Genes functionally characterized through RNAi in different tick species.
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.
- RNA interference
- tick-borne diseases
Ticks belong to the class of Arachnida together with spiders, scorpions, and mites. To date, there are about 900 species of ticks, majority of which are hard ticks belonging to the Ixodidae family, as well as about 200 species are soft ticks belonging to the Argasidae family, and a single species belonging to the Nuttalliellidae family . Most of the ticks of medical and veterinary importance are hard ticks. Through their blood-feeding behavior, ticks can directly affect their host by causing anemia, irritation, and allergic reactions particularly in heavy infestation. The saliva of some tick species may also contain neurotoxic substances that may cause the condition termed “tick paralysis” . Additionally, the transmission of pathogens including viruses, bacteria, and parasitic protozoa also occurs during blood feeding . Ticks are considered second to mosquitoes in terms of their impact on public health, but they are the most important vectors of different pathogens in both domestic and wild animals . Tick infestation and tick-borne diseases (TBDs) continue to have great economic impact on livestock production, particularly on cattle and small ruminants, in several continents . The annual loss in cattle production worldwide due to ticks and TBDs has been estimated to be worth billions of USD .
The complete dependence of ticks to host blood for the completion of their life cycle and generation of offspring is the reason for their notoriety as vectors of several diseases. Depending on the species, a tick may utilize one to three hosts during their life cycle. Most of the pathogens they transmit can be carried on throughout their life cycle through transstadial (from one stage to the next) transmission and to the next generation through transovarial (from adults to eggs) transmission . A single tick may carry multiple pathogens , thereby having the potential of infecting a host with a cocktail of pathogens. Most tick-borne infections are zoonotic in nature, and more of these are being recognized in recent years [1, 7]. Among the TBDs that are well-known in the veterinary and medical field are anaplasmosis, borreliosis, rickettsiosis, ehrlichiosis, babesiosis, theileriosis, and tick-borne encephalitis.
The significant impact of ticks and TBDs underscores the importance of tick control. For several decades, the application of chemical acaricides has been the primary tick control method, and acaricides were used extensively in livestock production. However, the continuous emergence of resistant tick strains makes most chemical acaricides ineffective . Moreover, the increasing concerns for animal product and environmental contamination set limitations for this control method. To search for new and more effective means of controlling ticks and TBDs, researchers have actively expanded the understanding on tick biology.
RNA interference (RNAi) is a reverse genetic approach for manipulation of genes that commonly utilizes double-stranded RNA (dsRNA) to induce post-transcriptional gene-specific silencing . RNAi has been extensively employed in many studies on tick biology and pathogen interaction since the first report of RNAi application in the hard tick
This chapter aims to show the extent of RNAi application in tick research, emphasizing the progress of advanced knowledge on tick biology and tick-pathogen interaction. We first discussed so far known RNAi mechanisms and the current RNAi inducing methods in ticks; then, briefly described the studies on tick physiology, immunity, and pathogen interaction that employed RNAi, highlighting the prospects of applications of RNAi in tick research.
