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.     

Evidence for a mounting sex pheromone in the brown ear tick Rhipicephalus appendiculatus,Neuman 1901 (Acari: Ixodidae)

The presence of a mounting sex pheromone was demonstrated on the surface of fed female Rhipicephalus appendiculatus. This pheromone, which is present on the female cuticle, allows the male to recognise the female. The pheromone was removed by cleaning the female in hexane, resulting in the loss of male mating behaviour in in vitro experiments. Male mating behaviour was resumed when extract made from fed female cuticle was replaced on cleaned females. When the extract was transferred to innanimate objects typical male mating behaviour was released. Preliminary chemical analyses indicated that the active component of the extract was contained in the sterol ester fraction of the extract.     

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.     

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     

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.     

Geographic variation in diapause response of adult Rhipicephalus appendiculatus ticks

Diapause in adults of the African brown ear tick Rhipicephalus appendiculatus is dependent on latitude and stock origin. Lower latitude populations, that is, those nearer the equator, show no diapause response to long or short day exposure. Diapause response to short day lengths is found in higher latitude populations, further south, and increases with increasing latitude. Nearly all individuals in the southernmost population, used in this study, enter diapause under short and long day exposure which implies that the onset of diapause in this population is obligatory. Diapause termination in higher latitude populations is induced by increasing photo period, further north, but still south of the equator, diapause is terminated by ageing. The differences in diapausing behaviour of the different populations are discussed in relation to their climatic conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diapause induction in adults of three Rhipicephalus appendiculatus stocks

Newly-moulted adults of three Rhipicephalus appendiculatus stocks, originating from Kenya, Zambia and Zimbabwe, were kept in different photoperiods (12h : 12h, 13h : 11h and 14h : 10h light : dark). The stock originating from Kenya showed almost no behavioural diapause in any of these day lengths, whereas virtually all individuals of the Zimbabwean stock entered a behavioural diapause irrespective of day length. Adults from the Eastern Province of Zambia, a transition zone between the multivoltine phenology in equatorial Africa and the univoltine in southern Africa, exhibited a photoperiod-dependent diapause response. The possible mechanisms of diapause regulation and their origin are 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.     

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.     

Repellent and acaricidal properties of Ocimum suave against Rhipicephalus appendiculatus ticks

An oil extracted from the leaves of a tropical shrub Ocimum suave was found to repel as well as kill all stages of the tick Rhipicephalus appendiculatus. In an in vitro bioassay for the larvae, the LC₅₀ of the oil in liquid paraffin was 0.024%. A 10% solution was found to kill all immatures and more than 70% of adults feeding on rabbits. Rabbits were protected for 5 days against attaching larvae using a 10% solution. Preliminary experiments undertaken with cattle kept in the field suggest that the oil may have potential in tick control, and a role in integrated tick management.     

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.     

Survival of Theileria parva-infected adult Rhipicephalus appendiculatus under laboratory and quasi-natural conditions

Adult Rhipicephalus appendiculatus Muguga, having high or low intensities of Theileria parva Muguga infection in their salivary glands, were exposed to 20 °C and 85% relative humidity in the laboratory or quasi-natural conditions. Survival of the ticks and T. parva infections in their salivary glands was then monitored over a two year period. Ticks, having an average infection level of 2 infected acini per female, survived for up to 70 or 106 weeks after moulting under the laboratory or quasi-natural conditions respectively. Those having an infection level of 26 infected acini per female, survived for a similar duration except that those under quasi-natural conditions survived for a slightly shorter duration (102 weeks). Similarly, T. parva parasites survived for much longer periods under quasi-natural conditions than under the laboratory conditions. They survived for up to 38 or 78 weeks post salivary gland infection under the laboratory or quasi-natural conditions respectively in both categories of infection levels. There was apparently a density dependent relationship in T. parva survival, with a dramatic fall in infection occurring in ticks with high levels of infection between weeks 10 and 18 or weeks 38 and 46 post salivary gland infection in those exposed to laboratory or quasi-natural conditions before levelling off. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Ecdysteroids in relation to adult development and reproduction in female Rhipicephalus appendiculatus (Acari; Ixodidae)

In view of the paucity of information on ecdysteroids during tick development, the profiles of the free ecdysteroids, together with the polar and apolar conjugates have been established by radioimmunoassay during development of adult females of the hard tick, Rhipicephalus appendiculatus. The free ecdysteroid titre increased sharply to a peak approximately 3 days post-engorgement, a day preceding beginning of oviposition. This titre decreased to a low level, which was maintained throughout oviposition. Although the titre of polar ecdysteroid conjugates was appreciably less than that of the free ecdysteroids during the peak, the general profile of such conjugates was similar to that of the free ecdysteroids. In the case of the apolar ecdysteroid conjugates, the titre increased simultaneously with production of free ecdysteroids, but was maintained at a relatively high level until the end of oviposition, when it sharply declined. The apolar conjugates were the predominant form of ecdysteroids present during most of oviposition. The free ecdysteroids as well as the polar and apolar conjugates were shown to contain 20-hydroxyecdysone accompanied by smaller amounts of ecdysone by high-performance liquid chromatography-RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS [SIM]). © 1996 Wiley-Liss, Inc.     

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.     

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.     

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.     

The Effect of Male Ticks on the Feeding Performance of Immature Stages of Rhipicephalus Sanguineus and Amblyomma Americanum (Acari: Ixodidae)

A unique group of immunoglobulin-binding proteins (IGBPs), produced by ixodid male ticks during the latter half of their prolonged feeding period, improves the feeding performance of co-feeding females. As a follow-up to this observation, we investigated whether male tick feeding also affects the feeding of other developmental stages. Immature stages of Rhipicephalus sanguineus (Latreeille) and Amblyomma americanum (L.) were fed on rabbits in the presence or absence of conspecific males. The mean weight of larvae and nymphs of both species that fed around males and detached from the host on the first day of dropping was significantly higher than when the immature ticks fed on rabbits in the absence of males. However, larvae of both species and nymphs of R. sanguineus that fed slower and detached on the second day of dropping did not show significant differences in weight. A similar pattern was observed for A. americanum nymphs although, unlike R. sanguineus, the presence of males also influenced the feeding performance of the nymphs that fed slowly and detached on the second day of drop-off. The improved feeding performance demonstrated by immature ticks in the presence of males may be due to immunomodulatory saliva proteins, such as immunoglobulin-binding proteins (IGBPs) that are introduced into the co-feeding site. The results are considered in relation to the distribution of ixodid tick species on their natural hosts.     

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.