Effect of α-tocopheryloxy acetic acid, a vitamin E derivative mitocan, on the experimental infection of mice with Plasmodium yoelii.

BACKGROUND: Malaria parasites are known to be vulnerable to oxidative stress. In this study, the effects of the administration of α-tocopheryloxy acetic acid (α-TEA), which is a vitamin E analogue mitocan, on Plasmodium yoelii infection in mice were examined. METHODS: Alpha-TEA was mixed with diet and fed to C57BL/6J mice before and/or after infection. For parasite infection, 4 × 104 red blood cells infected with P. yoelii (strain 17XL) were inoculated by intraperitoneal injection. In another series of experiment, the effect of the oral administration of α-TEA on P. yoelii 17XL infection in mice was examined. Finally, the combined effect of α-TEA and dihydroartemisinin or chloroquine on P. yoelii 17XL infection was examined. RESULTS: When 0.25% α-TEA was mixed with the diet for 7 days before infection and 14 days after infection (in total for 21 days), for 14 days after infection, and for 11 days from the third day after infection, all P. yoelii 17XL-infected mice survived during the observation period. However, all control mice died within 12 days after infection. These results indicated that α-TEA functions effectively even when administered post-infection. The oral administration of α-TEA for P. yoelii 17XL infection was also significant. Although the infected mice in the solvent control died within 10 days after infection, 90% of the mice infected with P. yoelii 17XL survived during the observation period when treated with 10 mg/head/day of α-TEA for 3 days from day 3 after infection. Although the combined effect of α-TEA and dihydroartemisinin (DHA) or chloroquine on P. yoelii 17XL infection was significant, no synergistic or additive effects were observed from the survival curve. CONCLUSIONS: This study showed the beneficial effects of α-TEA on the experimental infection of mice with P. yoelii 17XL. The stimulatory action of α-TEA on mitochondria and the accompanying reactions, such as reactive oxygen species production, and induction of apoptosis might have some effect on malarial infection.

Effect of vegetable oils on the experimental infection of mice with Trypanosoma congolense.

Vegetable oils are frequently used as solvents for lipophilic materials; accordingly, the effects of their components should be considered in animal experiments. In this study, the effects of various vegetable oils on the course of Trypanosoma congolense infection were examined in mice. C57BL/6J mice were orally administered four kinds of oils (i.e., coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil) with different fatty acid compositions and infected with T. congolense IL-3000. Oil-treated mice infected with T. congolense showed significantly higher survival rates and lower parasitemia than those of control mice. Notably, coconut oil, which mainly consists of saturated fatty acids, delayed the development of parasitemia at the early stage of infection. These results indicated that vegetable oil intake could affect T. congolense infection in mice. These findings have important practical implications; for example, they suggest the potential effectiveness of vegetable oils as a part of the regular animal diet for controlling tropical diseases and indicate that vegetable oils are not suitable solvents for studies of the efficacy of lipophilic agents against T. congolense.

Cryopreservation of canine spermatozoa using a skim milk-based extender and a short equilibration time.

Although useful spermatozoa cryopreservation techniques have been established, long-term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)-based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p < .05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p < .05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen-thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM-based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used.

Genetic mutations in sodium channel domain II and carboxylesterase genes associated with phenotypic resistance against synthetic pyrethroids by Rhipicephalus (Boophilus) decoloratus ticks in Uganda.