2. RNAi pathway in ticks
The mechanism of RNAi has been well studied in the nematode
2.1. dsRNA uptake
There are two recognized dsRNA uptake mechanisms in invertebrates: a transmembrane channel-mediated uptake through systemic RNA interference defective (SID) transmembrane proteins described in
SID homologues have not been identified in ticks. However, a homologue of ENTH, Epn-I, has been identified in the hard ticks
2.2. dsRNA processing and RISC assembly
The recommended length of dsRNA to effectively induce silencing of the target gene in non-mammalian systems is more than 200 bp . A study in
The RNAi inhibition of a target mRNA is accomplished by RISC formed by siRNAs and Argonaute (AGO) proteins. AGO proteins are highly conserved between species, encoded by multiple genes in most organisms. All AGO proteins are characterized by two domains: the PAZ domain and the PIWI domain . Upon ATP activation, AGO mediates RISC recognition of mRNA target that are homologous to siRNAs, subsequently leading to the cleavage of the mRNA target . In most insects, including
2.3. Amplification of RNAi signal
The ability to spread throughout the whole organism, inducing total systemic silencing of the target gene in spite of introducing only a relatively small amount of dsRNA, is an important aspect of RNAi observed in plants and invertebrates. This systemic RNAi-induced gene silencing in both plants and
3. Methods of introducing dsRNA in ticks
Direct injection is the most widely used technique for introducing dsRNA for in vivo gene silencing, not only in ticks but also in insects [11, 32]. Through this method, dsRNA is usually introduced directly into the hemocoel of ticks allowing the dsRNA to circulate within the hemolymph. In most reports, a high concentration of at least 1 µg dsRNA per tick has been shown to be effective in inducing gene silencing , but in some reports, lower concentration has been found to be similarly effective [34–36]. Injection has been accomplished using a 33–36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as
Soaking in dsRNA has been previously employed to study RNAi in the cell lines of
Electroporation is a technique that employs electric impulses to promote DNA uptake of cells and has been primarily used with in vitro cell transfection . In tick research, this technique has been first applied to facilitate the introduction of dsRNA in
Feeding dsRNA in insects has been achieved in different species using diets mixed with dsRNA, liposome-embedded or lipophilic siRNAs, and bacteria and transgenic plants that can synthesize dsRNA [32, 69]. Although in vitro feeding assays have been shown to be useful in studying different tick molecules and tick-pathogen interaction , its application in RNAi study in ticks has been limited. A study on the Lyme disease vector
4. RNAi and study of tick physiology
4.1. Genes related to salivary functions
The saliva is an important arsenal of ticks containing hundreds of pharmacologically potent substances that facilitate attachment to their hosts and blood-sucking . Different salivary proteins have redundant functions in counteracting the hemostatic , inflammatory, and immune mechanisms  of the host. Aside from its function in tick feeding, the salivary glands are also involved in osmoregulation and transmission of pathogens .
Many studies on characterization of salivary proteins in the recent years employed RNAi (Table 1). In fact, the first report on the application of RNAi in tick research described a tick inhibitor of inflammatory mediator, a salivary histamine-binding protein, wherein researchers induced in vitro RNAi by soaking salivary glands in dsRNA [10, 58]. Soluble N-ethylmaleimide-sensitive factor attachment receptors (SNARE) complex proteins, which mediate exocytosis in secretory pathways of the salivary glands, have been characterized in
Longistatin  and acidic chitinases  have been found to be important in the formation of blood pool and tick cement cone, respectively. The attachment site of
Other salivary proteins with immunomodulatory function, such as the anti-complement protein, isac , and two proteins that can inhibit neutrophil function, ISL 929 and 1373 , have also been knockdowned in
4.2. Genes related to digestion and midgut function
The midgut of ticks houses various kinds of enzymes that act on a large amount of ingested host blood, which contains great quantities of hemoglobin . Functional studies on these enzymes and other midgut proteins using RNAi have expanded the understanding of tick digestive physiology (Table 1). Silencing hemoglobinolytic enzymes, such as leucine aminopeptidase [91, 92], longipain , and cathepsin L [96, 97] had negative impact on tick feeding. Moreover, the longipain of
