Underwater survival of Rhipicephalus sanguineus (Acari: Ixodidae)

Rhipicephalus sanguineus (Acari: Ixodidae) is a worldwide distributed tick, also due to its adaptability to different environmental conditions. In order to assess its ability to survive and to lay eggs after water immersion, 150 engorged females from southern Italy were water immersed for 1-15 days whereas eggs were flooded for 1-5 days. All females survived water immersion for 48 h, some of them up to 72 h, but egg hatch rate was negatively correlated with female submersion period. All eggs flooded for up to 120 h hatched successfully. These findings suggest that R. sanguineus is able to survive underwater for some days without loosing any biological activity. This feature should be considered in relation to its potential to spread to new areas and to its role as a vector of pathogens also in consideration of changes in climate the Earth is currently experiencing.     

Crossbreeding between different geographical populations of the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae)

Brown dog ticks are distributed world-wide, and their systematics and phylogeny are the subject of an ongoing debate. The present study evaluates the reproductive compatibility between Rhipicephalus sanguineus ticks from North America, Israel, and Africa. Female ticks of the parent generation were mated with males from the same and alternate colonies. Every pure and hybrid cohort was maintained separately into the F2 generation with F1 females being allowed to mate only with males from the same cohort. The following survival parameters were measured and recorded for every developmental stage: feeding duration and success; engorgement weight, fertility, and fecundity of females; molting and hatching success. Ticks from North American and Mediterranean populations hybridized successfully. The survival parameters of all their hybrid lines were similar to those in pure lines throughout the F1 generation, and F1 adults were fully fertile. Parent adult ticks from the African population hybridized with either North American or Mediterranean ticks and produced viable progenies whose survival parameters were also similar to those in pure lines throughout the F1 generation. However, F1 adults in the four hybrid lines that included African ancestry were infertile. No parthenogenesis was observed in any pure or hybrid lines as proportion of males in F1 generation ranged from 40 to 60 %. Phylogenetic analysis of the 12S rDNA gene sequences placed African ticks into a separate clade from those of the North American or Mediterranean origins. Our results demonstrate that Rh. sanguineus ticks from North America and Israel represent the same species, whereas the African population used in this study is significantly distant and probably represents a different taxon.     

Gynandromorphism in Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae)

During a survey of ticks on horses in November 2000, at the University of S?o Paulo farm in Pirassununga county, S?o Paulo, Brazil, 1 gynandromorph of Amblyomma cajennense was collected from a naturally infested horse. In another survey on dogs in March 2001, in the urban area of Monte Negro, Rond?nia, Brazil, a gynandromorph of Rhipicephalus sanguineus was collected from a naturally infested dog. Both specimens are described and classified as perfect bipartite protogynanders.     

Cutaneous hypersensitivity induced in dogs and guinea-pigs by extracts of the tick Rhipicephalus sanguineus (Acari: Ixodidae)

The cutaneous hypersensitivity induced by Rhipicephalus sanguineus tick extract in dogs (natural host) and guinea-pigs (laboratory host) was evaluated. The left ear of infested and control (tick-bite naive) dogs and guinea-pigs was injected intradermally with an extract from unfed adult ticks and the right ear with phosphate buffered saline (PBS). Ear thickness variations were then measured after 10 min and 1, 2, 6, 18, 24, 48, 72 and 96 h post-injection. Results were expressed as percentual changes in the ear thickness in relation to pre-inoculation values. The final variation in ear thickness induced by the extract was given by subtracting, in each animal, the right ear percentual increase from that of the left ear. Guinea-pigs were tested at two different times following infestation and with two different doses of extract. Infested guinea-pigs from the three experiments developed an immediate (within the first 2 h post-inoculation) and a strong delayed reaction (24 h) to the extract. Dogs, unlike guinea-pigs, developed only a strong immediate reaction whereby an 80% increase in ear thickness was observed. Control animals, with the exception of one dog, did not develop any significant reaction to the extract. Only mild reactions were induced by PBS in the right ear of all animals. The correlation between the absence of a strong delayed type reaction to tick extract and the lack of resistance of the natural host to R. sanguineus tick is discussed.     

