REMIL LINGGATONG GALAY
University of Philippines Diliman
Dr. Galay, an associate professor at the UP Los Baños College of Veterinary Medicine, currently works on ticks and tick-borne diseases. Dr. Galay has led several research projects dealing with molecular detection of various tick-borne pathogens in livestock and companion animals. His work has led to discoveries on animal parasitic diseases, a significant contribution to his area of research. He is awarded as one of the Outstanding Young Scientists of 2020.
Sex: Male
Education: Yamaguchi University, Doctor of Philosophy in Veterinary Sciences, April 2011 - March 2015
Field of Specialization:
Zoology
Entomology
Parasitology
Researches:
Article title: Development of a Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting the Citrate Synthase Gene for Detection of Ehrlichia canis in Dogs
Authors: Remil Linggatong Galay, Tetsuya Tanaka
Publication Title: Veterinary Sciences 7(156), October 2020
Abstract:
Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples-after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 • C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar's test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.
Full text link: https://tinyurl.com/ycvuof26
Article title: Molecular Detection of Rickettsia Spp. and Coxiella Burnetii in Cattle, Water Buffalo, and Rhipicephalus (Boophilus) Microplus Ticks in Luzon Island of the Philippines
Authors: Remil Linggatong Galay, Melbourne R. Talactac, Bea V. Ambita- Salem, Maureen M. Dawn, Chu, Marie O. Lali, Dela Costa, Mae A. Cinnamon, Salangsang, Darwin Kyle, B. Caracas, Florante H. Generoso, Jonathan A. Babelonia, Joenil Vergano, Lena C. Berana, Kristina Andrea C. Sandalo, Billy P. Divina, Cherry Alvarez, Emmanuel R. Mago, Masako Andoh, Tetsuya Tanaka
Publication title: Tropical Medicine of Infectious Diseases 5(2):54, April 2020
Abstract:
Rickettsia and Coxiella burnetii are zoonotic, tick-borne pathogens that can cause febrile illnesses with or without other symptoms in humans, but may cause subclinical infections in animals. There are only a few reports on the occurrence of these pathogens in cattle and water buffalo in Southeast Asia, including the Philippines. In this study, molecular detection of Rickettsia and C. burnetii in the blood and in the Rhipicephalus (Boophilus) microplus ticks of cattle and water buffalo from five provinces in Luzon Island of the Philippines was done. A total of 620 blood samples of cattle and water buffalo and 206 tick samples were collected and subjected to DNA extraction. After successful amplification of control genes, nested PCR was performed to detect gltA of Rickettsia and com1 of C. burnetii. No samples were positive for Rickettsia, while 10 (cattle = 7, water buffaloes = 3), or 1.6% of blood, and five, or 1.8% of tick samples, were C. burnetii-positive. Sequence analysis of the positive amplicons showed 99-100% similarity to reported C. burnetii isolates. This molecular evidence on the occurrence of C. burnetii in Philippine ruminants and cattle ticks and its zoonotic nature should prompt further investigation and surveillance to facilitate its effective control.
Full text link: https://tinyurl.com/y8p3zyje
Article title: Molecular detection of Anaplasma spp. in blood and milk of dairy cattle in the Philippines
Authors: Remil Linggatong Galay, Kristina Andrea C. Sandalo, Flor Marie Immanuel Rafallo Pilapil-Amante, Tetsuya Tanaka
Publication title: Turkish Journal of Veterinary and Animal Sciences 43(4), July 2019
Abstract:
Anaplasmosis has become a major concern in the cattle industry throughout the world due to its great economic impact. The causative agents, Anaplasma species, are primarily transmitted by ticks, occurring intracellularly within blood cells, with some species being zoonotic. In this study, the presence of Anaplasma spp. was investigated in the blood and milk of dairy cattle in the Philippines. Blood and milk samples were collected from 98 dairy cattle from selected farms in five provinces in the southern part of Luzon Island in the Philippines. After DNA extraction, a conventional PCR for the control gene actin was performed, followed by nested PCR for Anaplasma spp. Selected amplicons were purified and subjected to sequence analysis. It was found that 97 (98.97%) blood samples and 6 (6.12%) milk samples were positive for Anaplasma. Sequence analysis revealed that the positive amplicons from milk samples and their corresponding blood samples shared a high identity (98%–100%) with reported Anaplasma marginale isolates. To the authors’ knowledge, this study provides the first molecular evidence of the presence of A. marginale in milk from dairy cattle under field conditions in the Philippines.
