Leucine aminopeptidase,HlLAP, from the ixodid tick Haemaphysalis longicornis,plays vital roles in the development of oocytes

Female ixodid ticks are amazing invertebrate animals which efficiently convert a large amount of nutrients derived from their ingested blood meals into eggs. Although oocyte development (vitellogenesis) in ticks is triggered by a blood meal and is assumed to be supported by nutrition derived from ovarian cells connecting oocytes, little is known about the ovarian molecules processing nutrient materials for the vitellogenesis. In this study, we have suggested a putative function of leucine aminopeptidase (HlLAP) in the ovary of parthenogenetic adult ixodid tick Haemaphysalis longicornis regarding a negative output of reproduction following disruption of HlLAP gene by RNA interference. Endogenous HlLAP was shown to be localized in the ovarian cells, including ovarian epithelial and pedicel cells which were assumed to provide nutrients for the developing oocytes. Histological studies demonstrated that a majority of immature oocytes in HlLAP gene knockdown ticks were transformed into abnormal morpho-histological oocytes with vacuolated cytoplasm and/or condensed nucleus. Taken together, a reduction of the numbers of laid eggs in the HlLAP gene knockdown ticks may be due to the degeneration of immature oocytes following deprivation of nutrients such as amino acids supplied not only by midgut HlLAP but also by the ovarian HlLAP. Regulation of the tick molecules involved in nutrient metabolism for the reproduction, including blood digestion and vitellogenesis, would help in controlling the tick population and tick-borne pathogens.     

No Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Ixodid Ticks Collected in Seoul

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.     

Detachment of hard ticks (Acari: Ixodidae) from hunted sika deer (Cervus nippon)

Ixodid ticks were collected from 13 sika deer, Cervus n. nippon, shot in the Boso Peninsula in central Japan from late February to early March 1999. Haemaphysalis megaspinosa was the most abundant species of the adults collected, although Haemaphysalis longicornis, H. flava, H. kitaokai, H. cornigera, Ixodes ovatus, and Amblyomma testudinarium were also collected. Males were more abundant than females for H. longicornis, H. megaspinosa, H. flava, and H. kitaokai. Ticks that had inserted their hypostome into its host skin (designated attached) were distinguished from those that were detached and on the host’s surface. A greater fraction of males than females of all four species were detached. Females were classified in three feeding stages (engorged, partially engorged, and unfed). More H. longicornis and H. megaspinosa unfed female ticks than engorged and partially-engorged female ticks were collected detached. Our results indicated that H. megaspinosa, H. longicornis, H. flava, and H. kitaokai male ticks detached sooner than female ticks after their host died.     

Association of the invasive Haemaphysalis longicornis tick with vertebrate hosts,other native tick vectors,and tick-borne pathogens in New York City,USA

Haemaphysalis longicornis, the Asian longhorned tick, is an invasive ixodid tick that has rapidly spread across the northeastern and southeastern regions of the United States since first reported in 2017. The emergence of H. longicornis presents a potential threat for livestock, wildlife, and human health as the host associations and vector potential of this invasive pest in the United States are poorly understood. Previous field data from the United States has shown that H. longicornis was not associated with natural populations of small mammals or birds, but they show a preference for medium sized mammals in laboratory experiments. Therefore, medium and large sized mammals were sampled on Staten Island, New York, United States, to determine H. longicornis host associations and vector potential for a range of human and veterinary pathogens. A total of 97 hosts were sampled and five species of tick (Amblyomma americanum, Dermacentor variabilis, H. longicornis, Ixodes scapularis, Ixodes cookei) were found feeding concurrently on these hosts. Haemaphysalis longicornis was found in the highest proportions compared with other native tick species on raccoons (55.4%), Virginia opossums (28.9%), and white-tailed deer (11.5%). Tissue, blood, and engorged larvae were tested for 17 different pathogens using a nanoscale PCR platform. Infection with five pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia spp., Mycoplasma haemocanis, and Bartonella spp.) was detected in host samples, but no pathogens were found in any larval samples. These results suggest that although large and medium sized mammals feed large numbers of H. longicornis ticks in the environment, there is presently a low potential for H. longicornis to acquire pathogens from these wildlife hosts.     