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.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Ovarian development and egg maturation are essential stages in animal reproduction. For bisexual ixodid ticks, copulation is an important prerequisite for the completion of the gonotrophic cycle. In this study, we aimed to characterize the morpho-histological changes in the ovary and oocytes of the tick Rhipicephalus sanguineus, together with the identification of feeding and reproductive parameters associated with mating. Virgin and cross-mated females (with R. turanicus males) weighed 60% less at full engorgement than females mated conspecifically. In addition, the oocytes of these females did not develop to the same advanced stages as those of the conspecifically mated females. Sequencing of a 250-bp ITS-2 fragment in eggs that originated from a cross between an R. sanguineus female and an R. turanicus male showed a genotype similar (except by a deletion of 1 thymine) to that observed in the mother, arguing against fertilization by a trans-specific male. These findings suggest that male sex peptides are species-specific molecules that influence both full engorgement and oocyte maturation. Mechanical stimulation of the gonopore alone was insufficient for the completion of the entire process of vitellogenesis.     

Abnormal development of Rhipicephalus sanguineus (Ixodidae)

Three malformed Rhipicephalus sanguineus specimenswere noticed in a tick laboratory colony. These specimens had different degreesof twining, from an almost fused individual (with two anal pores) to apartiallyfused adult male, with four spiracular plates. One female was a heart-shapedspecimen, with both body halves fused along a longitudinal axes, with fourspiracular plates and two anal pores.     

Markers of cell death in salivary glands of males of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae)

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.     

Activation of the ambusher tick Rhipicephalus microplus (Acari: Ixodidae) exposed to different stimuli

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO₂ alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO₂ combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO₂ was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO₂ activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO₂, larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO₂. The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.     

Cuticular fatty acid profile analysis of three Rhipicephalus tick species (Acari: Ixodidae)

Cuticular fatty acids (CFA) are important constituents of the arthropod exoskeleton, serving as structural and defense components, and participating in intra-species communication. Here we describe for the first time a comparative analysis of the CFA profiles of three tick species of the genus Rhipicephalus: R. annulatus, R. bursa and R. sanguineus. CFA profiles were determined for R. bursa and R. sanguineus grown both on rabbit or calf, and for R. annulatus grown on calf. CFA composition was compared for each species before and after ethanol treatment, for different hosts of each species, and between the different species. Our data suggest that adsorption of the host’s fatty acids changes the apparent CFA composition. Ethanol treatment efficiently removed the unbound fatty acids from the ticks and revealed the actual composition. Comparison between ticks grown on rabbit versus calf showed significant difference in the relative abundance of fatty acids C14 and 9,12-C18:2 for R. bursa, and a difference in the relative abundance of C14 for R. sanguineus. Comparison of the CFA between the three species revealed significant differences in the abundance of fatty acids C16, 9,12-C18:2, 9-C18:1, C18 and C20. Our results show that while the host had a minor effect on CFA composition within each species, significant differences were observed in the CFA profiles of different species. We suggest that CFA profiles may be used to distinguish between related species. CFA analysis can also be used in studies of communication and defense mechanisms in ticks and other arthropods.     

Natural occurrence of lethal aspergillosis in the cattle tick Rhipicephalus (Boophilus) microplus (Acari:Ixodidae)

The purpose of this study was to describe an unreported entomopathogenic fungus that naturally infects the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Engorged female ticks, showed symptoms of fungal infection after controlled tick infestation of cattle. Infected ticks developed a distinctive dark colour, a pale mould grew over the cuticle and the ticks eventually died covered with fungal conidiophores. The responsible fungus was isolated and cultured on mycological medium and submitted to microscopic morphology, biochemical phenotyping and 18S rRNA ribotyping analyses, which identified it as aflatoxin-producing Aspergillus flavus. Spores from the cultured fungus were experimentally sprayed over healthy engorged female ticks, obtaining an 80% prevalence of experimental infection of healthy ticks and their egg masses, the larval progeny after incubation under laboratory conditions was also infected. These results demonstrate that A. flavus is the causative agent of the natural fungal disease of the cattle tick R. microplus described here.     

Pathogenicity of entomopathogenic fungi to different developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae).