Full text link: https://tinyurl.com/y98pd7dd
Article title: Molecular detection of tick-borne pathogens in canine population and Rhipicephalus sanguineus (sensu lato) ticks from southern Metro Manila and Laguna, Philippines
Authors: Remil Linggatong Galay, Anna Angelica L. Manalo, Sidney Lindon Daquioag Dolores, Irene Pearl M. Aguilar, Kristina Andrea C. Sandalo, Kathlyn Cruz, Billy P. Divina, Masako Andoh, Tatsunori Masatani, Tetsuya Tanaka
Publication title: Parasites & Vectors 11(1), December 2018
Abstract:
Background The tropical climate of the Philippines and the high population of dogs, particularly in cities, favors the life-cycle of the brown dog tick, Rhipicephalus sanguineus (sensu lato), a vector of several canine tick-borne pathogens (TBPs) including zoonotic Rickettsia spp. Suspected cases of infections are commonly encountered in veterinary clinics, but the specific TBPs are rarely identified. Furthermore, infection with Rickettsia is not being clinically examined in dogs. In this study, the occurrence of TBPs in blood and ticks collected from household and impounded dogs in highly populated areas of the Philippines, Metro Manila, and the nearby province of Laguna, was examined. Results A total of 248 blood samples and 157 tick samples were subjected to PCR. First, samples were screened using primers for Anaplasma/Ehrlichia spp. and Babesia/Hepatozoon spp. Those that turned positive were further subjected to species-specific PCR. Rickettsia spp. were also detected through a nested PCR. Of the 248 blood samples, 56 (22.6%) were positive for Anaplasma/Ehrlichia spp., while 19 (7.6%) were positive for Babesia/Hepatozoon spp. Species-specific PCR revealed that 61 (23.4%) had a single TBP, with Ehrlichia canis being detected in 39 (15.7%) dogs, while 14 (5.6%) dogs were positive for different combinations of two to four TBPs. Rickettsia infection was detected in 6 (2.4%) dogs. In tick samples, 8 (3.2%) were positive for Ehrlichia/Anaplasma spp., while only 1 (0.63%) was positive for Babesia/Hepatozoon spp. As in the blood samples, E. canis was the most detected, being found in 5 (2%) samples. No tick samples tested positive for Rickettsia spp. Conclusion Ehrlichia canis is the most common TBP affecting dogs in the Philippines. Co-infection with TBPs is quite common, hence testing for multiple TBPs is necessary. Through nested PCR, Rickettsia infection was detected in dogs, and to the authors’ knowledge, this study provides the first molecular evidence of Rickettsia infection in dogs in the Philippines.
Article title: Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6)
Authors: Emmanuel Hernandez, Kudai Kusakisako, Melbourne R. Talactac, Remil Linggatong Galay, Kentaro Yoshii, Tetsuya Tanaka
Publication title: Scientific Reports 8(1) November 2018
Abstract:
Iron is a very important nutrient for cells; however, it could also cause fatal effects because of its capability to trigger oxidative stress. Due to high exposure to iron from their blood diet, ticks make use of several mechanisms to cope up with oxidative stress. One mechanism is iron sequestration by ferritin and its control protein (IRP). Since the IRP activity is dependent on the ferrous iron concentration, we tried to induce intracellular ferritin (FER1) protein expression by exposing Ixodes scapularis embryo-derived cell line (ISE6) to different concentrations of ferrous sulphate at different time points. We were able to induce FER1 protein after exposure to 2 mM of ferrous sulphate for 48 h, as observed in both Western blotting and indirect immunofluorescent antibody tests. This could indicate that the FER1 produced could be a product of the release of IRPs from the FER1 mRNA leading to its translation. The RNA interference of FER1, through the transfection of dsRNA, led to an increase in mortality and decrease in the cellular proliferation of ISE6 cells. Overall, ISE6 cells could be a good tool in further understanding the mechanism of FER1 action, not just in Ixodes ticks but in other tick species as well.
Full text link: https://tinyurl.com/yajqvaf9
Article title: Glutathione S-transferases play a role in the detoxification of flumethrin and chlorpyrifos in Haemaphysalis longicornis
Authors: Emmanuel Hernandez, Kudai Kusakisako, Melbourne R. Talactac, Remil Linggatong Galay, Takeshi Hatta, Kozo Fujisaki, Naotoshi Tsuji, Tetsuya Tanaka
Publication title: Parasites & Vectors 11(1) August 2018
Abstract:
Background: Haemaphysalis longicornis is a tick of importance to health, as it serves as a vector of several pathogens, including Theileria orientalis, Babesia ovata, Rickettsia japonica and the severe fever with thrombocytopenia syndrome virus (SFTSV). Presently, the major method of control for this tick is the use of chemical acaricides. The glutathione S-transferase (GST) system is one mechanism through which the tick metabolizes these acaricides. Two GSTs from H. longicornis (HlGST and HlGST2) have been previously identified. Results: Enzyme kinetic studies were performed to determine the interaction of acaricides with recombinant H. longicornis GSTs. Recombinant HlGST activity was inhibited by flumethrin and cypermethrin, while recombinant HlGST2 activity was inhibited by chlorpyrifos and cypermethrin. Using real-time RT-PCR, the upregulation of the HlGST gene was observed upon exposure to sublethal doses of flumethrin, while the HlGST2 gene was upregulated when exposed to sublethal doses of chlorpyrifos. Sex and strain dependencies in the induction of GST gene expression by flumethrin were also observed. Knockdown of the HlGST gene resulted in the increased susceptibility of larvae and adult male ticks to sublethal doses of flumethrin and the susceptibility of larvae against sublethal doses of chlorpyrifos was increased upon knockdown of HlGST2. Conclusions: HlGST could be vital for the metabolism of flumethrin in larvae and adult male ticks, while HlGST2 is important in the detoxification of chlorpyrifos in larval ticks.
