Amblyomma americanum (L): Tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.     

Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle

Cattle demonstrate divergent and heritable phenotypes of resistance and susceptibility to infestation with the cattle tick Rhipicephalus (Boophilus) microplus. Bos indicus cattle are generally more resistant to tick infestation than Bos taurus breeds although large variations in resistance can occur within subspecies and within breed. Increased tick resistance has been previously associated with an intense hypersensitivity response in B. taurus breeds; however, the mechanism by which highly resistant B. indicus cattle acquire and sustain high levels of tick resistance remains to be elucidated. Using the commercially available Affymetrix microarray gene expression platform, together with histological examination of the larval attachment site, this study aimed to describe those processes responsible for high levels of tick resistance in Brahman (B. indicus) cattle that differ from those in low-resistance Holstein-Friesian (B. taurus) cattle. We found that genes involved in inflammatory processes and immune responsiveness to infestation by ticks, although up-regulated in tick-infested Holstein-Friesian cattle, were not up-regulated in Brahman cattle. In contrast, genes encoding constituents of the extracellular matrix were up-regulated in Brahmans. Furthermore, the susceptible Holstein-Friesian animals displayed a much greater cellular inflammatory response at the site of larval R. microplus attachment compared with the tick-resistant Brahman cattle.     

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.     

Two-spotted spider mite reared on resistant eggplant affects consumption rate and life table parameters of its predator,Typhlodromus bagdasarjani (Acari: Phytoseiidae)

The blue tick, Rhipicephalus microplus, threatens cattle production in most tropical and subtropical areas of the world. Delayed skin hypersensitivity reactions are thought to cause Nguni cattle to be more resistant to R. microplus than Bonsmara cattle yet the cellular mechanisms responsible for these differences have not been classified. Tick counts and inflammatory cell infiltrates in skin biopsies from feeding sites of adult R. microplus ticks were determined in 9-month-old Nguni and Bonsmara heifers to determine the cellular mechanisms responsible for tick immunity. Nguni heifers (1.7 ± 0.03) had lower (P < 0.05) tick counts than the Bonsmaras (2.0 ± 0.03). Parasitized sites in Nguni heifers had higher counts of basophils, mast and mononuclear cells than those in the Bonsmara heifers. Conversely, parasitized sites in Nguni heifers had lower neutrophil and eosinophil counts than those in the Bonsmara heifers. Tick count was negatively correlated with basophil and mast cell counts and positively correlated with eosinophil counts in both breeds. In the Bonsmara breed, tick count was positively correlated with mononuclear cell counts. Cellular responses to adult R. microplus infestations were different and correlated with differences in tick resistance in Nguni and Bonsmara cattle breeds. It is essential to further characterise the molecular composition of the inflammatory infiltrate elicited by adult R. microplus infestation to fully comprehend immunity to ticks in cattle.     

Red deer (Cervus elaphus) as a host for the cattle tick Rhipicephalus microplus (Acari: Ixodidae) in Yucatan, Mexico

Rhipicephalus microplus is the most economically important cattle tick in the Mexican tropics. Wild ungulate species, including red deer (Cervus elaphus), are gaining popularity in diversified livestock ranching operations in Mexico. However, there is no information available on the susceptibility of red deer to infestation with the cattle tick, R. microplus, under hot, subhumid tropical conditions in Mexico. Biological data on R. microplus as an ectoparasite of cattle and red deer in a farm in the Mexican tropics are presented here. Ticks collected from red deer were identified as R. microplus (97 %) and Amblyomma cajennense (3 %), and tick species infesting cattle included R. microplus (95 %) and A. cajennense (5 %). Standard counts of R. microplus engorged females on red deer were 11 times higher than on cattle (428 ± 43 vs. 40 ± 18; p < 0.001). The reproductive efficiency index and larval hatching of R. microplus collected from cattle and red deer were similar (p > 0.05). Hemolymph samples of R. microplus collected from cattle were positive for Babesia spp. (10 %, 2/50) and all the samples from ticks infesting red deer were negative. Seventeen and ten percent of the blood samples from cattle and red deer were positive for Anaplasma marginale, respectively. The role of red deer as a host of R. microplus in Yucatan, Mexico and the importance of this host-parasite relationship relative to the epidemiology of R. microplus-borne diseases are discussed.     