The pathogenicity of 4 species of entomopathogenic fungi (Hyphomycetes species: Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, and Paecilomyces fumosoroseus) to various developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae) ticks was compared under laboratory conditions. The most virulent isolate, M. anisopliae-108, caused 92-96% mortality to unfed larvae and nymphs on day 7 postinfection (PI) and 100% mortality to unfed adults and engorged females on day 21 PI. The pathogenicity of M. anisopliae-108 to engorged larvae and nymphs was lower--82.6 and 60%, respectively. All tested B. bassiana, M. flavoviride, and P. fumosoroseus isolates were significantly less virulent (P < 0.05) or avirulent toward most life stages of R. sanguineus. The M. anisopliae and M. flavoviride isolates also prevented or reduced the ability of the ticks to lay eggs several days before their deaths. Female ticks infected by the fungi achieved only 11.3-60.8% of their egg-laying capacity compared with the controls.     

Toxicity of extract of Magonia pubescens (Sapindales: Sapindaceae) St. Hil. to control the brown dog tick, Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae)

The action of crude ethanol extract of the stem bark of the soapberry Magonia pubescens St. Hil. was studied upon larvae of the Brown Dog tick Rhipicephalus sanguineus (Latreille). Tick larvae were obtained by maintaining gravid females in an incubator, after collecting them from naturally infested kennels. The tick larvae were placed in envelopes of filter paper impregnated with different concentrations of the extract dissolved in dimethylsulfoxide (DMSO) and distilled water. Four tests were repeated with each solution (n > 120). The control was carried out in DMSO and distilled water. The bioassays were performed at 27 +/- 1 degrees C, RH > 80% and 12:12 light cycle. Mortality was observed after 48 h exposure. All motionless larvae were considered to be dead. The extract of M. pubescens showed larvicidal potential against R. sanguineus. The lethal concentrations of 1503 ppm (LC50) and 9991 ppm (LC99) were obtained. There was no mortality in the control group. Based on the results of the current study, M. pubescens should be recognized as an future alternative acaricide for the control of Brown Dog tick. These results reinforce the importance of the preservation of this soapberry in its natural biome.     

Fipronil-induced cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

The tick Rhipicephalus sanguineus is currently considered an urban plague. For this reason many studies are intended to find methods to control these ectoparasites. Thus, the present study analyzed the ultrastructural modifications of the salivary glands cells of semi-engorged females of R. sanguineus resulting from their exposition to Fipronil (active ingredient of Frontline®). The studied individuals were divided into four groups. Group 1 was exposed to distilled water (control) and groups 2, 3 and 4 were exposed to 1, 5 and 10 ppm of Fipronil, respectively. The salivary gland of ticks subjected to the acaricide showed accelerated process of cell death by atypical apoptosis, as well as augmented cell damages as the concentration of the chemical compound was increased. The acaricide toxicity at cellular level was demonstrated by remarkable changes of elements of the cytoskeleton and spherocrystals (extremely hard inorganic structures). However, tick defense mechanisms, such as the observed autofagic vacuoles proved the cells attempt to preserve their integrity and minimize the devastating action of this chemical compound on the salivary glands.     

Ecdysteroid levels changed by permethrin action in female Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

As recent studies have shown that ecdysteroids may play a major role in the regulation of vitellogenesis in Ixodidae, the present study quantified, by means of a radioimmunoassay, the levels of ecdysteroids present in the hemolymph of semi-engorged females of Rhipicephalus sanguineus ticks obtained from control females (exposed to distilled water) and those exposed to increasing concentrations of permethrin. The levels of ecdysteroids decreased significantly as the concentration of permethrin increased, suggesting that this compound could be an inhibitor of ecdysteroids secretion, and consequently interfering with the reproductive ability of these ticks, since this hormone is responsible for the synthesis and incorporation of vitellogenin by oocytes. This study complements the previous results with R. sanguineus semi-engorged females, showing that permethrin is a potent agent causing major morphological changes in tick oocytes, such as the appearance of large vacuoles in the cytoplasm, reduction in the amount of yolk granules and a decrease in oocyte size, thus culminating in cell death and consequently reducing or preventing reproduction in treated females. The findings that permethrin leads to a decrease in ecdysteroid titers could represent an entry step into this scenario.