Full text available: https://tinyurl.com/y753toe6
Article title: Hemolymph defensin from the hard tick Haemaphysalis longicornis attacks Gram-positive bacteria
Authors: Yurika Yada, Emmanuel Hernandez, Kudai Kusakisako, Melbourne R. Talactac, Remil Linggatong Galay, Masako Andoh, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Journal of Invertebrate Pathology 156, July 2018
Abstract:
Ticks are key vectors of some important diseases of humans and animals. Although they are carriers of disease agents, the viability and development of ticks are not harmed by the infectious agents due to their innate immunity. Antimicrobial peptides directly protect hosts against pathogenic agents such as viruses, bacteria, and parasites. Among the identified and characterized antimicrobial peptides, defensins have been considerably well studied. Defensins are commonly found among fungi, plants, invertebrates, and vertebrates. The sequence of the tick hemolymph defensin (HEdefensin) gene from the hard tick Haemaphysalis longicornis was analyzed after identification and cloning from a cDNA library. HEdefensin has a predicted molecular mass of 8.15 kDa including signal peptides and a theoretical isoelectric point of 9.48. Six cysteine residues were also identified in the amino acids. The synthetic HEdefensin peptide only showed antibacterial activity against Gram-positive bacteria such as Micrococcus luteus. A fluorescence propidium iodide exclusion assay also showed that HEdefensin increased the membrane permeability of M. luteus. Additionally, an indirect fluorescent antibody test showed that HEdefensin binds to M. luteus. These results suggested that HEdefensin strongly affects the innate immunity of ticks against Gram-positive bacteria.
Full text link: https://tinyurl.com/y7yghmm3
Article title: Vector competence of Haemaphysalis longicornis ticks for a Japanese isolate of the Thogoto virus
Authors: Melbourne R. Talactac, Kentaro Yoshii, Emmanuel Hernandez, Kudai Kusakisako, Remil Linggatong Galay, Kozo Fujisaki, Masami Mochizuki, Tetsuya Tanaka
Publication title: Scientific Reports 8(1), June 2018
Abstract:
Thogoto virus (THOV), a tick-borne arbovirus not previously reported in East Asia, was recently isolated from Haemaphysalis longicornis in Kyoto, Japan. In this study, we investigated the vector competence of H. longicornis ticks for a Japanese isolate of the Thogoto virus using anal pore microinjection and experimental virus acquisition. Our results showed that anal pore microinjection can readily infect adult ticks, and THOV-infected ticks can successfully transmit the virus to mice. Blood feeding was also critical in the distribution of the virus in tick organs, most especially in the salivary glands. Furthermore, co-feeding between an infected adult and naïve nymphs can also produce infected molted adults that can horizontally transmit THOV to mice. Altogether, our results suggest that H. longicornis is a competent vector for the Japanese THOV isolate and could be the primary tick vector of the virus in Japan.
Full text link: https://tinyurl.com/y8s9ne33
Article title: Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding
Authors: Emmanuel Hernandez, Kudai Kusakisako, Melbourne R. Talactac, Remil Linggatong Galay, Takeshi Hatta, Tomohide Matsuo, Kozo Fujisaki, Naotoshi Tsuji, Tetsuya Tanaka
Publication title: Parasites & Vectors 11(1), February 2018
Abstract:
Background: Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress. Methods: Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT). Results: We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands. Conclusions: In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.
Full text link: https://tinyurl.com/yc2n8hrv
Article title: Evaluation of vaccine potential of 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis
Authors: Kodai Kusakisako, Takeshi Miyata, Masashi Tsujio, Remil Linggatong Galay, Melbourne R. Talactac, Emmanuel Hernandez, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Experimental and Applied Acarology, 74(1), January 2018
Abstract:
Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity.
Full text link: https://tinyurl.com/y9qgbdgt
Article title: Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks
Authors: Remil Linggatong Galay, Tomohide Matsuo, Emmanuel Hernandez, Melbourne R. Talactac, Kudai Kusakisako, Rika Omemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasitology International 67(2), October 2017
Abstract:
Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.
