Inheritance of weight in Rhipicephalus appendiculatus ticks (Acari: Ixodidae) in the laboratory

A selection of the 10% lightest and 10% heaviest males and females of a population of individually weighed Rhipicephalus appendiculatus Neumann adults was made in two experiments. The offspring of homologous pairs were followed until the next adult stage (light x light, control x control and heavy x heavy). The engorged nymphal weights, unfed adult weights, engorged female weights of the parents, egg mass weights, egg weights, larval scutal lengths, engorged larval weights, unfed nymphal weights, engorged nymphal weights and adult weights of the progeny were determined. No significant differences could be demonstrated between the two lines for egg weight, larval scutal length, engorged larval weight and unfed nymphal weight. Significant differences were found between the egg masses, engorged nymphal weights and adult weights of the two lines. The heritability coefficients of body weight determined from adult to adult were 0.14 and 0.10, respectively, during the first and second experiments. Considering females and males separately, the coefficients were 0.10 and 0.18 during the frist experiment and 0.12 and 0.09 during the repeat experiment respectively.     

Comparison of Preservation Methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for Reliable DNA Amplification by PCR

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification

The closely related species Rhipicephalus appendiculatus and R. zambeziensis co-occur in a wide area in Zambia. In this area, specimens of both species have been collected on the same individual host at the same time. In addition, specimens that are morphologically intermediate between R. appendiculatus and R. zambeziensis have been found in this area. These observations cast some doubt on the species status of R. appendiculatus and R. zambeziensis. Because the two taxa have varied influences on the epidemiology of East Coast fever (ECF), a cattle disease for which they act as vectors, it is essential that their taxonomic status is clarified and their identification is accurate. Therefore a phylogenetic analysis of nucleotide sequences of the ITS2 and a fragment of the mitochondrial 12S rDNA was performed to reassess the specific status of both taxa. This revealed two well supported clades coinciding with R. appendiculatus and R. zambeziensis as two separate species. In order to facilitate species identification a PCR-RFLP diagnostic assay was developed based on BauI digestion of the ITS2 gene. This assay produced clear diagnostic banding patterns for the two species and is applicable over a wide range of both species’ distribution. Nucleotide sequence data reported in this paper are available in the GenBank™ under the accession numbers DQ849203–DQ849274.     

Cholesteryl esters on the body surfaces of the camel tick,Hyalomma dromedarii (Koch, 1844) and the brown dog tick,Rhipicephalus sanguineus (Latreille, 1806)

Cholesteryl esters were found to constitute a major component of the lipids coating the body cuticle of females of the camel tick, Hyalomma dromedarii and the brown dog tick, Rhipicephalus sanguineus. One or more cholesteryl esters, alone or in combination, have been shown to serve as the mounting sex pheromone of several species of ixodid ticks. Consequently, knowledge of these compounds is important for an understanding of the mating behavior of these ticks. Based on thin layer chromatography, cholesterol and cholesteryl esters were the most abundant neutral lipids found on the body surfaces of fed females of these two species. Analysis using HPLC demonstrated significant quantities of the following compounds, tentatively identified as cholesteryl esters (expressed in micrograms per female equivalent), in H. dromedarii: Cholesteryl acetate 18.2; cholesteryl laurate, 6.8; cholesteryl linoleate, 24.8; cholesteryl oleate, 12.9; cholesteryl palmitate, 0.3; and cholesteryl stearate 1.7.In contrast, the same method revealed only 3 cholesteryl esters in extracts of females of R. sanguineus: Cholesteryl acetate, 2.0; cholesteryl linoleate, 8.5; and cholesteryl oleate, 3.0. In both species, two unidentified peaks, with the spectral characteristics of cholesteryl esters, were also observed. Identification of the cholesteryl esters was confirmed: by (1) positive bioassay results with conspecific (H. dromedarii) males and heterospecific (Dermacentor variabilis) males; (2) similarity of ultraviolet spectra between identified sample peaks and authentic standards; and (3) demonstration of cholesterol and the corresponding free fatty acid following enzymatic digestion of each of the HPLC-separated fractions containing the different cholesteryl esters. Comparisons with the cholesteryl ester composition of the mounting sex pheromone of other metastriate Ixodidae are discussed. These findings, along with studies reported previously, suggest that differences in the mounting sex pheromones of ixodid ticks are an important factor in minimizing heterospecific matings in nature.Supported by Cooperative Agreement No. 263-0152-A-00-2207 with the U. S. Agency for International Development to the Department of Infectious Diseases. College of Veterinary Medicine, University of Florida, Gainesville, FL and a subcontract, ODURF No. 536521, to the Department of Biological Sciences, Old Dominion University, Norfolk, VA     

