric flasks until the applicationto the hosts. Triplicate was then performed (three repetitions)of each dose.

Experimental model

Nymphs were divided into three treated groups of threerabbits each: group II (20 mg of fluazuron/kg of bodyweight), group III (40 mg of fluazuron/kg of bodyweight), and group IV (80 mg of fluazuron/kg of bodyweight). Fluazuron was applied pour on to the backline.Three another rabbits, used as control (group I), receiveddistilled water (placebo) applied in the same way andvolume.

On the first day (24 h) after the administration of flua-zuron or distilled water, the artificial infestation was per-formed as described elsewhere (Bechara et al. 1995).Briefly, each rabbit was infested with R. sanguineus nymphs

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delivered inside feeding chambers glued to the shaved backof the hosts with the aid of a syringe.

The first observation was performed after 24 h, timeneeded for the nymphs’ attachment. The following obser-vations were done once a day until full tick engorgement.Soon after the feeding process was completed, theengorged nymphs (metanymphs) were dropped off thehosts, collected, and placed on labeled Petri dishes forfurther observations. The dishes containing the engorgednymphs (metanymphs) were placed inside a BOD incuba-tor and monitored for 7 days. This period was establishedto complete 14 days after treatment with fluazuron (fromthe day of application of the chemical on the host), 7 daysto complete the engorgement, and 7 days of observation,considering that a maximum of 14 days should be enoughfor the chemical to cause death of the ectoparasites,according to Acatak®’s manufacturer information leaflet.After the monitoring for 7 days, all the engorged nymphs(metanymphs) were forwarded to histological and histochem-ical techniques.

Methods

Histology

All the nymphs were fixed for 24 h in 4 % paraformalde-hyde, dehydrated in ethanol, embedded in Leica resin for24 h at 4 °C, and transferred to plastic molds previouslyfilled with polymerized Leica resin. After resin polymeriza-tion, all the blocks were sectioned at 3-μm thickness slicesusing a Leica RM 2255 microtome (Bio Rad) and stainedwith hematoxylin and eosin, following routine histologicalprocedures. The glass slides were examined in a MoticBA300 photomicroscope. This device and other equipmentwere from the Histology Laboratory of the Biology Depart-ment at the Biosciences Institute, UNESP, campus of RioClaro, SP, Brazil.

Histochemistry

To detect changes such as presence or absence, frequency,and distribution of proteins, polysaccharides, and lipids inthe nymphs of control and fluazuron-treated groups, histo-logical sections were prepared for the histochemical techni-ques listed below.

Periodic acid-Schiff technique for polysaccharide detection(according to Junqueira and Junqueira 1983) The engorgednymphs were fixed with aqueous Bouin. Slides with sec-tions were immersed for 10 min in 0.4 % periodic acid,washed with distilled water, and stained with Schiff’sreagent for 1 h in the dark. The material was then washedthrice with sulfur water for 3 min each and rinsed with tap

water for 30 min. After drying, slides were clarified withxylol and mounted in Canada balsam.

Bromophenol blue staining for protein detection (accordingto Pearse 1985) The engorged nymphs were fixed with 4 %paraformaldehyde. All slides were stained with bromophe-nol blue for 2 h at room temperature. Afterwards, they werewashed with 0.5 % acetic acid for 5 min and tap water for15 min; slides were quickly immersed in tertiary butylalcohol, allowed to dry at room temperature, clarified, andmounted in Canada balsam.

Baker’s method for lipid detection (according to Baker,1946) The engorged nymphs were fixed with formol calci-um for 15 h and transferred to dichromate calcium for 18 h.Afterwards, they were washed with distilled water, and theslides were immersed in hematein for 5 h. The material wasrinsed, differentiated in Weigert’s solution, and washed withdistilled water. After drying, slides were mounted with glyc-erin and covered with cover slips.

Results

Histology

Integument

Group I (control) The histological results obtained showedthat the integument of engorged R. sanguineus nymphs iscomposed of a cuticle and an epithelial layer below it(Fig. 1a, b). The cuticle is subdivided into two main regions:the outer and thinner region is called epicuticle, and theinner and thicker region is the procuticle. The latter stillpresents two distinct and well-defined areas: the exocuticle(close to the epicuticle) and the endocuticle (adjacent to theepithelial layer) (Fig. 1a, b).