We previously reported emergence of super synthetic pyrethroid (SP) resistant Rhipicephalus (Boophilus) decoloratus ticks in Uganda. This study investigated the genetic basis of phenotypic resistance against SP in R. (B.) decoloratus and sought to identify novel diagnostic mutations for rapid detection of SP resistance in the above tick species. Genomic DNA was extracted from pooled larvae of 20 tick populations (19 of known SP susceptibility and 1 unknown susceptibility). The voltage sensitive sodium channel (VSSC) domain II S4-5 linker (SP target) and partial carboxylesterase (SP metabolizing enzyme) genes were amplified by PCR, cloned and sequenced. The resultant sequences were analyzed to determine single nucleotide polymorphisms (SNPs) associated with phenotypic resistance in the various tick populations investigated. Novel SNPs that introduced Eco RI and Eco RII restriction sites in carboxylesterase gene were identified in silco and validated with restriction fragment length polymorphism (RFLP) against 18 tick populations of known SP susceptibility. The study identified a super knock down resistance (kdr) mutation T58C in R. (B.) decoloratus VSSC associated with stable SP resistance. We further identified multiple nonsynonymous mutations in carboxylesterase of SP resistant ticks; one of which conferred novel EcoRII (G195C) restriction site for PCR-RFLP detection of SP resistance. In conclusion, this study is the first to report super kdr mutation in sodium channel domain II and multiple mutations in carboxylesterase genes that may concurrently mediate stable resistance against synthetic pyrethroids in R. (B.) decoloratus ticks from Uganda. The Eco RII based PCR-RFLP is a useful tool for rapid detection of stable SP resistant R. (B.) decoloratus ticks.

First report of dog ticks and tick-borne pathogens they are carrying in Malawi.

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi.

Acaricidal activity of Erigeron acer L. root against Haemaphysalis longicornis and phytochemical profiling by liquid chromatography-tandem mass spectrometry.

The present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Inhibitory effect of cyclophilin A from the hard tick Haemaphysalis longicornis on the growth of Babesia bovis and Babesia bigemina.

Haemaphysalis longicornis is known as one of the most important ticks transmitting Babesia parasites in East Asian countries, including Babesia ovata and Babesia gibsoni, as well as Theileria parasites. H. longicornis is not the natural vector of Babesia bovis and Babesia bigemina. Vector ticks and transmitted parasites are thought to have established unique host-parasite interaction for their survival, meaning that vector ticks may have defensive molecules for the growth control of parasites in their bodies. However, the precise adaptation mechanism of tick-Babesia parasites is still unknown. Recently, cyclophilin A (CyPA) was reported to be important for the development of Babesia parasites in ticks. To reveal a part of their adaptation mechanism, the current study was conducted. An injection of B. bovis-infected RBCs into adult female H. longicornis ticks was found to upregulate the expression profiles of the gene and protein of CyPA in H. longicornis (HlCyPA). In addition, recombinant HlCyPA (rHlCyPA) purified from Escherichia coli exhibited significant inhibitory growth effects on B. bovis and B. bigemina cultivated in vitro, without any hemolytic effect on bovine RBCs at all concentrations used. In conclusion, our results suggest that HlCyPA might play an important role in the growth regulation of Babesia parasites in H. longicornis ticks, during natural acquisition from an infected host. Furthermore, rHlCyPA may be a potential alternative chemotherapeutic agent against babesiosis.

Draft genome sequence data of Haemaphysalis longicornis Oita strain.

Haemaphysalis longicornis Neumann, 1901 is one of the most well-known hard ticks because of its medical and veterinary importance. Haemaphysalis longicornis transmit a wide range of pathogens among vertebrates, affecting humans and animals in Asia and Oceania. In Japan, the tick species is a major pest of cattle because it can spread a protozoan parasite Theileria orientalis, which causes theileriosis and produces economic losses to the livestock industry (Yokoyama et al. 2012 [1]). Apart from bovine theileriosis, H. longicornis is a vector of bovine babesiosis caused by Babesia ovata, canine babesiosis caused by Babesia gibsoni, and rickettsiosis and viral diseases in humans. Its habitats are mainly Japan, Australia, New Zealand, New Caledonia, the Fiji Islands, Korea, China, and Russia (Oliver et al. 1973 [2]). In the United States, heavy H. longicornis infestations on cattle and white-tailed deer were reported in 2019, making it now one of the tick species to be an increasing threat to livestock animals and humans globally. Ticks reproduce offspring after mating with female and male ticks, however, interestingly, there are two races of H. longicornis: bisexual (diploid) and parthenogenetic (triploid) races [2]. Parthenogenetic H. longicornis is distributed throughout Japan, while the northern limit of the bisexual race is believed to be Fukushima Prefecture on Honshu Island (Fujita et al. 2013 and Kitaoka et al. 1961 [3,4]). This tick species is also considered to be of great scientific importance, and the parthenogenetic race collected in Okayama prefecture has been reared since 1961, while the bisexual race collected in Oita prefecture has been reared since 2008 under laboratory conditions in Japan (Boldbaatar et al. 2010 and Fujisaki et al. 1976 [5,6]). Namely, the "Okayama strain" and "Oita strain" of H. longicornis have been maintained for more than six decades and 15 years, respectively, stably under laboratory conditions. To obtain reference data of bisexual H. longicornis, we sequenced unfed females with haploid genomes using Illumina and MinION Q20 kit then obtained a draft genome consisting of 2.48 Gbp. The number of the contig was 98,529 and N50 was 46.5 Kb. Genome information derived from our laboratory colony of bisexual H. longicornis ticks would provide fundamental insight into understanding how different reproductive lineages occur within the same species of the tick.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