Aversion of the invasive Asian longhorned tick to the white-footed mouse,the dominant reservoir of tick-borne pathogens in the U.S.A.

The Asian longhorned tick (Haemaphysalis longicornis) was reported for the first time in the U.S.A. in 2017 and has now spread across 12 states. The potential of this invasive tick vector to transmit pathogens will be determined through its association to hosts, such as the white-footed mouse (Peromyscus leucopus), which is the primary reservoir for the causative agent of Lyme disease (Borrelia burgdorferi) and other zoonotic pathogens. Larval H. longicornis were placed on P. leucopus; 65% of the larvae (n = 40) moved off the host within a short period of time, and none engorged. By contrast, larval blacklegged ticks (Ixodes scapularis) did not move from where they were placed in the ear of the mouse. A laboratory behavioural assay was then conducted to assess the interaction of H. longicornis with the hair of potential mammalian host species in the U.S.A. H. longicornis larvae were significantly less likely to enter the hair zone of P. leucopus and humans compared to the hair of domestic cats, domestic dogs and white-tailed deer. This study identifies a tick–host interaction behaviour, which can be quantified in a laboratory assay to predict tick–host associations and provides insights into how ticks select a host.     

Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis

Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria.     

Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis

Ticks grow rapidly during blood feeding, and their body weight may ultimately increase 100-fold more than that before feeding. The molecular mechanisms controlling growth during blood feeding in ticks remain largely unknown. The conserved insulin/PI3K/Akt signaling pathway regulates growth and metabolism in eukaryotes. Here, we show evidence for the involvement of Akt in growth during blood feeding in the parthenogenetic strain of the hard tick Haemaphysalis longicornis. We identified a homolog of the Ser/Thr kinase Akt (HlAkt) from the EST database of the H. longicornis embryo. HlAkt cDNA had a 1,590 bp ORF that encodes 529 amino acids with a predicted molecular weight of 60 kDa. HlAkt possesses a PH domain, a Ser/Thr kinase domain, a hydrophobic motif, and dual phosphorylation residues (Thr 338 and Ser 503) that are essential for kinase activation. Knockdown of HlAkt by RNA interference caused inhibition of blood feeding in female ticks. Histological observation demonstrated that HlAkt knockdown led to the arrest of growth in internal organs. HlAkt knockdown also affected the expressions of blood meal-induced genes that are essential for blood digestion, development, and reproduction in the female tick. These results strongly indicate that HlAkt is essential to complete the blood feeding process accompanied by the growth of internal organs in adult ticks. This is the first report of identification and characterization of Akt in Chelicerata, including ticks.     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

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.     

Ticks collected from migratory birds,including a new record of Haemaphysalis formosensis,on Jeju Island,Korea

Migratory birds may disperse parasites across ecological barriers, and recent climate change may alter the pattern of ectoparasite dispersal via changed patterns of bird migration. In order to document the parasitization of migratory birds by Ixodidae ticks on Jeju Island in Korea, we examined 934 migratory birds comprising 75 species for ticks from 2010 to 2012. In total, 313 ticks were collected from 74 migratory birds across 17 avian species and identified based on morphological keys. These ticks represented six species: Haemaphysalis flava, H. formosensis, H. longicornis, H. concinna, Ixodes turdus and I. nipponensis. Of particular note was the presence of H. formosensis, a species not previously reported to have been found in Korea, and H. concinna, which had not been previously reported on Jeju Island. The dominant tick species found were H. flava (226 ticks, 72.2 %) and I. turdus (54 ticks, 17.3 %), and ground-dwelling thrushes such as Pale thrushes (Turdus pallidus; 39 birds, 52.7 %) were the most important hosts. Although H. longicornis is the most abundant and prevalent terrestrial tick on Jeju Island, the species accounted for only 3.8 % of the total ticks collected in this study, suggesting that ticks on migratory birds may differ from the local tick fauna and that exotic ticks may be introduced via migratory birds. Therefore, long-term programs for tick and tick-borne disease surveillance are recommended to understand the role of migratory animals in the introduction of exotic species and associated pathogens and in life cycles of ticks at different stages in this region.     

Hemolymph proteins in ticks

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.     