A simple layer of cells supported by a basal mem-brane forms the epithelial layer. These epithelial cells,which involve all the individuals’ bodies, are small andflat, with flat and strongly stained nuclei (Fig. 1a, b). Insome regions, the separation between the cuticle andepithelial cells can be observed, with the emergence ofa space between these layers, probably an exuvial space(Fig. 1a, b).

Group II The nymphs fed on rabbits treated with 20 mg offluazuron/kg of body weight present the integument withalready significant histological alterations compared withthe ones form the control group (Fig. 1c). The epicuticleand procuticle are thinner than the ones found in the controlgroup. The two subregions (exocuticle and endocuticle) aresmaller; however, the endocuticle is much smaller (Fig. 1C).

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The epithelial cells are larger, flat, or cubic, and the nucleivary from flat to round-shaped (Fig. 1c).

Group III The individuals from group III present morehistological alterations in the integument when compared withthe ones from the previous groups (Fig. 1d). These alterationsare related to the difficulty to distinguish the subdivisions of

the cuticle, as well as the disorganization and even disappear-ance of some regions of the integument (Fig. 1d). The epicu-ticle presents the same characteristics found in this same layerin the group II (Fig. 1d).

There are several segments between the epicuticle andthe epithelial layer in which the procuticle is not subdivided,forming a single layer (Fig. 1d). The epithelial cells are

Fig. 1 Histological sections offluazuron-treated nymphs tickR. sanguineus. a–j Hematoxy-lin and eosin staining. a–bControl group, c group II(20 mg/kg), d group III(40 mg/kg), e–f group IV(80 mg/kg), g–j control group;c cuticle, edc empty digest cells,ep epithelial cells, epc epicuti-cle, enc endocuticle, excexocuticle, l lumen, m musculartissue, prc procuticle, stc stemcells (generative cells), spcspent cells. Scale bars: a–g0.02 mm, h 0.1 mm, i 0.02 mm,j 0.1 mm

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larger than the ones from the control group, however,smaller than the ones from group II (Fig. 1d).

Group IV The integument of the nymphs in the group IVpresents significant modifications when compared tothose observed in the individuals from the other groups(Fig. 1e, f). These alterations are mainly found in theorganization and in the size of the cuticle, which is thinnerand does not present subdivisions. The epicuticle and theprocuticle with exocuticle and endocuticle seem to be fus-ing into a single layer (Fig. 1e, f). The epithelial layer is notcontinuous around the body of the nymphs, showing somepoints of interruption. The shape of the cells varies fromcubic to flat (Fig. 1e, f).

Midgut

Group I (control) An epithelial wall supported by a basalmembrane and a thin layer of muscular tissue forms themidgut of engorged R. sanguineus nymphs after 7 days ofobservation (Fig. 1g). The epithelial wall is pseudostratifiedand formed by digestive and generative cells (Fig. 1g–j).

There are few very small generative cells (stem cells), withsmall, central, and round-shaped nuclei, and totally supportedby the basal membrane. Their cytoplasm and borders are rarelyseen once the large digestive cells cover them (Fig. 1g).

The digestive cells predominate in the midgut of R.sanguineus nymphs. These cells go through several devel-opment stages during the process of engorgement andphases of the individuals’ life cycle. The digestive cells ofthe nymphs from this study are found in two stages: thelarge spent cells and the small empty digest cells. The firstones are full of large and round-shaped endosomes withblood (mainly red cells), ingested from the host duringengorgement; digestive vacuoles; and large and alsoround-shaped haematin residual bodies (final product ofthe intracellular digestion, which is being accumulated inthe cellular interior). The latter presents few and smallvacuoles in the cytoplasm, which probably have alreadyrelease haematin residues towards the lumen. Nuclei of bothtypes of cells are large, round-shaped, central, and stronglystained; however, the cell border was only detected in thetype of digestive cells, where their cubic or triangular shapecould be observed (Fig. 1g–j).

The lumen of the midgut could not be observed, since itis completely filled with blood and cells from the basalmembrane and/or with the residues released during the celllysis present in other digestion phase or life cycle, and isincorporated to the fecal material (Fig. 1g–j).

