Introduction
Ticks are obligate haematophagous ectoparasites within the order Acari and are classified into three families: Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae (Barker and Barker, Reference Barker and Barker2023). Approximately 900 tick species have been described globally, with around 10% implicated in the transmission of bacterial, protozoal, and viral pathogens of medical and veterinary importance (Beati and Klompen, Reference Beati and Klompen2019; Dennis and Piesman, Reference Dennis, Piesman, Goodman, Dennis and Sonenshine2005; Guglielmone et al., Reference Guglielmone, Robbins, Apanaskevich, Petney, Estrada-Peña, Horak, Shao and Barker2010; Jongejan and Uilenberg, Reference Jongejan and Uilenberg2004). This includes Anaplasma, Babesia, Borrelia, Coxiella, Ehrlichia, Francisella, Rickettsia, and Theileria spp., as well as viruses such as tick-borne encephalitis virus (TBEV) and Crimean-Congo haemorrhagic fever virus (Choi et al., Reference Choi, Pyzocha and Maurer2016; Wood et al., Reference Wood, Roberts, Kerr, Wasden, Hammer, McCreadie and Rayner2023).
The complex life cycles of both soft and hard ticks, comprising egg, larva, nymph, and adult stages, require one or more blood meals at each stage of development. While hard ticks have one instar per stage, the nymphal stage of soft ticks may pass through multiple instars (up to eight). Notably, hard ticks feed for extended durations, ranging from several days to weeks, depending on the developmental stage and species. For example, larvae and nymphs of hard ticks generally feed for several days, whereas adults, which require a larger blood meal to support reproduction, feed for more extended periods (typically around 5–12 days) (Balashov, Reference Balashov1972; Lees, Reference Lees1948; Needham and Teel, Reference Needham and Teel1991), resulting in an increase in body weight of females by several hundred to over a thousand times (Kitaoka, Reference Kitaoka1967). Based on host interaction patterns, hard ticks can be classified as (i) one-host ticks, which feed on a single host throughout all stages, (ii) two-host ticks, where larvae and nymphs feed on one host and adults on another, and (iii) three-host ticks, which feed on three different hosts across the larva, nymph, and adult stages (Sonenshine and Roe, Reference Sonenshine, Roe, Sonenshine and Roe2013).
The significance of ticks and tick-borne diseases (TTBDs) has necessitated various control measures, primarily involving chemotherapeutic agents such as acaricides and, to a lesser extent, vaccines (Estrada-Peña et al., Reference Estrada-Peña, Szabó, Labruna, Mosqueda, Merino, Tarragona, Venzal and de la Fuente2020). However, the escalating development of resistance to multiple acaricide classes has created an urgent need for alternative strategies, including biological control (Sullivan et al., Reference Sullivan, Parker and Skinner2022), improved animal husbandry practices (e.g., proper fencing of animals and restricting animal movements) (Sliwa et al., Reference Sliwa, Baumgardt, DeYoung, Ortega-S, Hewitt, Goolsby and Lohmeyer2023) and rotational grazing (Cruz-González et al., Reference Cruz-González, Pinos-Rodriguez, Alonso-Diaz, Romero-Salas, Vicente-Martinez, Fernandez-Salas, Jarillo-Rodriguez and Castillo-Gallegos2023; Rapiya et al., Reference Rapiya, Hawkins, Muchenje, Mupangwa, Marufu, Dzama and Mapiye2019).
Advancing these approaches hinges on a deeper understanding of tick physiology, vector competence, tick-borne pathogens (TBPs), and host–pathogen interactions. To this end, laboratory studies commonly use experimental animals, such as rabbits, cattle, sheep, mice, guinea pigs, and gerbils, to maintain tick colonies and evaluate interventions. However, such studies are costly, logistically demanding, and ethically contentious (Allan, Reference Allan, Sonenshine and Roe2013). It has been estimated that tens of thousands of animals are used annually for acaricide testing alone (Kröber and Guerin, Reference Kröber and Guerin2007a). For instance, producing 20,000 Dermacentor reticulatus adults requires 15 rabbits and 20 guinea pigs (Slovák et al., Reference Slovák, Labuda and Marley2002), while developing a trivalent Theileria parva vaccine involves approximately 130 cattle and 500 rabbits (Di Giulio et al., Reference Di Giulio, Lynen, Morzaria, Oura and Bishop2009). Repeated use of animals is further constrained by the development of acquired resistance to tick infestation (Allan, Reference Allan, Sonenshine and Roe2013; Enigk and Grittner, Reference Enigk and Grittner1953; Rechav et al., Reference Rechav, Heller‐Haupt and Varma1989). Growing concerns regarding animal welfare and the ethical justification of such practices have intensified calls for more humane and sustainable research methodologies (Allan, Reference Allan, Sonenshine and Roe2013; Fontana et al., Reference Fontana, Figueiredo, Martins and Santos2021).