Further evidence for geographic differentiation in R. appendiculatus (Acari: Ixodidae) from Eastern and Southern provinces of Zambia

Studies in the biology, ecology and behaviour of R. appendiculatus in Zambia have shown considerable variation within and between populations often associated with their geographical origin. We studied variation in the mitochondrial COI (mtCOI) gene of adult R. appendiculatus ticks originating from the Eastern and Southern provinces of Zambia. Rhipicephalus appendiculatus ticks from the two provinces were placed into two groups on the mtCOI sequence data tree. One group comprised all haplotypes of specimens from the Eastern province plateau districts of Chipata and Petauke. The second group consisted of a single haplotype of specimens from the Southern province districts and Nyimba, an Eastern province district on the fringes of the valley. This variation provides additional evidence to the earlier observations in the 12S rDNA and ITS2 data for the geographic subdivision of R. appendiculatus from Southern province and Eastern province plateau. The geographic subdivision further corresponds with differences in body size and diapause between R. appendiculatus from these geographic areas. The possible implications of these findings on the epidemiology of East Coast fever (ECF) the disease for which R. appendiculatus is one of the vectors are discussed. Nucleotide sequence data reported in this paper are available in the GenBank^TM under the Accession Numbers DQ859259–DQ859266     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

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.     

Questing activity of Rhipicephalus appendiculatus (Acari: Ixodidae) nymphs: a random process?

Abstract.  In Zambia, an experiment under quasi-natural conditions indicated that Rhipicephalus appendiculatus nymphs react to prevailing climatic conditions. Higher temperatures and higher vapour pressure deficits lead to decreased activity. The majority of nymphs (>75%) were recorded at ground level. Simulations showed that larval phenology and temperature during the nymphal premoulting period largely explain the seasonal abundance patterns of nymphs, as observed on cattle, given the absence of a behavioural diapause. Consequently, the effect of climate, as observed in our studies, is masked. However, the results of the present study indicate that daily climatic conditions probably have a much larger effect on the transmission dynamics of Theileria parva . The vertical distribution of questing instars is a function of temperature and humidity. In years of unfavourable conditions, nymphs might feed mainly on hosts other than cattle, and this could govern the infection prevalence in the adult population. This suggestion is supported by previous epidemiological studies.     

Experimental evidence for a female sex pheromone in Arrenurus manubriator (Acari: Hydrachnida; Arrenuridae)

We present experimental evidence for a water-borne female-produced sex pheromone in aquatic parasitengonine mites. Water that has contained adult female Arrenurus manubriator Marshall will elicit arrestant behaviour in conspecific adult males, and if the cue is sufficiently strong, the males will assume a readiness posture (with 4th pair of legs held over the back, bent anteromedially at the genuotibial joint) that is typically a precursor to coupling. Water that has not been exposed to female mites does not induce any behavioural response from male mites. Female-conditioned water that has been passed through a C-18 column does not elicit any response from male A. manubriator, while the rehydrated residue from the column does induce arrestant behaviour and may result in the readiness posture. The results from the C-18 extraction indicate that the pheromone is nonpolar in nature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of ricinoleic acid esters from castor oil of Ricinus communis on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

This study examines the effects of ricinoleic acid esters from Ricinus communis castor oil on the vitellogenesis of Rhipicephalus sanguineus ticks attached to hosts that were fed with commercial rabbit food containing these esters. The oocytes of ticks from the treatment group (TG) showed cytoplasmic changes that inhibited the development of oocytes I and II to the advanced stages (IV and V) in addition to preventing the maturation of oocytes V, resulting in small ones. In addition, sperm was not observed in ampoules. Our findings confirm the acaricide potential of ricinoleic acid esters.     

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.     

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 LC₅₀ (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 LC₅₀ and 95% confidence interval were calculated, g (95): LC₅₀ = 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.     