Group II The midgut of the nymphs engorged on rabbitstreated with 20 mg of fluazuron/kg of body weight did not

present significant modifications either in the morphologyor number of cells. The generative cells and empty digestcells, as well as the muscular layer around the lumen, remainwith the same characteristics found in the control group(group I) (Fig. 2a, b). Only the digestive spent cells seemto accumulate less blood in the endosome and digestivevacuoles, as well as residual bodies of haematin whencompared with the ones of the individuals from the controlgroup (Fig. 2a, b).

Group III The nymphs from group III presented more alter-ations in the midgut compared with the individuals from theprevious group and control group (group I). These altera-tions occurred in both stages of digestive cells (spent cellsand empty digest cells), as well as in the lumen of themidgut (Fig. 2c, d).

The digestive spent cells are smaller and with moderatenumber of endosomes, digestive vacuoles, and residual bod-ies of haematin of various sizes distributed in the cytoplasm(Fig. 2c, d). Some empty digest cells present small wavingin their apical plasma membrane (Fig. 2c, d). The lumen ofthe midgut of this group’s nymphs can be detected, showingless blood being stored, and residues that are formed afterthe hosts’ treatment (Fig. 2c, d).

Group IV The midgut of the nymphs engorged on rabbitstreated with 80 mg of fluazuron/kg of body weight showssignificant histological alterations when compared withthose from control groups II and III. These alterations arefound in both the digestive spent cells and empty digest cellsand also in the midgut lumen (Fig. 2e–h).

The digestive spent cells are much smaller when com-pared with those of the individuals from previous groupsand present small and few endosomes, digestive vacuoles,and residual bodies of haematin in the cytoplasm (Fig. 2e–h). The empty digest cells are rarely found. When present,they show large invaginations in the plasma membranetowards the lumen. Large vacuolated regions are observedin the cytoplasm, mainly close to the lumen (Fig. 2e–h). Thelumen of the midgut is easily seen, containing few residuesfrom the digestive processes (Fig. 2e–h).

Histochemistry

Periodic acid-Schiff staining for neutral polysaccharidedetection

Integument: group I (control) The periodic acid-Schiff(PAS) technique revealed the presence of polysaccharidesin the integument of the individuals from the control group.The concentration of this element gradually increases fromthe epicuticle to the epidermal cells; i.e., the epidermal cells

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are strongly stained; the endocuticle is moderately stained,and the outer layers of the cuticle (epicuticle and exocuticle)are weakly stained by the test (Fig. 2i).

Integument: group II This histochemical test reveals thesmall number of polysaccharides in the integument of theindividuals from this group. All the layers of the cuticle

(epicuticle and procuticle with exocuticle and endocuticle)are weakly stained by PAS. Only the epidermal cells reactmoderately due to the presence of PAS-positive granulation(Fig. 2j).

Integument: group III The application of this histochemicaltest revealed the presence of little polysaccharide in the

Fig. 2 Histological sections offluazuron-treated nymphs tickR. sanguineus. a–h Hematoxy-lin and eosin staining. a–bGroup II (20 mg/kg), c–d groupIII (40 mg/kg), e–h group IV(80 mg/kg). i–o PAS staining todetect polysaccharides. i Controlgroup, j group II (20 mg/kg),k group III (40 mg/kg), l groupIV (80 mg/kg), m control group,n group II (20 mg/kg) and groupIII (40 mg/kg), o group IV(80 mg/kg). c cuticle, edc emptydigest cells, ep epithelial cells,epc epicuticle, enc endocuticle,exc exocuticle, prc procuticle,l lumen, spc spent cells. Scalebars: a–g 0.02 mm, h 0.1 mm,i–o 0.02 mm

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integument of the individuals. The distribution of this ele-ment is similar to the one found in the integument of theindividuals from the previous group (group II): weaklystained and/or no reaction to PAS in all the layers of thecuticle and epidermal cells moderately stained (Fig. 2k).

Integument: group IV The histochemical test for the detec-tion of polysaccharides shows the small number of theseelements in the integument of the individuals from group IV.The cuticle (without subdivisions) is negative or weaklystained by the test, and the epidermal cells are moderatelystained (Fig. 2l).

Midgut: group I (control) This histochemical test revealsfew polysaccharides in the midgut of engorged R. sanguineusnymphs, demonstrated by the weak reaction of most cells(Fig. 2m). The digestive spent cells do not react or reactweakly to the test (Fig. 2m). However, the empty digest cellspresent few and small PAS-positive vacuoles (Fig. 2m). Asthis technique is not specific for the detection of nuclei, thegenerative cells (stem cells), muscular cells, and basal mem-brane (identified in light microscopy mainly by the nuclei) arenot observed (Fig. 2m).