4.3. Genes related to reproductive function
Ticks are known for their high fecundity, laying hundreds of eggs per batch in the case of soft ticks and up to thousands in the case of hard ticks. A series of physiological events takes place in female ticks during and after blood feeding that initiate ovarian maturation and subsequent oviposition. Vitellogenesis, the synthesis and oocyte deposition of the yolk protein precursor (vitellogenin), is a key process for ovarian development and oocyte maturation induced by blood meal in ticks . Three genes encoding vitellogenin have been identified and characterized in
|Histamine-binding protein (HBP)||Altered feeding pattern and longer feeding period; decreased histamine-binding activity in the salivary glands||[10, 58]|
|Salp14/ Salp9pac||Impaired feeding, decreased post-blood meal weight, decreased anticoagulant activity of salivary gland extract|||
|Neuronal isoform munc18-1 (nSec1)||Decreased post-blood meal weight and prolonged feeding time, decreased anticoagulant secretion of salivary gland|||
|Synaptobrevin||Inhibited secretion of anticoagulant stimulated by PGE|||
|Cystatin||Decreased post-blood meal weight, mortality during feeding, low feeding success rate|||
|Anticomplement protein (Isac)||Decreased post-blood meal weight, decreased |||
|Sialostatin L (cystatin)||Failure to feed on the host, decreased post-blood meal weight and failed oviposition|||
|Aquaporin||Decreased post-blood meal weight, decreased volume of ingested blood|||
|HlYkt6 (SNARE)||Decreased post-blood meal weight, high mortality, supressed salivary secretion and anticoagulant activity|||
|ISL 929 and 1373||Supressed PMN inhibitory activity of saliva from knockdowned ticks|||
|Rhipilin (Kunitz type protease inhibitor)||Prolonged attachment time, decreased post-blood meal weight|||
|Longistatin||Mortality after attachment, failure to engorge, poor blood pool formation|||
|Serine protease inhibitor (serpin)||No effect on tick attachment, feeding and oviposition|||
|Decreased attachment rate and engorgement weight|||
|Reprolysin||Decreased egg weight and egg conversion ratio|||
|N-ethylmaleimide sensitive fusion protein (NSF)||Inhibition of engorgement, failure of oviposition|||
|Synaptosomal Associated Protein of 25 kDa (SNAP-25)||Decreased post-blood meal weight, decreased egg weight, failure in hatching|||
|Vti (SNARE)||Decreased post-blood meal weight and survival, failed oviposition|||
|Glutaminyl cyclase (QC)||Decreased post-blood meal weight, egg weight and hatch|||
|AV422||Decreased post-blood meal weight|||
|Acidic chitinase (Ach)||Leakage of blood from the mouthparts in late feeding phase, loose attachment in the host’s skin|||
|Longepsin||No effects reported|||
|Leucine aminopeptidase||Extended pre-oviposition period, decreased egg weight and egg conversion ratio, morphological abnormalities in the oocytes||[91, 92]|
|Hemalin (thrombin inhibitor)||Longer blood feeding period, failure to engorge, decreased inhibitory activity of fibrinogen clot formation in the midgut|||
|Boophilin (thrombin inhibitor)||Decreased oviposition|||
|Serine proteinase||Suppressed erythrocyte degradation; decreased post-blood meal weight|||
|Longipain||Impaired blood feeding, decreased post-blood meal weight, increased |||
|Cathepsin L||Decreased weight gain|||
|Decreased post-blood meal weight|||
|Astacin||Decreased egg weight and egg conversion ratio|||
|Follistatin-related protein (FRP)||Decreased egg conversion ratio|||
|Vitellogenin receptor (VgR)||Failure of Vg uptake by oocytes; failed oviposition|||
|Suppressed oocyte maturation and failed oviposition, failure of |||
|Suppressed oocyte maturation, long pre-oviposition period,|||
|Voraxin||Failure to engorge and lay eggs in females fed with males injected with a combination of subolesin and voraxin dsRNA|||
|Vitellogenin (Vg)||Decreased post-blood meal weight, abnormal oocytes, decreased egg conversion ratio|||
|GATA factor||Disrupted egg development|||
|S6 kinase||Disrupted egg development|||
|Target of rapamycin (TOR)||Decreased post-blood meal weight, mortality after engorgement, Failure of oocytes to mature and failure to lay eggs|||
|Glutamine:fructose-6-phosphate aminotransferase (HlGFAT)||Decreased post-blood meal weight and survival|||
|β-Actin||Decreased post-blood meal weight and oviposition|||