Article title: Synchronous Langat Virus Infection of Haemaphysalis longicornis Using Anal Pore Microinjection
Authors: Melbourne R. Talactac, Kentaro Yoshii, Emmanuel Hernandez, Kodai Kusakisako, Remil Lingggatong Galay, Kozo Fujisaki, Masami Mochizuki, Tetsuya Tanaka
Publication title: Viruses 9(7): 189, July 2017
Abstract:
The tick‐borne encephalitis virus (TBEV) serocomplex of flaviviruses consists of arboviruses that cause important diseases in animals and humans. The transmission of this group of viruses is commonly associated with tick species such as Ixodes spp., Dermacentor spp., and Hyalomma spp. In the case of Haemaphysalis longicornis, the detection and isolation of flaviviruses have been previously reported. However, studies showing survival dynamics of any tick‐borne flavivirus in H. longicornis are still lacking. In this study, an anal pore microinjection method was used to infect adult H. longicornis with Langat virus (LGTV), a naturally attenuated member of the TBEV serocomplex. LGTV detection in ticks was done by real‐time PCR, virus isolation, and indirect immunofluorescent antibody test. The maximum viral titer was recorded at 28 days post-inoculation, and midgut cells were shown to be the primary replication site. The tick can also harbor the virus for at least 120 days and can successfully transmit LGTV to susceptible mice as confirmed by detection of LGTV antibodies. However, no transovarial transmission was observed from the egg and larval samples. Taken together, our results highly suggest that anal pore microinjection can be an effective method in infecting adult H. longicornis, which can greatly assist in our efforts to study tick and virus interactions.
Full text link: https://tinyurl.com/y97efeaj
Article title: Ticks' antioxidant complex: A defense stronghold and a potential target for their control
Authors: Remil Linggatong Galay, Emmanuel Hernandez, Kodai Kusakisako, Melbourne R. Talactac, K. Fujisaki, Tetsuya Tanaka
Abstract:
The parasitic blood-sucking lifestyle of ticks makes them efficient transmitters of various diseases in humans and animals worldwide. All developmental stages of ticks exclusively feed on blood, and female hard ticks in particular can ingest large volume of blood relative to their unfed size. Their nutritional dependence for blood is extraordinary because it is potentially toxic, with blood containing high iron and pro-oxidant content. Iron and pro-oxidants may react and produce reactive oxygen species that in high levels may cause oxidative stress. To counteract the negative effects that may result from consumption of blood, ticks are armed with a complex antioxidant system. Several components of their antioxidant defense have been already identified and characterized using various molecular biological techniques. These antioxidant molecules were found to have significant roles in various physiological processes such as blood feeding and reproduction. Others were found to be involved in detoxification mechanisms against environmental toxins including chemical acaricides. A few studies also demonstrated the significant role of tick antioxidant molecules in microbial population and pathogen transmission. This chapter will review our current understanding of tick antioxidant defense with emphasis on its potential as a target for tick and tick-borne pathogen control.
Full text link: https://tinyurl.com/y9ogmumt
Article title: Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis
Authors: Melbourne R. Talactac, Yurika Yada, Kentaro Yoshii, Emmanuel Hernandez, Kodai Kusakisako, Maeda Hiroki, Remil Linggatong Galay, Kozo Fujisaki, Masami Mochizuki, Tetsuya Tanaka
Publication title: Developmental and comparative immunology v68, November 2016
Abstract:
Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.
Article title: 2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis
Authors: Kodai Kusakisako, Remil Linggatong Galay, Rika Umemiya-Shirafuji, Emmanuel Hernandez, Hiroki Maeda, Melbourne R. Talactac, Naotoshi Tsuji, Masami Mochizuki, Kozo Fujisaki, Tetsuka Tanaka
Publication title: Parasites & Vectors 9(1): 457, August 2016
Abstract:
Background Ticks are obligate hematophagous arthropods that feed on vertebrate blood that contains iron. Ticks also concentrate host blood with iron; this concentration of the blood leads to high levels of iron in ticks. The host-derived iron reacts with oxygen in the tick body and this may generate high levels of reactive oxygen species, including hydrogen peroxide (H2O2). High levels of H2O2 cause oxidative stress in organisms and therefore, antioxidant responses are necessary to regulate H2O2. Here, we focused on peroxiredoxin (Prx), an H2O2-scavenging enzyme in the hard tick Haemaphysalis longicornis. Methods The mRNA and protein expression profiles of 2-Cys peroxiredoxin (HlPrx2) in H. longicornis were investigated in whole ticks and internal organs, and developmental stages, using real-time PCR and Western blot analysis during blood-feeding. The localization of HlPrx2 proteins in tick tissues was also observed by immunostaining. Moreover, knockdown experiments of HlPrx2 were performed using RNA interference to evaluate its function in ticks. ResultsReal-time PCR showed that HlPrx2 gene expression in whole ticks and internal organs was significantly upregulated by blood-feeding. However, protein expression, except in the midgut, was constant throughout blood-feeding. Knockdown of the HlPrx2 gene caused significant differences in the engorged body weight, egg weight and hatching rate for larvae as compared to the control group. Finally, detection of H2O2 after knockdown of HlPrxs in ticks showed that the concentration of H2O2 significantly increased before and after blood-feeding. Conclusion Therefore, HlPrx2 can be considered important for successful blood-feeding and reproduction through the regulation of H2O2 concentrations in ticks before and after blood-feeding. This study contributes to the search for a candidate target for tick control and further understanding of the tick’s oxidative stress coping mechanism during blood-feeding.
Full text link: https://tinyurl.com/y7yvxr3j
Article title: Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control
Authors: Remil Linggatong Galay, Takeshi Miyata, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Methods in microbiology 1404: 261-273, April 2016
Abstract:
Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.
