Hard tick calreticulin (CRT) gene coding regions have only one intron with conserved positions and variable sizes

Calreticulin (CRT) is a unique eukaryotic gene. The CRT gene product, calreticulin, was first identified as a calcium binding protein in 1974, but further investigations have indicated that CRT protein performs many functions in cells, including involvement in evading the host's immune system by parasites. Many studies of CRT have been published since the molecule was first discovered; however, the CRT gene exon-intron structure is only known for a limited number of ectoparasite species. In this study, we compared tick CRT genomic sequences to the corresponding cDNA from 28 species and found that 2 exons and 1 intron are present in the tick CRT gene. The intron position is conserved in 28 hard ticks, but intron size and nucleotide sequences vary. Three tick introns possess duplicated fragments and are twice as long as other introns. All tick CRT introns obey the GT-AG rule in the splice-site junctions and are phase 1 introns. By comparing tick CRT introns to those of fruit fly, mouse, and human, we conclude that tick CRT introns belong to the intron-late type. The number and size of CRT introns have increased through the evolution of eukaryotes.     

Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.     

A comparison of infestation patterns by Ixodes ticks in urban and rural populations of the Common Blackbird Turdus merula

Although spatial variation in the patterns of parasite infestations among host populations may have important ecological and epidemiological consequences, the causes underlying such variation are poorly known. In the context of a long-term study on the population biology of Common Blackbirds Turdus merula , we examined the prevalence and intensity of infestation by Ixodes ticks between birds living in rural vs. urban habitats. The overall prevalence of tick infestations was significantly higher in the rural habitat where 74% of individuals ( n  = 130) were infested. This result contrasted markedly with the situation in the urban habitat where less than 2% of individuals ( n  = 360) carried ticks. There was no significant effect of the sex of the host on the intensity or prevalence of tick infestations. There was a significant effect of the age of the host on tick infestations essentially due to the absence of ticks on nestlings. Possible mechanisms responsible for the differences between habitats could include differences in tick survival and/or host resistance towards ticks. Previous studies have shown higher population densities and suggested longer survival for Blackbirds in urban than in rural habitats. Given that ixodid ticks are known to transmit pathogens like Borrelia spp. to wild birds, and that Blackbirds can act as reservoirs for these pathogens, the infection patterns observed in our study area provide a suitable situation to study the interrelations between ticks, Blackbirds and pathogens.     

African swine fever virus multigene family 360 genes affect virus replication and generalization of infection in Ornithodoros porcinus ticks

Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host, Ornithodoros porcinus porcinus, an MGF360/530 gene deletion mutant (Pr4Delta35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Delta35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Delta3-C2, which lacked three MGF360 genes (3HL, 3Il, and 3LL), exhibited reduced viral growth in ticks. Pr4Delta3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Delta3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Delta3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.     

A conserved basic residue cluster is essential for the protein quality control function of the Arabidopsis calreticulin 3

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca²⁺ homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca²⁺-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3′s C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3^M transgenes showed that a highly conserved basic tetrapeptide Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ is essential but a less conserved basic tetrapeptide Arg⁴⁰¹Arg⁴⁰²Arg⁴⁰³Arg⁴⁰⁴ is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Host detection by four Australian tick species

The behavior of 4 tick species in the presence of their dominant host species was examined. Nymphs and adult ticks could locate a host from greater distances than the larvae. Adult Aponomma concolor were able to locate their mammalian host (echidna) from distances greater than 3 reptile-infesting species could locate their hosts. The results suggested that all 4 tick species detect hosts passively, which may be related to the relative scarcity of hosts and their irregular use of refuge sites.     

Salivary glands in ixodid ticks: control and mechanism of secretion

The salivary glands are vital to the biological success of ixodid ticks and the major route for pathogen transmission. Important functions include the absorption of water vapor from unsaturated air by free-living ticks, excretion of excess fluid for blood meal concentration, and the secretion of bioactive protein and lipid compounds during tick feeding. Fluid secretion is controlled by nerves. Dopamine is the neurotransmitter at the neuroeffector junction regulating secretion via adenylate cyclase and an increase in cellular cAMP. Dopamine also affects the release of arachidonic acid which is subsequently converted to prostaglandins. Prostaglandin E(2) (PGE(2)) is secreted at extremely high levels into tick saliva for export to the host where it impacts the host physiology. Additionally, PGE(2) has an autocrine or paracrine role within the salivary gland itself where it interacts with a PGE(2) receptor to induce secretion (exocytosis) of bioactive saliva proteins via a phosphoinositide signalling pathway and an increase in cellular Ca(2+). Regulation of fluid secretion has been extensively studied, but little is known about the mechanism of fluid secretion. Continuing advances in tick salivary gland physiology will be made as key regulatory and secretory gland proteins are purified and/or their genes cloned and sequenced.     