Localization of secreted ferritin (FER2) in the embryos of the tick Haemaphysalis longicornis.

Despite the absence of a blood meal, embryogenesis involves many processes that require nutrients and other essential elements, including iron. Due to the lack of an external source of these nutrients, these requirements are acquired maternally. Because of the toxic nature of iron, they are transferred through iron transport molecules such as secreted ferritin (FER2). Here we tried to follow the trail of the FER2 through indirect immunofluorescence, and we observed an apparent shift of FER2 from the germ layer at the early part of development to the appendages during the late stage of embryogenesis. FER2 is also found in the middle part of the legs of the embryo. The apparent movement not only sheds light on iron processing events during embryogenesis but also indirectly guides organogenesis in the tick.

Investigation of vertical and horizontal transmission of Spiroplasma in ticks under laboratory conditions.

Many arthropods harbour bacterial symbionts, which are maintained by vertical and/or horizontal transmission. Spiroplasma is one of the most well-known symbionts of ticks and other arthropods. It is still unclear how Spiroplasma infections have spread in tick populations despite its high prevalence in some tick species. In this study, Ixodes ovatus, which has been reported to harbour Spiroplasma ixodetis at high frequencies, was examined for its vertical transmission potential under experimental conditions. Next, two isolates of tick-derived Spiroplasma, S. ixodetis and Spiroplasma mirum, were experimentally inoculated into Spiroplasma-free Haemaphysalis longicornis colonies and the presence of Spiroplasma in their eggs and larvae was tested. Our experimental data confirmed that S. ixodetis was transmitted to eggs and larvae in a vertical manner in the original host I. ovatus. In the second experiment, there was no significant difference in engorged weight, egg weight, and hatching rate between Spiroplasma-inoculated and control H. longicornis groups. This suggested that Spiroplasma infection does not affect tick reproduction. Spiroplasma DNA was only detected in the eggs and larvae derived from some individuals of S. ixodetis-inoculated groups. This has demonstrated the potential of horizontal transmission between different tick species. These findings may help understand the transmission dynamics of Spiroplasma in nature and its adaptation mechanism to host arthropod species.

Identification and Characterization of Rhipicephalus microplus ATAQ Homolog from Haemaphysalis longicornis Ticks and Its Immunogenic Potential as an Anti-Tick Vaccine Candidate Molecule.

Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased (p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72-3.46) and 1.46% (CI 0.87-2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43-14.90) and 0.33% (CI 0.02-1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01-0.51) and 2.08% (CI 1.25-3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey.

RNA activation in ticks.

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3'- untranslated region (3'-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3'-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

RNAi of the translation inhibition gene 4E-BP identified from the hard tick, Haemaphysalis longicornis, affects lipid storage during the off-host starvation period of ticks.