Article title: RNA Interference — A Powerful Functional Analysis Tool for Studying Tick Biology and its Control
Authors: Remil Linggatong Galay, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: RNA Interference
Abstract:
Ticks (Acari: Ixodida) are blood-sucking arthropods globally recognized as vectors of numerous diseases. They are primarily responsible for the transmission of various pathogens, including viruses, rickettsiae, and blood parasites of animals. Ticks are second to mosquitoes in terms of disease transmission to humans. The continuous emergence of tick-borne diseases and acaricide resistance of ticks necessitates the development of new and more effective control agents and strategies; therefore, understanding of different aspects of tick biology and their interaction with pathogens is very crucial in developing effective control strategies. RNA interference (RNAi) has been widely used in the area of tick research as a versatile reverse genetic tool to elucidate the functions of various tick proteins. During the past decade, numerous studies on ticks utilized RNAi to evaluate potentially key tick proteins involved in blood feeding, reproduction, evasion of host immune response, interaction with pathogens, and pathogen transmission that may be targeted for tick and pathogen control. This chapter reviewed the application of RNAi in tick research over the past decade, focusing on the impact of this technique in the advancement of knowledge on tick and pathogen biology.
Full text link: https://tinyurl.com/yd42xjku
Article title: Induction of gene silencing in Haemaphysalis longicornis ticks through immersion in double-stranded RNA
Authors: Remil Linggatong Galay, Emmanuel Hernandez, Melbourne Talactac, Hiroki Maeda, Kodai Kusakisako, Rika-Umemiya Shirafuji Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Tick and tick-borne diseases 7(5), April 2016
Abstract:
The Continuous Emergence of tick-borne diseases and chemical acaricide-resistant tick strains necessitates the development of new and more effective control strategies. RNA interference through the injection of double-stranded RNA (dsRNA) has been a very useful tool in tick research for evaluating gene function. However, this technique can be sophisticated due to the required equipment and technique. Here we studied the feasibility of an immersion technique to induce gene silencing in Haemaphysalis longicornis ticks. We targeted the Hlfer1 gene, previously shown to be crucial in successful blood feeding and reproduction. Larval, nymphal, and adult female H. longicornis ticks were immersed in Hlfer1 or Luciferase dsRNA for control. The dsRNA dissolving medium, incubation temperature and time were varied to establish the optimum conditions. RT-PCR was performed to confirm gene silencing.It was found that immersing the ticks in dsRNA dissolved in nuclease-free water at 15 ◦ C for 12h resulted in clear gene silencing. The phenotypes of adult ticks immersed in dsRNA were then compared with those of adult ticks injected with dsRNA. Similar to dsRNA injection, the post–blood meal weight of ticks immersed in Hlfer1 dsRNA was significantly lower than the control group. Moreover, high post–blood meal mortality and low egg output was observed both from ticks injected with and immersed in Hlfer1 dsRNA. Our results here suggest that Immersion in dsRNA can effectively induce gene silencing and not only offers an alternative method to dsRNA injection but also opens the possibility of applying dsRNA for tick control
Article title: Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis virus surrogate Langat virus
Authors: Melbourne R. Talactac, Kentaro Yoshii, Hiroki Maeda, Kodai Kusakisako, Emmanuel Hernandez, Naotoshi Tsuji, Kozo Fujisaki, Remil Linggatong Galay, Tetsuya Tanaka, Masami Mochizuki
Publication title: Parasites & Vectors 9(1), February 2016
Abstract:
Longicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat virus (LGTV), a tick-borne flavivirus. Longicin P1 and P4 peptides were chemically synthesized. Antiviral activity of the longicin peptides against LGTV was evaluated through in vitro virucidal assays, wherein the antiviral efficacy was determined by reduction in number of viral foci and virus yield. Additionally, longicin P4 was also tested for its activity against human adenovirus, a non-enveloped virus. Lastly, to assess the importance of longicin on the innate antiviral immunity of H. longicornis ticks, gene silencing through RNAi was performed. Longicin P4 produced significant viral foci reduction and lower virus yield against LGTV, while longicin P1 failed to demonstrate the same results. Conversely, both longicin partial analogs (P1 and P4) did not show significant antiviral activity when tested on adenovirus. In addition, longicin-silenced ticks showed significantly higher virus titer after 7 days post-infection but a significantly lower titer was detected after an additional 14 days of observation as compared to the Luc dsRNA-injected ticks. Mortality in both groups did not show any significant difference. Our results suggest that longicin P4 has in vitro antiviral activity against LGTV but not against a non-enveloped virus such as adenovirus. Likewise, though most cationic antimicrobial peptides like longicin act directly on target membranes, the exact mechanism of membrane targeting of longicin P4 in enveloped viruses, such as LGTV, requires further investigation. Lastly, while the in vitro virucidal capacity of longicin P4 was confirmed in this study, the role of the endogenous tick longicin in the antiviral defense of H. longicornis against LGTV still remains to be demonstrated.