Detection of Wolbachia in the tick Ixodes ricinus is due to the presence of the hymenoptera endoparasitoid Ixodiphagus hookeri

The identification of micro-organisms carried by ticks is an important issue for human and animal health. In addition to their role as pathogen vectors, ticks are also the hosts for symbiotic bacteria whose impact on tick biology is poorly known. Among these, the bacterium Wolbachia pipientis has already been reported associated with Ixodes ricinus and other tick species. However, the origins of Wolbachia in ticks and their consequences on tick biology (known to be very diverse in invertebrates, ranging from nutritional symbionts in nematodes to reproductive manipulators in insects) are unknown. Here we report that the endoparasitoid wasp Ixodiphagus hookeri (Hymenoptera, Chalcidoidea, Encyrtidae)--strictly associated with ticks for their development--infested at almost 100% prevalence by a W. pipientis strain belonging to a Wolbachia supergroup that has already been reported as associated with other hymenopteran parasitoids. In a natural population of I. ricinus that suffers high parasitism rates due to I. hookeri, we used specific PCR primers for both hymenopteran and W. pipientis gene fragments to show that all unfed tick nymphs parasitized by I. hookeri also harbored Wolbachia, while unparasitized ticks were Wolbachia-free. We demonstrated experimentally that unfed nymphs obtained from larvae exposed to I. hookeri while gorging on their vertebrate host also harbor Wolbachia. We hypothesize that previous studies that have reported W. pipientis in ticks are due to the cryptic presence of the endoparasitoid wasp I. hookeri. This association has remained hidden until now because parasitoids within ticks cannot be detected until engorgement of the nymphs brings the wasp eggs out of diapause. Finally, we discuss the consequences of this finding for our understanding of the tick microbiome, and their possible role in horizontal gene transfer among pathogenic and symbiotic bacteria.     

Metagenomic Profile of the Viral Communities in Rhipicephalus spp. Ticks from Yunnan,China

Besides mosquitoes, ticks are regarded as the primary source of vector-borne infectious diseases. Indeed, a wide variety of severe infectious human diseases, including those involving viruses, are transmitted by ticks in many parts of the world. To date, there are no published reports on the use of next-generation sequencing for studying viral diversity in ticks or discovering new viruses in these arthropods from China. Here, Ion-torrent sequencing was used to investigate the presence of viruses in three Rhipicephalus spp. tick pools (NY-11, NY-13, and MM-13) collected from the Menglian district of Yunnan, China. The sequencing run resulted in 3,641,088, 3,106,733, and 3,871,851 reads in each tick pool after trimming. Reads and assembled contiguous sequences (contigs) were subject to basic local alignment search tool analysis against the GenBank database. Large numbers of reads and contigs related to known viral sequences corresponding to a broad range of viral families were identified. Some of the sequences originated from viruses that have not been described previously in ticks. Our findings will facilitate better understanding of the tick virome, and add to our current knowledge of disease-causing viruses in ticks living under natural conditions.     

Ticks from a Morelet's crocodile in Belize.

Parasitism of crocodilians by ticks has rarely been reported, and to our knowledge only seven published accounts exist. On 3 July 1999, we collected four ticks from a subadult Morelet's crocodile (Crocodylus moreletii) captured in northern Belize. These were identified as Amblyomma dissimile (one female), and Amblyomma sp. (two nymphs, one larva). The crocodile was captured on land approximately 100 m from water, and all four ticks were attached to loose skin on the lateral surface of the tail. Crocodilians are most susceptible to terrestrial ectoparasites, including ticks, during overland movements. However, most such movements occur in response to drought, when tick questing activity is suppressed, which likely accounts for the small numbers of tick specimens recorded from crocodilians and the absence of any noticeable impact of parasitism on host fitness.     

Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis

Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT-PCR with the 3' and 5' RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His(57) and Ser(195) conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT-PCR products were used to prime the 3' and 5' RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT-PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conserved in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research.     

Four serine proteinase inhibitors (serpin) from the brown ear tick,Rhiphicephalus appendiculatus; cDNA cloning and preliminary characterization

While development of an anti-Boophilus microplus vaccine is advanced and practical, work on other economically important ticks such as Rhipicephalus appendiculatus is still in its infancy. Guess PCR primers, designed from a consensus amino acid sequence (NAVYKFG) motif were used with rapid amplification of cDNA ends (RACE) to clone four cDNAs encoding serine proteinase inhibitors (serpin) from the brown ear tick Rhipicephalus appendiculatus. The four genes designated as R. appendiculatus serpin (RAS) -1 to -4 encode polypeptides of 378, 380, 398 and 486 amino acids long, respectively. Sequence comparison of RAS-1 to -4 predicted amino acid sequences to the serpin-like hypothetical protein from Ixodes ricinus (Leboulle et al., 2002) revealed closer structural similarities among tick serpins. Expression analysis by RT-PCR showed that RAS-1 to -4 are expressed in other tick organs in addition to salivary glands and midguts. Except for RAS-3 whose expression level appears to be equivalent in all tick organs, RAS-1, -2 and -4 are predominantly expressed in the salivary glands. We have discussed our findings with reference to development of vaccines against R. appendiculatus.     

Competition,facilitation or mediation via host? Patterns of infestation of small European mammals by two taxa of haematophagous arthropods

1. We studied the effect of flea infestation on the pattern of tick (Ixodes ricinus and Ixodes trianguliceps) infestation on small mammals. 2. We asked (1) whether the probability of an individual host being infested by ticks was affected by its infestation of fleas (number of individuals and species) and (2) whether the abundance and prevalence of ticks in a host population was affected by the abundance, prevalence, level of aggregation, and species richness of fleas. 3. The probability of a host individual being infested by ticks was affected negatively by flea infestation. At the level of host populations, flea abundance and prevalence had a predominantly positive effect on tick infestation, whereas flea species richness had a negative effect on tick infestation. 4. The effect of flea infestation on tick infestation was generally greater in I. ricinus than in I. trianguliceps, but varied among host species. 5. It can be concluded that the effect of fleas on tick infestation of small mammals may be either negative or positive depending on the level of consideration and parameters involved. The results did not provide support for direct interactions between the two ectoparasite taxa, but suggested population and community dynamics and the defence system of the hosts as possible factors.     

Emerging rickettsioses

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.     

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100?bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2–HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.