Proteomic changes in various organs of Haemaphysalis longicornis under long-term starvation

Haemaphysalis longicornis (Neumann), a tick of public health and veterinary importance, spend the major part of their life cycle off-host, especially the adult host-seeking period. Thus, they have to contend with prolonged starvation. Here, we investigated the underlying molecular mechanism of tick starvation endurance in the salivary glands, midguts, ovaries, and Malpighian tubules of starved H. longicornis ticks using the data-independent acquisition quantitative proteomic approach to study the proteome changes. Essential synthases such as glutamate synthase, citrate synthase, and ATP synthase were up-regulated probably due to increased proteolysis and amino acid catabolism during starvation. The up-regulation of succinate dehydrogenase, ATP synthase, cytochrome c oxidase, and ADP/ATP translocase closely fits with an increased oxidative phosphorylation function during starvation. The differential expression of superoxide dismutase, glutathione reductase, glutathione S-transferase, thioredoxin, and peroxiredoxin indicated fasting-induced oxidative stress. The up-regulation of heat shock proteins could imply the activation of a protective mechanism that checks excessive protein breakdown during starvation stress. The results of this study could provide useful information about the vulnerabilities of ticks that could aid in tick control efforts.     

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.     

The temperature and humidity preferences of Haemaphysalis longicornis, Ixodes Holocyclus and Rhipicephalus sanguineus (Ixodidae): studies on eggs

The eggs of Haemaphysalis longicornis, Ixodes holocydus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to compare the temperature and humidity requirements of ticks from climatically dissimilar habitats. The response to changes in humidity was also studied with H. longicornis. Hatching in I. holocydus eggs occurred from 18 to 28°C and at a saturation deficit of 2 mm Hg or less. The eggs of R. sanguineus hatched between 18 and 38°C and tolerated saturation deficits up to 20 mm Hg. The eggs of H. longicornis hatched between 18 and 35°C and at saturation deficits up to 8 mm Hg. When eggs of H. longicornis were exposed to moderately dry conditions for different periods of time and then transferred to moist conditions, incubation periods and mortality were reduced in comparison with eggs kept continuously under dry conditions. On the other hand, mortality of H. longicornis eggs was reduced in moderately dry conditions, provided that they had been exposed to a moist environment for about 10 days previously. Weight loss of eggs in dry air and the rate of development of eggs were examined and are discussed. The eggs of I. holocylus lost weight very rapidly in dry air, whereas weight loss from R. sanguineus eggs was much slower. Weight loss from H. longicornis eggs fell between these two rates. The rate of development was fastest in R. sanguineus and slowest in I. holocydus.     

Antiviral RNA interference in disease vector (Asian longhorned) ticks

Disease vectors such as mosquitoes and ticks play a major role in the emergence and re-emergence of human and animal viral pathogens. Compared to mosquitoes, however, much less is known about the antiviral responses of ticks. Here we showed that Asian longhorned ticks (Haemaphysalis longicornis) produced predominantly 22-nucleotide virus-derived siRNAs (vsiRNAs) in response to severe fever with thrombocytopenia syndrome virus (SFTSV, an emerging tick-borne virus), Nodamura virus (NoV), or Sindbis virus (SINV) acquired by blood feeding. Notably, experimental acquisition of NoV and SINV by intrathoracic injection also initiated viral replication and triggered the production of vsiRNAs in H. longicornis. We demonstrated that a mutant NoV deficient in expressing its viral suppressor of RNAi (VSR) replicated to significantly lower levels than wildtype NoV in H. longicornis, but accumulated to higher levels after knockdown of the tick Dicer2-like protein identified by phylogeny comparison. Moreover, the expression of a panel of known animal VSRs in cis from the genome of SINV drastically enhanced the accumulation of the recombinant viruses. This study establishes a novel model for virus-vector-mouse experiments with longhorned ticks and provides the first in vivo evidence for an antiviral function of the RNAi response in ticks. Interestingly, comparing the accumulation levels of SINV recombinants expressing green fluorescent protein or SFTSV proteins identified the viral non-structural protein as a putative VSR. Elucidating the function of ticks’ antiviral RNAi pathway in vivo is critical to understand the virus-host interaction and the control of tick-borne viral pathogens.