Midgut: group II Through this histochemical test, the pres-ence of little polysaccharide in the midgut of nymphs fromgroup II is detected, as in the control group. All the cellspresent similar reaction to the control group (Fig. 2n).

Midgut: group III The application of PAS shows the pres-ence of few polysaccharides in the midgut of the nymphsfrom this group. The staining is similar to the one found inthe individuals from the control group (Fig. 2n).

Midgut: group IV The midgut of nymphs engorged onrabbits treated with 80 mg of fluazuron/kg of body weight isweakly stained to PAS in all the cells. Even the empty digestcells, which previously presented PAS-positive vacuoles,started to react weakly and homogenously to the test in alltheir extension (Fig. 2o).

Bromophenol blue staining for protein detection

Integument: group I (control) Through this histochemicaltest, the presence of large amounts of proteins in the integu-ment of nymphs from control group is detected. The epicuti-cle, the exocuticle, and the epidermal cells react strongly. Theendocuticle is weakly stained (Fig. 3a–c).

Integument: group II The results show the large amounts ofprotein, demonstrated by the strong staining in the integumentof nymphs from group II. All the cuticle layers are stronglystained, and the epidermal cells react moderately (Fig. 3d).

Integument: group III The application of this histochemicaltest shows the presence of large amounts of protein in theintegument of nymphs fed on rabbits treated with 40 mg offluazuron/kg of body weight. The cuticle layers react strong-ly, and the epidermal cells react moderately (Fig. 3e).

Integument: group IV This histochemical test shows a largenumber of protein elements in the integument of nymphs.The cuticle (without subdivisions) reacts strongly to the test,while epidermal cells react moderately (Fig. 3f).

Midgut: group I (control) This histochemical test showslarge amounts of protein in the midgut cells of engorged R.sanguineus nymphs after 7 days of observation (Fig. 3g, h).The digestive spent cells are strongly stained, which wasdemonstrated by large and numerous endosomes, digestivevacuoles, and haematin residual bodies in the cytoplasm,preventing the nuclei observation. The empty digest cellsshowed strongly stained nuclei and few and small positivevacuoles in the cytoplasm (Fig. 3g, h). The nuclei of stem cellsand muscular cells, as well as the basal membrane, were alsostrongly stained (Fig. 3g, h).

Midgut: group II Histochemical test bromophenol bluereveals that the nymphs’ midgut is strongly stained forprotein, although the digestive spent cells are less stainedthan the ones found in nymphs from the control group(Fig. 3i, j).

Midgut: group III Results show large amounts of protein inthe midgut of R. sanguineus nymphs. However, the indi-viduals from the previous groups showed more positive-ness (Fig. 3k, l). The digestive spent cells are stronglystained, with positive endosomes, digestive vacuoles, andhaematin residual bodies in their cytoplasm. As the num-ber of cytoplasm elements is smaller, the positive nucleican now be observed. There are no histochemical alter-ations for the empty digest cells, but only histologicalones (Fig. 3k, l). The lumen can be observed, adjacentto the apical region of the digestive cells, and is weaklystained (Fig. 3k, l).

Midgut: group IV The midgut of nymphs from this grouppresents a great decrease in the amounts of protein whencompared with the previous groups (Fig. 3m, n). The diges-tive spent cells present few and small positive endosomes,digestive vacuoles, and haematin residual bodies, as well asdetectable and positive nuclei, while the empty digest cellspresent large vacuolated regions negative to the test(Fig. 3m, n). The lumen presents a different reactiondepending on the region involved: the regions next to thedigestive cells react moderately, while the central regionsare weakly stained (Fig. 3m, n).

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Baker’s technique for lipid detection

Integument: group I (control) Through the application ofBaker histochemical test, a small amount of lipid in theintegument of the individuals can be observed. The tworegions of the procuticle (endocuticle and exocuticle) arenegative to the test; however, the epicuticle and epidermal

cells are positive. In addition, the nuclei of epidermal cellsare stained due to hematein, solution obtained through theoxidation of hematoxylin. The Fig. 4a illustrates thesechanges.