|Na+-K+-ATPase||Decreased post-blood meal weight and oviposition|||
|Valosin-containing protein (HlVCP)||Decreased post-blood meal weight|||
|Cyclophilins (Immunophilin)||Lower post-blood meal weight, low survival after blood feeding and failure to lay eggs after silencing cyclophilin A|||
|Ribosomal protein P0||Decreased post-blood meal weight, low engorgement rate, and high mortality|||
|Protein disulphide isomerases (PDI)||Mortality after engorgement, leakage of blood from the midgut, little egg output|||
|Organic anion transporter polypeptide (OATP)||Decreased post-blood meal weight, oviposition and egg conversion ratio|||
|Ferritins (FER)||Decreased post-blood meal weight, oviposition and hatch|||
|Decreased post-blood meal weight, survival, oviposition and hatch||[112, 113]|
|Iron regulatory protein (IRP1)||Decreased post-blood meal weight and hatching of eggs|||
|Elongation factor 1-α||High post-blood meal mortality, decreased post-blood meal weight and failure of oviposition|||
|Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH)||Longer blood feeding period, decreased post-blood meal weight, longer pre-oviposition period, decreased oviposition and hatch after SDH silencing; higher volume of hemolymph after LKR silencing|||
|Ubiquitin||Shorter post-blood meal survival, decreased or absence of egg output, impaired embryogenesis||[14, 29, 45, 72];|
|Glycogen synthase kinase-3 (GSK-3)||Decreased oviposition and hatching|||
|CD147 receptor||Inhibited feeding, low post-blood meal weight tender cuticle|||
|Insulin-like growth factor binding protein-related proteins||Decreased post-blood meal weight|||
|Putative 5.8S, ITS2 and 28S rRNA||High mortality and very low post-blood meal weight|||
|Putative 2B7 60S ribosomal protein L13e||High mortality and very low post-blood meal weight|||
|Putative interphase cytoplasm foci protein 45||High mortality and very low post-blood meal weight|||
|Putative threonyl-tRNA synthetase||High mortality and very low post-blood meal weight|||
|Putative 60S ribosomal protein L13a||100% mortality|||
|Putative mitochondrial 12S rRNA||High mortality and very low post-blood meal weight|||
|Chymotrypsin inhibitor (HlChI)||Mortality after attachment, retarded blood feeding and longer feeding period, decreased post-blood meal weight, decreased egg weight and egg conversion ratio|||
|Scavenger receptor||Decreased post-blood meal weight, mortality after engorgement, decreased oviposition and hatch; inhibited bacterial phagocytosis of granulocytes||[26, 28]|
|4E-BP (eIF4E-binding protein)||Decreased lipid accumulation in the midgut and fat bodies after long starvation period|||
|Protein kinase B (AKT)||Inhibition of engorgement and growth of organs during blood feeding; decreased expression of |||
|Decreased cell glycogen content and viability, and altered cell membrane permeability|||
|Spook (Spo)||Arrested development and molting|||
|Shade (Shd)||Abnormal ecdysis and delayed molting|||
|Cystatin (RHCyst)||Decreased attachment and hatching rate|||
|Tropomyosin||Longer feeding time, decreased engorgement rate and post-blood meal weight, high mortality after blood feeding, failed oviposition|||
|Subolesin (4D8)||Decreased post-blood meal weight, oviposition and survival; failure of embryogenesis; silencing in eggs and larvae when dsRNA injected to engorged females||[43, 124]|
|Decreased post-blood meal weight, oviposition and survival||[43, 101, 124]|
|Decreased post-blood meal weight, oviposition and survival|||
|Decreased post-blood meal weight, oviposition and survival; decreased fertility; silencing in eggs and larvae when dsRNA injected to engorged females||[43, 124]|
|Decreased post-blood meal weight, oviposition and survival; more dramatic effect when simultaneously silenced with Rs86||[124, 125]|
|High mortality, decreased post-blood meal weight, oviposition and hatch in dsRNA-injected adults and progeny of dsRNA-injected adults||[42, 45, 46]|
|Decreased post-blood meal weight||[45, 126]|
|Decreased egg output|||
|Decreased egg output|||
|Midgut protein Rs86||Decreased post-blood meal weight and oviposition|||
|Midgut protein Hl86||Decreased post-blood meal weight|||
|Midgut protein Bm86||Decreased number of engorging ticks, lower post-blood meal body weight and survival after feeding in |||
|Midgut protein Ree86||No significant effect|||
|Midgut protein ReeATAQ||No significant effect|||
|Longicin||Decreased post-blood meal weight, increased |||
|α2-macroglobulin proteins||Decreased phagocytic action of hemocytes||[132, 133]|