Full text link: https://tinyurl.com/y8p3nsdk
Article title: Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis
Authors: Remil Linggatong Galay, Rie Takechi, Rika Umemiya-Shirafuji, Melbourne R. Talactac, Hiroki Maeda, Kodai Kusakisako, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasitology International 65(3), January 2016
Abstract: No abstract
Article title: A novel C-type lectin with triple carbohydrate recognition domains has critical roles for the hard tick Haemaphysalis longicornis against Gram-negative bacteria
Authors: Hiroki Maeda, Takeshi Miyata, Kodai Kusakisako, Remil Linggatong Galay, Melbourne R. Talactac, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Developmental and comparative Immunology 57, December 2015
Abstract:
C-type lectins (CLecs) play an important role in innate immunity against invaders. In this study, a novel CLec was identified from Haemaphysalis longicornis ticks (HlCLec). HlCLec contains a signal peptide and a transmembrane region. Interestingly, HlCLec possesses three dissimilar carbohydrate recognition domains (CRDs). Each CRD contains the mutated motif of Ca2+-binding site 2. HlCLec mRNA was up-regulated during blood feeding, and had highest expression in the midgut and ovary. HlCLec localization was also confirmed by immunofluorescent antibody test (IFAT). HlCLec was found on the cell membrane and basal lamina of midgut and ovary. In addition, the recombinant HlCLec and individual CRDs demonstrated direct binding activity to Escherichia coli and Staphylococcus aureus; however, no growth inhibition activity was observed. Furthermore, E. coli injection after silencing of HlCLec caused drastic reduction in survival rate of ticks. These results strongly suggest the key role of HlCLec in tick innate immunity against Gram-negative bacteria.
Article title: Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis
Authors: Kodai Kusakisako, Tatsutori Masatani, T Miyata, Remil Linggatong Galay, H. Maeda, Melbourne R. Talactac, N. Tsuji, Masami Mochizuki, F. Fujisaki, Tetsuya Tanaka
Publication title: Insect Molecular Biology, 25(1) October 2015
Abstract:
Ticks are obligate haematophagous arthropods that feed on vertebrate blood containing high levels of iron. The host-derived iron reacts to oxygen in the tick's body, and then high levels of reactive oxygen species, including hydrogen peroxide (H2 O2 ), may be generated. High levels of H2 O2 cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H2 O2 . We focused on peroxiredoxins (Prxs), H2 O2 -scavenging enzymes. The sequence of Haemaphysalis longicornis 2-Cys Prx (HlPrx2) was identified from fat body cDNA libraries of this tick and recombinant HlPrx2 was then prepared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two conserved cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitution, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H2 O2 was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrated that only the wild-type HlPrx2 formed homodimers and that all of the proteins that we made had a high molecular weight peak. These results indicate that both Cys51 and Cys172 are essential for the dimerization of HlPrx2, whereas only the Cys51 residue is necessary for antioxidant activity.
Article title: Ticks and Tick-borne Diseases
Authors: Rie Takechi, Remil Linggatong Galay, Tomohide Matsuo, Hiroki Maeda, Kudai Kusakisako, Melbourne R. Talactac, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Ticks and tick-borne diseases 7(1), August 2015
Abstract: no abstract
Article title: Identification of the Babesia-responsive leucine-rich repeat domain-containing protein from the hard tick Haemaphysalis longicornis
Authors: Hiroki Maeda, Koshi Kurisu, Takeshi Miyata, Kodai Kusakisako, Remil Linggatong Galay, Melbourne R. Talactac, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasitology Research 114(5), February 2015
Abstract:
Haemaphysalis longicornis is a tick known for transmitting Babesia parasites, including Babesia gibsoni, in East Asian countries. The vector tick must have strategies to control Babesia parasites, while Babesia parasites are also considered to establish an evasive mechanism from the tick's innate immunity. Due to this mutual tolerance, H. longicornis is considered to be a vector of Babesia parasites. Recent studies have shown the important roles of leucine-rich repeat (LRR) domain-containing proteins in innate immunity in many living organisms. Some LRR domain-containing proteins were identified in ticks; however, their functions are still unknown. In this study, a novel LRR domain-containing protein was identified from H. longicornis (HlLRR). HlLRR contains two LRR domains, and the expression levels of mRNA and proteins were upregulated during blood feeding, particularly in the salivary glands and midgut. In addition, recombinant HlLRR (rHlLRR) demonstrated growth inhibition activity against B. gibsoni in vitro without a hemolytic effect at any concentration used. Moreover, the diameters of Babesia merozoites treated with rHlLRR were significantly larger than those of the control group. These results strongly indicate the key roles of HlLRR in the tick's innate immunity against Babesia parasites. Furthermore, HlLRR might be a potential alternative drug to treat babesiosis.