Integument: group II The histochemical test for the detec-tion of lipids reveals a large number of these elements in

Fig. 3 Histological sections offluazuron-treated nymphs tickR. sanguineus. a–n Bromophe-nol blue staining to detect pro-teins. a–c Control group, dgroup II (20 mg/kg), e group III(40 mg/kg), f group IV(80 mg/kg), g–h control group,i–j group II (20 mg/kg),k–l group III (40 mg/kg), m–ngroup IV (80 mg/kg). c cuticle,edc empty digest cells, epepithelial cells, epc epicuticle,enc endocuticle, exc exocuticle,l lumen, m muscular tissue, prcprocuticle, stc stem cells (gener-ative cells), spc spent cells. Scalebars: a–m 0.02 mm, n 0.1 mm

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the nymphs’ epicuticle. The procuticle, much thinner thanthe epicuticle, and the epidermal cells reacted moderately(Fig. 4b).

Integument: group III This test reveals strong staining forlipid elements in the epicuticle of the individuals fed onrabbits treated with 40 mg of fluazuron/kg of body weight.

Other characteristics are similar to the ones observed in theprevious group: procuticle and epidermal cells reactingmoderately to the test (Fig. 4c).

Integument: group IV Through this histochemical test, largeamounts of lipid are observed in the cuticle of individualsfrom group IV. Here, the staining of epidermal cells decreases

Fig. 4 Histological sections offluazuron-treated nymphs tickR. sanguineus. a–l Baker stain-ing to detect lipids. a Controlgroup, b group II (20 mg/kg),c group III (40 mg/kg), d groupIV (80 mg/kg), e–f controlgroup, g–h group II (20 mg/kg),i–j group III (40 mg/kg), k–l group IV (80 mg/kg). c cuti-cle, edc empty digest cells, epepithelial cells, epc epicuticle,enc endocuticle, exc exocuticle,l lumen, m muscular tissue, prcprocuticle, stc stem cells(generative cells), spc spentcells. Scale bars:a–k 0.02 mm, l 0.1 mm

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significantly, indicating a decrease in the amount of lipidfound in these cells (Fig. 4d).

Midgut: group I (control) Through this histochemical test,the strong staining of lipid elements in the midgut ofengorged nymphs can be detected after 7 days of observa-tion (Fig. 4e, f). The digestive spent cells present numerousand large and strongly stained residual bodies coveringthe nuclei and border (Fig. 4e, f). The empty digestcells present few and small negative vacuoles in theircytoplasm (Fig. 4e, f). The nuclei of all the cells, stem cellsand muscular and digestive (empty digest cells and spentcells) cells, are stained due to the use of hematein, a solutionobtained through the oxidation of hematoxylin (Fig. 4e, f).

Midgut: group II The histochemical test for the detection oflipids shows strong staining in the midgut cells of nymphsengorged on rabbits treated with 20 mg of fluazuron/kg ofbody weight. Most cells present a similar reaction to thatfound in the control group. Only the digestive spent cellsstart being altered, showing a smaller number of stronglystained residual bodies in the cytoplasm (Fig. 4g, h).

Midgut: group III This test reveals less staining than the testin the midgut cells on nymphs from group III, indicating adecrease in the amounts of lipid in this organ when com-pared with previous groups (Fig. 4i, j). The digestive spentcells show a smaller number of strongly stained residualbodies in the cytoplasm, and the empty digest cells do notpresent histochemical alterations, only histological altera-tions as described above (Fig. 4i, j). The lumen, located inthe interior of the midgut, can be observed and is weaklystained to the test (Fig. 4i, j).

Midgut: group IV The results show smaller amounts of lipidin the midgut cells of treated nymphs from group IV(Fig. 4k, l). In the digestive spent cells, few and smallpositive residual bodies are found, while in the empty digestcells, large, negative, and irregular-shaped vacuoles occupymost part of the cell (Fig. 4k, l). The lumen can be observedand is weakly stained (Fig. 4k, l).

Discussion

This study demonstrated the effects of different doses of theAGR fluazuron on the integument and midgut epithelium ofR. sanguineus nymphs through morphological, histological,and histochemical studies, by comparing the resultsobtained from those individuals from the control groupand evaluating the effectiveness of the chemical in theformation of the integument and in the digestion processesof R. sanguineus.