|Macrophage migration inhibitory factor||No effect on phenotypes|||
|Janus kinase ( JAK)–signaling transducer activator of transcription (STAT) pathway||Increased |||
|Dual oxidase (Duox)||Decreased level of |||
|Peroxidase ISCW017368||Decreased level of |||
|Glutathione S-transferase||Decreased tick attachment and post-blood meal weight|||
|Increased susceptibility to permethrin|||
|Selenoprotein W||Decreased tick attachment and post-blood meal weight|||
|Selenoprotein K||Decreased oviposition|||
|Selenoprotein M||Decreased oviposition||[138, 139]|
|Thioredoxin reductase||Decreased native microbial load in midguts and salivary glands|||
|Rmcystatin3 (cysteine protease inhibitor)||Increased resistance to bacteria|||
|Subolesin||Inhibited ||[141, 142]|
|Salp14||No effect on acquisition of |||
|Salp25D||Decreased acquisition of |||
|Immunophilin||Decreased hatch, decreased larval survival, increased |||
|Kunitz-type serine protease inhibitor (Spi)||Inhibition of engorgement, decreased egg weight|||
|Glutathione S-transferase||Inhibited |||
|H+ transporting lysosomal vacuolar proton pump (vATPase)||Inhibited |||
|Selenoprotein M||Inhibited |||
|Putative von Willebrand factor (94Will)||Decreased |||
|Flagelliform silk protein (100Silk)||Decreased |||
|Putative metallothionein (93 Meth)||Increased |||
|Tick salivary lectin pathway inhibitor (TSLPI)||Decreased load of |||
|Kunitz-type serine protease inhibitor (DvKPI)||Increased rickettsial infection in the midgut|||
|Kunitz-type protease inhibitor 5 (KTPI)||Decreased post-blood meal weight|||
|Histamine release factor||Decreased post-blood meal weight, decreased |||
|Serum amyloid A||Decreased |||
|Ricinusin||Decreased post-blood meal weight in |||
|Chitin deacetylase-like protein (IsCDA)||No significant effect on |||
|Antifreeze glycoprotein||Decreased survival and mobility of ticks in extremely cold temperature; decreased |||
inhibitor of apoptosis protein (E3 ubiquitin ligase, XIAP)
|Cytochrome c oxidase subunit III||Failure in transmission of |||
Silencing these genes through RNAi greatly reduced the reproductive capacity of female ticks, which showed immature and light-colored oocytes. The uptake of vitellogenin in the oocytes is facilitated by vitellogenin receptor, which has been characterized in
4.4. Genes related to structural and metabolic functions
Various gene encoding proteins important in cellular structure and metabolism have been characterized using RNAi. Due to their wide distribution and systemic function, knockdown of these proteins caused detrimental effects on different tick physiological functions and some even proved to be lethal (Table 1). Among these proteins is the multifunctional ubiquitin, which has been first targeted based on a homologous gene of
In the hard tick
The significance of proteins involved in iron metabolism to tick feeding and reproduction has been also demonstrated using RNAi in two hard tick species,
5. RNAi studies on tick protective antigens and immunity
The immune system of ticks has a vital role of protecting them from harmful substances in the blood, including components of their host’s immune system, and from various pathogens that they acquire in their blood feeding activity. Tick protective antigens, therefore, gain wide interest due to their potential as target for tick control. The highly conserved tick protective antigen subolesin, previously known as 4D8, was first identified from
The membrane-bound glycoprotein Bm86 expressed mainly in the midgut of
The function of some components of immunity, such as α2-macroglobulin proteins [132, 133], antimicrobial peptides , Janus kinase (JAK)-signaling transducer activator of transcription (STAT) pathway , dityrosine network , and cysteine protease inhibitor in the hemocytes  have been analyzed using RNAi. The α2-macroglobulin proteins of
The obligatory blood feeding lifestyle of ticks exposes them to high levels of pro-oxidants that may trigger oxidative stress. Antioxidant enzymes function to protect them from the harmful effects of oxidative stress. Furthermore, these antioxidant enzymes provide detoxification mechanisms to counteract toxins that they encounter in the environment, such as chemical acaricides. RNAi has been very useful in evaluating the function of these antioxidants. Silencing a selenoprotein in
6. Understanding tick-pathogen interaction through RNAi