Article title: Iron metabolism in hard ticks (Acari: Ixodidae): The antidote to their toxic diet
Authors: Remil Linggatong Galay, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasitology International 64(2), December 2014
Abstract: No Abstract
Article title: Evaluation and comparison of the potential of two ferritins as anti-tick vaccines against Haemaphysalis longicornis
Authors: Remil Linggatong Galay, Takeshi Miyata, Rika Umemiya-Shirafuji, Hiroki Maeda, Kodai Kusakisako, Naotoshi Tsuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasites & Vectors 7(1): 482, October 2014
Abstract:
Background Tick control is an essential aspect of controlling the spread of tick-borne diseases affecting humans and animals, but it presently faces several challenges. Development of an anti-tick vaccine is aimed at designing cost-effective and environmentally friendly protection against ticks and tick-borne diseases as an alternative to the use of chemical acaricides. A single vaccine from the tick midgut protein Bm86 is currently available for field applications, but its efficacy is limited to only some tick species. Identification of candidate vaccine antigens that can affect multiple tick species is highly desirable. The hard tick Haemaphysalis longicornis has two kinds of the iron-binding protein ferritin (HlFER), an intracellular HlFER1 and a secretory HlFER2, and RNA interference experiments showed that these are physiologically important in blood feeding and reproduction and in protection against oxidative stress. Here we investigated the potential of targeting HlFERs for tick control by immunizing the host with recombinant HlFERs (rHlFER1 and rHlFER2).Methods Rabbits were immunized with rHlFERs three times subcutaneously at two-week intervals. Antisera were collected before the first immunization and a week after each immunization to confirm the antigen-specific serum antibody titer by serum ELISA. Two weeks after the final immunization, the rabbits were challenged with tick infestation. After dropping, tick feeding and reproduction parameters were evaluated to determine vaccine efficacy. To demonstrate the effects of antibodies, oxidative stress was detected in the eggs and larvae.ResultsThe antibody titer of rHlFER-immunized rabbits greatly increased after the second immunization. Antibodies exhibited cross-reactivity with rHlFERs and reacted with tick native HlFERs in Western blot analysis. Significantly lower bodyweight was observed in the ticks infested from the rHlFER2-immunized rabbit compared to those from the control rabbit. Reduced oviposition and hatching rate were observed in both rHlFER-immunized groups. rHlFER2 showed a higher vaccine efficacy. The antibodies against rHlFERs were detected in the eggs, and higher levels of oxidative stress biomarkers in the eggs and larvae, of ticks from rHlFER vaccinated rabbits.Conclusion Collectively, these results showed that HlFER2 has a good potential as an anti-tick vaccine antigen that may affect multiple tick species.
Full text link: https://tinyurl.com/y9nz35xu
Article title: Two Kinds of Ferritin Protect Ixodid Ticks from Iron Overload and Consequent Oxidative Stress
Authors: Remil Linggatong Galay, Rika Umemiya-Shirafuji, Eugene T. Bacolod, Hiroki Maeda, Hiroki Maeda, Kodai Kusakisako, Jiro Koyama, Naotoshi Tsuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: PLoS ONE 9(3):e90661, March 2014
Abstract:
Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts' immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick Haemaphysalis longicornis has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after Hlfer knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron-mediated oxidative stress during blood feeding.
Full text link: https://tinyurl.com/y7c445ow
Article title: Expression analysis of autophagy-related genes in the hard tick Haemaphysalis longicornis
Authors: Rika Umemoya-Shirafuji, Remil Linggatong Galay, Hiroki Maeda, Suguro Kawano, Tetsuya Tanaka, Shinya Fukumoto, Hiroshi Suzuki, Naotoshi Tsuji, Kozo Fukisaki
Publication title: Veterinary Parasitology 201(1-2), February 2014
Abstract: No abstract
Article title: Inhibitory effect of cyclophilin A from the hard tick Haemaphysalis longicornis on the growth of Babesia bovis and Babesia bigemina
Authors: Hiroki Maeda, Damdinsuren Boldbaatar, Koda Kusakisako, Remil Linggatong Galay, Kyaw Min Aung, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Parasitology Research 112(6), March 2013
Abstract:
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.
Article title: Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis
Authors: Remil Linggatong Galay, Kyaw Min Aung, Rika Umemiya-Shirafuji, Hiroki Maeda, Tomohide Matsuo, Hiroaki Kawaguchi, Niroaki Miyoshi, Hiroshi Suzuki, Xuenan Xuan, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Journal of experimental Biology 216(10), February 2013
Abstract:
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 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 on blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy (TEM). 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 egg despite the low mRNA transcript. RNAi induced reduced post-blood meal body weight, high mortality, and decreased fecundity. The expression of vitellogenin genes were affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results showed that the iron storage and protective functions of ferritin are critical to successful blood feeding and reproduction of H. longicornis.