The histological analysis of R. sanguineus from the con-trol group revealed that the integument of these individualsconsists of a layer of epithelial cells and a cuticle subdividedin: epicuticle (outer) and procuticle (inner), corroboratingdata obtained by Balashov (1983) in Hyalomma asiaticum,Sonenshine (1991) in Dermacentor andersoni and Derma-centor variabilis, and Harrison and Foelix (1999) in Orni-thodoros moubata. The procuticle presented two distinctareas: the exocuticle (close to the epicuticle) and the endo-cuticle (close to the epithelial layer), as observed in previousstudies by Balashov (1983) on H. asiaticum, Sonenshine(1991) on D. andersoni and D. variabilis, Dotson et al.(1995) on Amblyomma hebraeum, Harrison and Foelix(1999) on O. moubata, and Montasser and Amin (2010)on Argas persicus.

In the integument of nymphs from the control group,regions could be found where there is a separation of thecuticle and adjacent epithelial layer, suggesting that theecdysis would be starting. Once the epidermal cellswould already be secreting the molting fluid, which con-tains proteases and chitinases to digest the endocuticle ofthe old cuticle and the exuvial space, space that separatesthe epidermal cells of the preceding cuticle would alreadybe in formation (Chapman 1982; Merzendorfer andZimoch 2003).

The integument of nymphs fed on rabbits treated with20 mg of fluazuron/kg of body weight presented smallerepicuticle and procuticle (exocuticle and endocuticle). How-ever, the endocuticle is significantly smaller than thosefound in the individuals from the control group, whichallows us to infer that the growth of the procuticle duringthe engorgement process, fundamental for the accommoda-tion of all the blood ingested by the tick (Sonenshine 1991;Dotson et al. 1995; Harrison and Foelix 1999), will not beoccurring due to the inhibition of the synthesis and/or deposi-tion of chitin caused by fluazuron. These alterations couldjustify the reduced size of the nymphs fed on host treated withfluazuron, a characteristic found by Da Glória (1988) andOliveira et al. (2011), in studies on Rhipicephalus (Boophilus)microplus and R. sanguineus, respectively.

There was no evidence that the process of ecdysis hadstarted in nymphs from group II, as in the ones fromcontrol group, which indicates that fluazuron is inhibitingthe processes related to the molting of nymphs engorgedon treated rabbits, probably by preventing the synthesisand/or deposition of the necessary chitin to form a newcuticle which will cover their body during periodic ecdy-sis. Splinder (1990), Chen (1987), and Kemp et al. (1990)found similar data for other tick species fed on hoststreated with fluazuron.

The interference of fluazuron in the synthesis and/ordeposition of chitin lead to abnormal endocuticular deposi-tion which affects the cuticle’s elasticity and stiffness,

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preventing the normal formation of a new cuticle and thecompletion of ecdysis (Mikolajczyk et al. 1994; Oberlanderand Silhacek 1998; Palli and Retnakaran 1999; Oberlanderand Smagghe 2001). According to Palli and Retnakaran(1999), the inhibition of the synthesis and/or deposition ofchitin would occur by the inhibition of certain biochemicalprocesses, such as the inhibition of chitin synthase, theactivation of chitinases involved in the catabolism of chitin,and the inhibition of proteases which activate chitin synthe-tase, among others.

A greater number of alterations were detected in theintegument of the nymphs from groups III and IV. Alter-ations in relation to the absence of subdivisions show that,as the doses of fluazuron increase, damages to the integu-ment of the individuals exposed to the chemical also in-crease. These data could be justified by the chitin, necessaryfor the formation of the cuticle layers. As it is not beingsynthetized and/or deposited in the integument of the indi-viduals due to the action of fluazuron, a single layer ofprocuticle or even a single cuticle was formed. Other studiesby Mommaerts et al. (2006) and Saenz-De-Cabezon et al.(2006) on Ctenocephalides felis fleas also reported lessthickness and loss of subdivisions in the cuticle after treat-ment with lufenuron, other chemical from the same class asfluazuron—benzoylphenylurea.

The histochemical techniques showed the presence ofpolysaccharides, proteins, and lipids in the integument ofnymphs from the control group and treated groups. Thepresence of polysaccharides in the cuticle of nymphs fromthe control group was reduced; the epicuticle and exocuticleare weakly positive, while the endocuticle reacted moder-ately to the test that corroborates data obtained by Oliveiraet al. (2011) for engorged R. sanguineus nymphs. In thecuticle of the treated individuals (groups II, III, and IV),there was a progressive decrease in the number of polysac-charides as the dose of fluazuron increased. This can beoccurring due to fluazuron once this chemical would beinterfering in the synthesis and/or deposition of chitin, apolysaccharide that is present in the cuticle of arthropods.Gangishetti et al. (2009) found similar results for Drosoph-ila larvae exposed to lufenuron and diflubenzuron.