In alignment with the 3Rs principles (Replacement, Reduction, and Refinement) of animal use, artificial tick feeding systems (ATFS) offer a promising alternative. These systems support the rearing and experimental feeding of ticks under controlled conditions without live hosts, thus minimising the use of animals. Capillary-based and membrane-based ATFS have been employed for various tick species, providing novel avenues for research into tick physiology, vector competence, pathogen transmission, and the efficacy of acaricides or vaccines (Kröber and Guerin, Reference Kröber and Guerin2007a; Sojka et al., Reference Sojka, Pytelková, Perner, Horn, Konvičková, Schrenková, Mareš and Kopáček2016).
Despite growing interest in ATFS and an increasing number of published studies exploring their use, a critical synthesis of the literature remains lacking. Therefore, this systematic review aims to (i) evaluate and summarise the current literature on artificial tick-feeding techniques for both soft and hard ticks; (ii) assess the scope and application of ATFS in studies of tick biology, pathogen transmission, and tick control; and (iii) identify methodological challenges, knowledge gaps, and opportunities for future research and innovation.
Materials and methods
A comprehensive literature search was conducted, and four main electronic databases, Web of Science, Scopus, CAB Direct, and PubMed, were systematically searched to retrieve peer-reviewed journal articles, conference proceedings, and postgraduate theses published in any language between 1912 and 2024 (searches conducted up to 31 May 2024). A combination of keywords and Boolean operators was used to capture the breadth of relevant literature, including terms such as ‘tick feeding’, ‘tick in vitro feeding’, ‘tick artificial feeding’, ‘hard tick artificial feeding’, ‘soft tick in vitro feeding’, and related genus-specific queries for both hard and soft ticks. In addition, the reference lists of all retrieved articles were manually screened to identify further eligible studies.
Following the database search, all identified records were imported into Covidence (https://www.covidence.org/), a systematic review management tool, for screening and deduplication. Titles and abstracts were independently screened against predefined inclusion and exclusion criteria. Studies were eligible for inclusion if they met the following criteria: (i) original research articles, conference proceedings or postgraduate theses published in peer-reviewed sources; (ii) studies investigating artificial tick feeding using membrane-based or capillary-based systems; and (iii) experiments involving the use of ATFS to assess tick feeding, development, pathogen transmission or control measures. In addition to the English language, articles published in other languages (Bulgarian, French, German, Portuguese, and Russian) were also included.
Where duplicate content existed between a peer-reviewed article and a corresponding thesis, only the published article was included. Studies were excluded if they were review articles, book chapters, commentaries, editorials, letters, abstract-only publications, preprints, or grey literature. Studies focusing exclusively on natural host tick feeding or non-tick arthropods were also excluded. Full-text versions of eligible studies were accessed through the University of Melbourne library and interlibrary loan services.
Relevant data were systematically extracted into a Microsoft Excel® spreadsheet from all studies meeting the inclusion criteria. Extracted variables included: study title, author(s), year and country of publication, study objectives, tick species and developmental stages, feeding technique employed (membrane feeding or capillary feeding), membrane type (animal-derived or synthetic), feeding media composition, use of pheromones, kairomones and phagostimulants, and a summary of key outcomes and findings.
On the basis of the techniques mentioned in publications, the studies were broadly classified into capillary feeding (CF), membrane feeding (MF), and semi-automated MF systems. Our analysis has included studies documenting the applications of ATFS in studying various aspects of ticks, e.g., tick biology/physiology, pathogen transmission, and testing the efficacy of various drugs and vaccine targets. Finally, we have also discussed the future prospects and potential applications of the ATFS for improved understanding of TTBDs and their control.
Results
Study selection and an overview of ATFS
A systematic search of four databases for ATFS resulted in 11,252 articles (PubMed: n = 2,185; Scopus: n = 1,646; CAB Direct: n = 4,697; Web of Science: n = 2,724). Following title and abstract screening, and full-text assessment, 206 studies met the predefined inclusion criteria and were included in the final analysis (Fig. 1).
Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. This flowchart summarises the PRISMA-guided study identification, screening, eligibility and inclusion processes, showing that 206 studies met the inclusion criteria and were included in the review.
Figure 1 Long description
The flowchart illustrates the process of study identification, screening, eligibility and inclusion. It begins with 'Articles identified through online database searching' totaling 11,252. 'Duplicates removed' accounts for 8,976 articles. 'Articles screened' totals 2,276, with 'Articles excluded' numbering 1,912. 'Full-text articles assessed' are 364, with 'Full-text articles excluded' being 158 due to various reasons: book chapter (1), review article (9), not cell culture (10), no full text available (9), thesis article included (1), tick feeding on animals only (93) and no artificial feeding of ticks (35). Finally, 'Articles included in qualitative syntheses' are 206.