Article title: Host-derived transferrin is maintained and transferred from midgut to ovary in Haemaphysalis longicornis ticks
Authors: Hiroyuki Mori, Remil Linggatong Galay, Hiroki Maeda, Tomohide Matsuo, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki, Tetsuya Tanaka
Publication title: Ticks and ticke-borne diseases 5(2), January 2013
Abstract: No abstract
Article title: Target of rapamycin (TOR) controls vitellogenesis via activation of the S6 kinase in the fat body of the tick, Haemaphysalis longicornis
Authors: Rika Umemiya-Shirafuji, Damdinsuren Boldbaatar, Min Liao, Battur Banzragch, Morshedur Rahman, Thasaneeya Kuboki, Remil Linggatong Galay, Tetsuya Tanaka, Kozo Fujisaki
Publication title: International Journal of Parasitology 42(11), September 2012
Abstract:
Vitellogenin (Vg) synthesis, vitellogenesis, is an essential process for the development and reproduction of ticks. Our previous finding led to the hypothesis that target of rapamycin (TOR) pathway is important for vitellogenesis in the hard tick, Haemaphysalis longicornis. The TOR pathway controls cellular activity according to nutrient availability in eukaryotes. TOR, a member of the phosphatidylinositol 3-kinase family, is a central player in this pathway. Here, we present preliminary evidence that H. longicornis TOR (HlTOR) controls vitellogenesis via activation of S6 kinase (S6K) in the fat body. RNA interference (RNAi)-mediated gene silencing of HlTOR was undertaken to elucidate the involvement of HlTOR in the vitellogenesis of the tick. HlTOR-RNAi caused inhibition of S6K phosphorylation in the fat body. HlTOR-RNAi also altered not only the expression levels of GATA mRNA and protein but also the intracellular localisation of GATA in the fat body. The expression levels of Vg mRNA and protein in the fat body of HlTOR-RNAi ticks were significantly lower than those in control ticks. In the pre-ovipositional stage, the ovaries of control ticks had brown oocytes developing, but those of HlTOR-RNAi ticks were white and immature. The haemolymph colour indicated that the amount of Vg was lower in HlTOR-RNAi ticks than in the controls. Furthermore, rapamycin inhibited S6K phosphorylation and reduced the expression levels of Vg mRNA and protein in the fat bodies. Vg proteins were not detected in rapamycin-treated fat bodies in the presence of 20-hydroxyecdysone. These results suggest that HlTOR activity is critical for vitellogenesis stimulated by 20-hydroxyecdysone.
Article title: HlSRB, a Class B Scavenger Receptor, Is Key to the Granulocyte-Mediated Microbial Phagocytosis in Ticks
Authors: Kyaw Min Aung, Damdinsure Boldbaatar, Rika Umemiya-Shirafuji, Min Liao, Naotoshi Tsuji, Xuan Xuenan, Hiroshi Suzuki, Aiko Kume, Remil Linggatong Galay, Tetsuya Tanaka, Kozo Fujisaki
Publication title: PLoS ONE 7(3): E33504 March 2012
Abstract:
Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods.
Full text link: https://tinyurl.com/yd4wktbb
Article title: Scavenger Receptor Mediates Systemic RNA Interference in Ticks
Authors: Kyaw Min Aung, Damdinsuren Boldbaatar, Rika Umemiya-Shirafuji, Min Liao, Xuan Xuenan, Hiroshi Suzuki, Remil Linggatong Galay, Tetsuya Tanaka, Kozo Fujisaki
Publication title: PLoS ONE 6(12): E28407 December 2011
Abstract:
RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.
Full text link: https://tinyurl.com/ycze5r57
Article title: Anti-babesial activity of a potent peptide fragment derived from longicin of Haemaphysalis longicornis
Authors: Remil Linggatong Galay, Hiroki Maeda, Kyaw Min Aung, Rika Umemiya Shirafuji, Xuenan Xuan, Ikuo Igarashi, Naotoshi Tsuji, Tetsuya Tanaka, Kozo Fujisaki
Publication title: Tropical Animal Health and Production 44(2): 343-8, November 2011
Abstract:
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.
Article title: Tick Longicin Implicated in the Arthropod Transmission of Toxoplasma Gondii
Authors: Tetsuya Tanaka, Remil Linggatong Galay, Rika Umemiya-Shirafuji, Hiroshi Suzuki
Publication title: Journal of Veterinary Science and Technology 3(2), January 2011
Abstract:
Antimicrobial peptides are major components of host innate immunity, a well-conserved evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to have evolved to produce specific defense peptides implicated in controlling the infection and transmission of various pathogens. Longicin, a defensin peptide identified from the hard tick, Haemaphysalis longicornis , is known to have a significant deadly effect against both Gram-negative and Gram-positive bacteria and other microorganisms. In this study, female H. longicornis ticks were experimentally injected with Toxoplasma gondii tachyzoite parasites, and the transcription profiles of longicin in ticks demonstrating the amplification of T. gondii B-1 gene fragments were examined to determine whether and how ticks may respond immunologically in controlling T. gondii infections. As a result, 10 days after parasite injection, ticks indicated the upregulation of the longicin gene, consistently with the presence of T. gondii The effects of recombinant longicin on the morphology of T. gondii tachyzoites were also examined in vitro . Tachyzoite parasites incubated with recombinant longicin induced pathological changes in cell morphology followed by a marked reduction in the number of parasites. These findings suggested that recombinant longicin could impair parasite membranes, leading to the destruction of Toxoplasma parasites.
Full text link: https://tinyurl.com/ycfr683h