Proteins were also analyzed. Large amounts of proteinwere found in the cuticle of nymphs from the control group.These data corroborate Balashov (1983) for Hyalommaasiaticum, Sonenshine (1991) for D. andersoni and D. var-iabilis, Dotson et al. (1995) for Amblyomma hebraeum, andHarrison and Foelix (1999) for O. moubata. Similar distri-bution of this element was detected in the treated groupsshowing that the synthesis of protein would not be impairedby the fluazuron doses used here.

This study also allowed investigating the amount anddistribution of the lipid. Nymphs from the control grouppresented few lipids in the integument; only the epicuticle is

strongly stained by the test. The individuals fed on rabbitstreated with 20, 40, or 80 mg of fluazuron/kg of body weightpresented great number of lipids in the cuticle showed bythe strong reaction to Baker in the epicuticle (groups II andIII) and/or in the outer layers of the cuticle (group IV). Infact, there is not an increase in the number of lipids in all thetreated groups, but a decrease in thickness and even non-formation of other layers of the cuticle formed by otherelements, mainly endocuticle of glycoprotein constitution.Thus, the epicuticle, which is formed by lipid and notcontaining chitin, is evidenced. These data can indicate thatfluazuron would not be affecting the processes related tosynthesis and storage of lipids in the integument of theexposed nymphs.

Oliveira et al. (2011), in studies on engorged R. sangui-neus nymphs, reported that the epithelial layer of the integ-ument of these individuals is formed by a simple layer ofcells which react strongly to the three elements (glycolipo-protein composition) (Sonenshine 1991). These data corrob-orate the present study once the epidermal cells of thenymphs’ integument from the control group show the sameorganization and affinity to the histochemical tests for thedetection of protein, polysaccharides, and lipids.

The epidermal cells of the integument of nymphs fromtreated groups presented similar reaction to bromophenolblue and Baker, however, reacted weakly to PAS, demon-strating a decrease in the number of polysaccharides. Thesedata allow us to infer that the synthesis of polysaccharides,probably chitin, which would participate in the formation ofthe cuticle by the epidermal cells, would be impaired by theaction of fluazuron.

The histological analysis of R. sanguineus nymphs fromthe control group also showed that the midgut of theseindividuals consist of an epithelial wall formed by differentcell types supported by a basal membrane and a thin layer ofmuscular tissue, corroborating data by Till (1961), Balashov(1983), Agbede and Kemp (1985), Sonenshine (1991),Agyei and Runham (1995), and Harrison and Foelix (1999),for other tick species.

Studies by Agbede and Kemp (1985) on Boophilusmicroplus and Harrison and Foelix (1999) on O. moubatareported that generative stem cells maintain the originalmorphology and the location close to the basal membraneduring all the phases of the digestion process, and that theyare responsible to form other types of cells in the midgut. Inthis study, the generative cells (stem cells are representedmainly by their nuclei) occupied the same region in themidgut; however, they were rarely seen once the digestivecells frequently covered them.

The digestive cells predominate in the midgut of R. san-guineus nymphs from the control group. These cells werefound in two stages: the large spent cells, filled with endo-somes with blood ingested from the host during engorgement;

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digestive vacuoles (with blood cells which are being storedand lysed in the vacuoles); and haematin residual bodies (finalproduct of the intracellular digestion, which is being accu-mulated in the cellular interior), as well as small emptydigest cells which present small and few vacuoles in thecytoplasm, probably nondigested food due to the reduceddigestive capacity (Agyei and Runham 1995). Similarresults were obtained by Koh et al. (1991), forHaemaphysalislongicornis nymphs, and Agyei and Runham (1995), for B.microplus and Rhipicephalus appendiculatus nymphs, oncethey found these same stages of digestive cells in the midgutof these ectoparasites.