Biological aspects of <Emphasis Type="Italic">Amblyomma brasiliense</Emphasis> (Acari: Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20°C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.     

The life cycle of Hyalomma rufipes (Acari: Ixodidae) under laboratory conditions

Biological characteristics of Hyalomma rufipes parasitising on rabbits and sheep were compared under laboratory conditions in Gansu, China. Mature ticks could parasitize both rabbits and sheep, while immature ticks only fed on rabbits successfully. Adults sucked blood on sheep significantly longer than on rabbits (16 and 13 days, respectively). Other adult parasite characteristics fed on the two hosts were similar, including the weight of engorged adult, female daily oviposition, and the weight and amount of the egg mass laid. Those indicated that this tick species showed little host specificity between sheep and rabbits during its adult stage. In total, the life cycle of H. rufipes was completed in an average period of 179 days. The average developmental periods were 59 days for egg incubation, 3 and 21 days for immature tick prefeeding and feeding, 2, 12 and 40 days for adult prefeeding, female preoviposition and oviposition. The longer female fed for engorgement, the shorter preoviposition period of engorged female needed, although when the feeding period was less than 15 days, this relationship was not obvious. The results confirmed the correlation between the weight of the engorged female and the number of eggs laid (r = 0.909). The reproductive efficiency index (REI) and reproductive fitness index (RFI) in females was 10.63 and 7.22, respectively. Engorged nymphs moulting to females were significantly heavier (27.6 ± 0.89 mg) than those moulting to males (22.3 ± 0.52 mg). Males outnumbered females by 1.4:1.     

The leucokinin-like peptide receptor from the cattle fever tick,Rhipicephalus microplus,is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes

Waladde S. M., Kemp D. H. and Rice M. J. 1979. Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes. International Journal for Parasitology9: 89–95. Newly moulted adult females of the cattle tick Boophilus microplus readily attach on a modified Baudruche membrane. Apparatus design permits the media offered below the membrane to be kept at 37°C and to be changed easily. Patterns of feeding activity were recorded within 2 h of placing the ticks on the membrane by monitoring the changes in electrical resistance between a tick and the medium with a high input impedance electric circuit. Differences in patterns of sucking and salivation were related to the chemical composition of media presented below the membrane. These observations suggest that the newly discovered cheliceral taste receptors of B. microplus are able to mediate changes in feeding patterns in response to stimulation by different chemical solutions in the feeding lesion. Incorporation of phosphorus-32 into the medium allows the volume ingested by ticks to be measured. The techniques described here facilitate the study of host factors that influence attachment, engorgement and detachment of the cattle tick.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.     

Goats,hares, and rabbits as hosts for the New Zealand cattle tick,Haemaphysalis longicornis

Abstract

Feral goats and hares were commonly infested by immature stages of the New Zealand cattle tick, Haemaphysalis longicornis. No explanation could be found for the low prevalence of adult ticks on these hosts. The ears of both host species were almost the exclusive feeding site of the ticks and this may be a consequence of grooming behaviour. Another potential host, the rabbit, was examined but few were found to be infested.

The less restricted range of non-domesticated hosts, together with feeding habits that differ from domestic stock, make them an important additional source of information on the ecology and seasonal pattern of activity of H. longicornis. Also, they are a source of contamination for tick-free pasture, and could possibly maintain the tick population in the absence of sheep and cattle. It is important that their role as alternative hosts be understood and considered in tick-control programmes.     

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman^® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.     

Boophilus microplus: digestion of hemoglobins by the engorged female tick

When the cattle tick Boophilus microplus, after dropping from its host, was maintained at 27 C, the digestion of hemoglobins in the gut proceeded at a steady rate and was virtually complete by the 13th day. The rate was essentially the same whether the ticks were strains susceptible (Yeerongpilly) or resistant (Biarra) to organophosphorous compounds or whether the hosts were British breed, Brahman or banteng cattle, or buffalo. Ferrihemoglobin appeared in the gut contents and hematin was deposited. About 10% of the hematin released from the hemoglobin was transferred to the eggs and from them to the larvae. Translucent ticks, sometimes found on heavily infested hosts and referred to as “serum” ticks, had about half the normal hemoglobin content.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.