The role of 2,6-dichlorophenol as sex pheromone of the tropical horse tick Anocentor nitens (Acari: Ixodidae)

The objective of this study was to evaluate the role of2,6-dichlorophenol (2,6-DCP) as sex pheromone of Anocentornitens. Sex pheromones were extracted by submerging 150 females fed for 6 days in hexane (3 ml) for two hours and sonicating them for15 min. The extract was analyzed by GC-MS employing the Single Ion Monitoring (SIM) method. Identification of 2,6-DCP was based on the comparison of mass spectra with a computer search using the NIST library and by matching the mass spectrum of the peak at the retention time of 2,6-DCP from extract sand the synthetic product. In an olfactometer, males were released at 2.5cm from females, control rubber septa and impregnated rubber septa with increasing concentrations of 2,6-DCP (50, 500 and 5000 ng). A higher percentage of orientation and higher frequencies of angles between 0° and 10° were observed for males tested with females than with controls. Attraction of males to dummies impregnated with 2,6-DCP was observed with highest response (100%) at 50 ng. There was a gradual decrease of responsiveness of males with increasing concentration of 2,6-DCP (70 to 75%).This compound stimulated a behavior of mounting and ventral positioning ofA. nitens males at the lowest 2,6-DCP concentration where as dummies impregnated with 2,6-DCP higher than 50 ng concentration inhibited these behaviors. Therefore, we can conclude that 2,6-DCP can elicit the complete behavior sequence of orientation, location, mounting and ventral positioning behaviors and plays a role as an attractant and, as a mounting sex pheromone in A. nitens. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Cytotoxic effects of permethrin in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

Because of the medical and veterinary importance of ticks and the wide use of synthetic chemical substances such as permethrin (active ingredient of Advantage® Max3 – Bayer)for their control, this study evaluated the effects of different concentrations (206, 1031 and 2062 ppm) of the acaricide on the salivary glands of Rhipicephalus sanguineus semi-engorged females. Results showed that permethrin is a potent substance that acts morpho-physiologically in the tick glandular tissue, causing changes in the acini shape intense vacuolation in acinar cells, and disruption of the tissue by cell death process, with subsequent formation of apoptotic bodies, especially at higher concentrations, thus precluding the accurate identification of different types of acini. Importantly, it is demonstrated that permethrin acts on salivary gland tissue, as well as affecting the nervous system, accelerating the process of glandular degeneration, and interfering with the engorgement process of female ticks, preventing them from completing the feeding process.     

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.     

Effects of Ricinus communis oil esters on salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study showed the interference of esters extracted from Ricinus communis in the secretory cycle of salivary glands of Rhipicephalus sanguineus ticks, which consequently caused collateral effects on their feeding process. Ticks attached on hosts which were fed with commercial feed containing different concentrations of R. communis oil esters suffered damages such as cytoplasmic changes in their salivary glands, notably in the acinar cells, impairing the functioning of the acini and accelerating the organs degeneration as a whole. It was found that esters interfered with the activity of cellular secretion by changing the glycoprotein of salivar composition especially in acini II cells. It was also shown that the damages caused by esters in the salivary glands cells of these ectoparasites increased in higher concentrations of the product and degenerative glandular changes were more pronounced.     

Feeding electrogram studies on the African cattle brown ear tick Rhipicephalus appendiculatus: evidence for an antifeeding effect of tick resistant serum

Abstract. Feeding behaviour of partially engorged Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae) on rabbit serum held in capillary tubes and placed over the tick mouthparts was studied using the feeding electrogram technique with simultaneous macro video photography. Correlation of electrical events with fluid movement in the vicinity of the tick's mouthparts and the capillary meniscus, permitted the characterization of an orderly sequence of signals, termed the 'Feeding Complex', associated with highest weight gains. This complex consisted of a 3–8 Hz fast-sucking waveform typically lasting 4–5 min, a sharp drop in potential at salivation, and rest lasting 1 or 2 min where no waveform or fluid movements occur. Very high impedence recordings from within the tick capitulum indicate that the fast-sucking waveform coincides with bursts of potentials corresponding to contraction of the pharyngeal dilator muscles, whereas during rest a tonic series of spikes signifies that the floor of the salivarium is actively lowered. Feeding electrograms of ticks fed on serum from tick-resistant rabbits showed significantly fewer feeding complexes. The weight gains achieved by these ticks were reduced correspondingly. This suggests that some of the humoral effectors of immunity have an antifeedant effect on this unusual parasite of rabbits.