The midgut of the nymphs engorged on rabbits treatedwith 20 mg of fluazuron/kg of body weight presented alter-ations only in the digestive cells, spent cells, once these cellsaccumulate less blood in the endosomes and digestivevacuoles, as well as haematin residues which demonstratethat fluazuron already interferes in the digestive processes,probably for interfering in the formation of the peritrophicmembrane, found in the apical surface of the midgut andessential for the selective permeability of the plasma mem-brane of the midgut cells (Zhu et al. 1991; Terra 2001), todelimit the area where the digestive events occur (Shen et al.1999; You et al. 2003) and to protect the microvilli presentin the epithelial cells from ingested microorganisms andfrom their own digestive enzymes (Matsuo et al. 2003). Asfluazuron is inhibiting the synthesis of chitin and the peri-trophic membrane is mainly formed by this component(Peters 1992; Lehane 1997; Matsuo et al. 2003), this mem-brane can be not properly formed, which would leave themidgut cells totally exposed and vulnerable to their owndigestive enzymes. Thus, the enzymes could be damagingspecializations of the midgut membrane, such as the micro-villi, and consequently decreasing and impairing the obtain-ing of the blood ingested during feeding on the treated host.Other studies by Vasuki (1999) on two species of Aedesaegypti and Anopheles stephensi mosquitoes also observedthe reduction in the amount of blood ingested and digestedfrom hosts treated with other arthropod growth regulators,the hexaflumuron.

Alterations were found in the nymphs from group III inboth stages of digestive cells, spent cells, and empty digestcells as well as in the midgut lumen. The spent cells aresmaller and with fewer endosomes, digestive vacuoles, andresidual bodies; the empty digest cells presented small api-cal waving, and the lumen is now observed. These resultscould once more be justified by the inhibition of the syn-thesis of the necessary chitin to form the peritrophic mem-brane. In addition, the dose of fluazuron was higher;therefore, the damage in the peritrophic membrane wasprobably higher. These data corroborate Glebic et al.(2002), for Culex quinquefasciatus larvae, where a decreasein hemolysis (lysis of blood elements) can occur in the midgut

of the individuals submitted to other arthropod growth regu-lator, the methoprene.

Important damages caused by this acaricide were foundin the midgut of nymphs engorged on rabbits treated with80 mg of fluazuron/kg of weight; the spent cells are muchsmaller and present few and small endosomes, digestivevacuoles, and residual bodies in their cytoplasm, and theempty digest cells are rarely present and have large invagi-nations in the apical plasma membrane, as well as largevacuolated regions, mainly close to the midgut lumen,which contains few residues from digestive processes.This allows us to infer that fluazuron, inhibiting the syn-thesis of chitin, would be preventing the formation of theperitrophic membrane and indirectly damaging the diges-tive cells, which would not be performing their functions.Thus, the blood ingested from the treated hosts is notbeing captured to the interior of the cell, not sufferinglysis, not forming the digestive residues, and not releasingthe necessary nutrients for the development of the ecto-parasites. Nishiura et al. (2005) also reported the occur-rence of little developed midgut cells in A. aegypti larvaetreated with methoprene.

The use of bromophenol blue, Baker, and PAS histo-chemical techniques allowed the observation of largeamounts of protein and lipid, as well as the reduced numberof polysaccharides in the midgut of nymphs from the controlgroup. In nymphs engorged on treated rabbits (group II, III,and IV), a gradual decrease occurs in the protein and lipidelements from group II to IV, i.e., level of dose-dependentalteration. Even the concentration of polysaccharides, whichwas not high in the control group, was smaller in the midgutof nymphs engorged on rabbits fed with 80 mg of flua-zuron. These data confirm that fluazuron, which woulddecrease the efficiency of the organ in performing itsfunctions and consequently affecting the whole individual,would indirectly impair the processes that would be performedby digestive cells.

Thus, it was verified by this study that the arthropodgrowth regulator fluazuron (active ingredient of Acatak) isinterfering in the integument and in the midgut cells of R.sanguineus nymphs fed on treated rabbits. The doses offluazuron applied are impairing the polymerization of newcuticles and the processes of absorption and digestion ofthe host’s blood, which prevents the development of thenymphs and emergence of adults after periodic ecdysis,indicating the possibility to use this arthropod growthregulator in the control of this stage of the biological cycleof R. sanguineus.

Acknowledgments We would like to thank to FAPESP (grant no.2010/50827-0) for financial support and CNPQ academic careerresearch fellowship to G.H. Bechara and M.I. Camargo-Mathias.

Conflict of interest None.

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