RNAi has undoubtedly paved a way to better understand the different aspects of ticks' association with various pathogens. Numerous tick proteins with different functions have been found to be involved in the acquisition, establishment, and transmission of pathogens. Several proteins have been studied through RNAi to determine their importance in the development cycle of different pathogens. The knockdown of subolesin [142, 156], GST, vATPase, and selenoprotein M  in
RNAi also demonstrated that the Lyme disease agent
An interesting study on
Several reports also demonstrated the interaction of
7. Future directions in tick research and application in tick control
Indeed, great progress in understanding tick biology has been already accomplished in the past. However, many aspects of tick physiology and host-tick-pathogen interaction need to be unraveled yet. Moreover, several optimizations can still be done to improve RNAi in tick research. While being the most widely used method of introducing dsRNA, the injection method (particularly microinjection) that requires elaborate equipment may not be accessible to all laboratories. Moreover, injection is mostly applicable to adult and sometimes nymphal stages, and may be injurious to the ticks, especially when performed by an inexperienced researcher. The soaking method is simpler, less invasive, and less laborious. Electroporation has been recently shown to be effective in introducing dsRNA in eggs  and may be useful in studying the function of genes that are involved in embryogenesis and physiology of immature tick stages.
RNAi may also prove to be a promising tick control method and not just a research tool. In pest insect management, the possibility of using RNAi as a novel tool of pest control is already being explored by feeding liposome-coated dsRNA or dsRNA expressed in transgenic plants or bacteria . RNAi targeting several genes have been accomplished by feeding plants expressing dsRNA in several species of economically important crop pests . Feeding dsRNA to ticks is still an underdeveloped approach, which has been yet accomplished only by artificial feeding. Coating dsRNAs with liposomes or nanocarriers may increase dsRNA stability that may make it feasible for administration to the host. Genes that are highly conserved across different tick species, and are of importance in tick survival are good candidate targets. These include proteins with structural and metabolic functions, such as ubiquitin, tropomyosin, and ferritin. However, the specificity of dsRNA to the tick gene should be highly considered. Additional consideration would be the establishment of a minimum effective dose, since the synthesis of dsRNA is costly.
Additionally, RNAi has been proposed as an alternative method for the sterile insect technique in blood-sucking mosquitoes that will produce sterile males by feeding dsRNA in mosquito larvae . Quite similarly, the application of RNAi for tick control was also proposed in a single report on
In this chapter, we have reviewed the application of RNAi in tick research and described the significant contribution of RNAi in advancing our knowledge on tick biology and tick-pathogen interaction. RNAi has revolutionized the advancement of our understanding of various aspects of tick blood feeding and digestion, reproduction, metabolism, and immunity. As a functional analysis tool, RNAi has become very handy in elucidating the functions of different proteins from more than 10 hard tick species and a few soft tick species. It has been particularly helpful in screening potential target antigens for anti-tick and tick-borne pathogen vaccine development . Several methods of introducing dsRNA in ticks have been employed but injection has remained to be the most widely used technique. The number of published research on ticks that involves the application of RNAi has been continuously increasing through the years, and it is expected to continue doing so. A great majority of the published reports focused on hard ticks, but due to some physiological differences, more research using RNAi on soft ticks should be conducted. Finally, with numerous potential target genes already identified, the application of RNAi as a tick control method should be investigated in the future, starting with optimization of dsRNA delivery method for practical use.
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 22580335, 25292173, and 26660229. We also thank our colleague, Melbourne R. Talactac, at the Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, for his assistance in writing this chapter.
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