4E-BP, an eIF4E-binding protein, is well known as a cap-dependent translation inhibitor. Here, the 4E-BP homolog, Hl4E-BP, was isolated and identified from the hard tick Haemaphysalis longicornis. Hl4E-BP transcripts were ubiquitously expressed in the active stages, including the larvae, nymphs, and female adults, and the transcription levels were found to be higher in unfed than engorged ticks. In contrast, the expression levels of non-phosphorylated Hl4E-BP, which is a 13.4-kDa protein detected by the anti-recombinant Hl4E-BP antibody, were the highest in engorged ticks and significantly decreased progressively during the unfed starvation period of ticks. The functional role of Hl4E-BP as a metabolic brake was verified by histochemical observations on the lipid storage in midguts and fat bodies during the starvation period using ticks injected with dsHl4E-BP. The results indicate that Hl4E-BP is highly relevant to the lipid storage of ticks during the non-feeding starvation period. Our results suggest, for the first time, that Hl4E-BP may have a crucial role in the starvation resistance of ticks in an off-host condition via lipid metabolism control, although it was unclear whether Hl4E-BP might be involved in lipid synthesis regulation and/or lipid consumption inhibition.

Anti-babesial activity of a potent peptide fragment derived from longicin of Haemaphysalis longicornis.

Babesiosis is one of the most important tick-borne diseases affecting livestock that can cause major economic losses worldwide particularly in the tropics. Control relies on controlling both the protozoan parasite and the tick vector. Antiprotozoal drugs are most commonly used for treatment, but problems on emergence of resistant strains and food residues are encountered. Longicin, a defensin-like peptide identified from the hard tick, Haemapysalis longicornis, as well as one of its synthetic partial analogs (P4), were previously reported to exert antimicrobial, fungicidal, and parasiticidal activity. Both longicin and P4 showed babesiacidal activity, in vitro and in vivo. Here, peptide fragments of P4 were studied for in vitro activity against bovine Babesia parasites. One of the peptide fragments, antimicrobial peptide 1 (AMP1), reduced the parasitemia of Babesia bigemina. No peptide had significant effect on Babesia bovis. The sequence of AMP1 corresponded to the longicin sequence which is associated with antiparasitic activity. Although AMP1 caused reduction in parasitemia of B. bigemina, the difference in morphology of the parasite compared with the control group was not statistically significant. However, the percentage occurrence of piroplasms decreased, whereas the abnormal pycnotic form increased. The results demonstrated that this shorter peptide retained the anti-babesial activity of the parent peptide, exerting an antiparasitic effect against a bovine Babesia species. Therefore, this short peptide can be considered for chemical synthesis as an alternative therapeutic agent for babesiosis.

Effect of α-Tocopheryloxy Acetic Acid on the Infection of Mice with Plasmodium berghei ANKA In Vivo and Humans with P. falciparum In Vitro.

PURPOSE: Malarial parasites are susceptible to oxidative stress. The effects of α-tocopheryloxy acetic acid (α-TEA), a vitamin E analog, on infection by Plasmodium berghei ANKA and P. falciparum in mice and human red blood cells (RBCs), respectively, were examined in this study. METHODS: For in vivo studies in mice, RBCs infected with P. berghei ANKA were inoculated via intraperitoneal injection and α-TEA was administered to C57BL/6 J male mice after infection. The blood-brain barrier (BBB) permeability was examined by Evans blue staining in experimental cerebral malaria at 7 days after infection. The in vitro inhibitory effect of α-TEA on P. falciparum 3D7 (chloroquine-sensitive strain) and K1 (multidrug-resistant strain) was tested using a SYBR Green I-based assay. RESULTS: When 1.5% α-TEA was administered for 14 days after infection, 88% of P. berghei ANKA-infected mice survived during the experimental period. Nevertheless, all the control mice died within 12 days of infection. Furthermore, the Evans blue intensity in α-TEA-treated mice brains was less than that in untreated mice, indicating that α-TEA might inhibit the destruction of the BBB and progression of cerebral malaria. The in vitro experiment revealed that α-TEA inhibited the proliferation of both the 3D7 and K1 strains. CONCLUSION: This study showed that α-TEA is effective against murine and human malaria in vivo and in vitro, respectively. Although α-TEA alone has a sufficient antimalarial effect, future research could focus on the structure-activity relationship to achieve better pharmacokinetics and decrease the cytotoxicity and/or the combined effect of α-TEA with existing drugs. In addition, the prophylactic antimalarial activity of premedication with α-TEA may also be an interesting perspective in the future.