A critical appraisal revealed that the principal techniques used for artificial feeding of ticks were (i) CF (n = 68), (ii) MF (n = 126), (iii) a combination of both methods (n = 4), and (iv) semi-automated feeding using membranes (n = 8). While the earliest attempt at artificial tick feeding (ATF) was recorded in 1912, the majority of included studies were published within the last 30 years (Fig. 2), originating predominantly from Australia, Brazil, the Czech Republic, France, Germany, Kenya, Switzerland, the UK, and the USA (Fig. 3).
Timeline of studies reporting artificial feeding of ticks using capillary feeding (CF) and membrane feeding (MF) systems. The figure shows a marked increase in the number of artificial tick feeding system (ATFS) studies over time, with the most pronounced growth in the past decade. MF accounts for the majority of studies and has driven much of the recent increase, whereas CF has remained consistently represented but at lower annual frequencies. This recent expansion reflects the growing adoption of ATFS in research on tick-borne diseases.
Figure 2 Long description
A bar graph displays the number of studies on membrane and capillary feeding systems from 1912 to 2024. The x-axis is labeled 'Publication year' and the y-axis is labeled 'Number of studies'. Green bars represent membrane feeding system studies, while red bars represent capillary feeding system studies. The graph shows an increase in studies over time, with a notable rise in membrane feeding system studies in recent years, particularly after 2010. Capillary feeding system studies are consistently lower in number compared to membrane feeding system studies.
Geographical distribution of studies on artificial tick feeding systems. The map highlights global research activity, with the largest number of studies from the USA, multiple European countries and Brazil, reflecting established tick-research centres and ongoing work on tick-borne diseases.
Figure 3 Long description
A world map illustrates the distribution of studies on artificial tick feeding systems by country. The United States leads with 48 studies, followed by Germany with 26, Brazil with 20 andKenya with 13. European countries such as France, UK and the Czech Republic have 11, 10 and 9 studies respectively. Other countries like Australia and Switzerland have 10 and7 studies respectively . The map uses a gradient to represent the number of studies, ranging from 1 to 48.
Influence of tick feeding behaviour on ATFS success
Feeding behaviours differ significantly between soft and hard ticks, impacting ATFS success. For instance, soft ticks usually feed for several minutes to hours while hard ticks require extended feeding periods from several days to weeks, requiring larger volumes and a continuous supply of blood or nutrient media. At 37°C, rapid blood deterioration necessitates frequent replacement, typically twice daily (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). Furthermore, as hard ticks exhibit complex pre-feeding behaviour (requiring heat, tactile stimuli, i.e., animal hair and kairomones emitted by the host, i.e., carbon dioxide [CO2]) and feeding for several days, they require a controlled environment (e.g., in an incubator or climatic chamber) where specific temperature and humidity levels (typically >80%) are maintained under controlled hygienic conditions to avoid fungal growth and increase tick survival (Sebastian et al., Reference Sebastian, Król, Novoa, Nijhof, Pfeffer, Nava and Obiegala2023; Sonenshine and Roe, Reference Sonenshine, Roe, Sonenshine and Roe2013).
Capillary feeding
Overview and applications
CF involves the placement of a blood-filled capillary tube over the tick's hypostome to facilitate forced feeding. First developed by Gregson for feeding Dermacentor (D.) andersoni and later adapted by Chabaud to study the nutrient uptake by Ornithodoros (O.) erraticus and O. talaje, this technique is suitable for both hard and soft ticks (Chabaud, Reference Chabaud1950; Gregson, Reference Gregson1937). It offers a simpler and faster alternative to MF and has been employed to introduce molecular markers, drugs, or pathogens into ticks (Sebastian et al., Reference Sebastian, Król, Novoa, Nijhof, Pfeffer, Nava and Obiegala2023).
Studies on tick physiology
Twenty studies have used CF to investigate physiological parameters of ticks under controlled laboratory conditions. These included factors such as feeding media composition, blood (with/without anticoagulants), serum, and plasma from different animals, and various capillary tube dimensions (Table 1). The technique has been used for various species of hard and soft ticks (Fig. 4A).
Number of studies reporting artificial feeding of ticks using capillary feeding (CF; A) and membrane feeding (MF; B). Bars are stratified by tick species and coloured by soft ticks (magenta) and hard ticks (green). CF studies were dominated by hard ticks, particularly Rhipicephalus microplus (n = 14), followed by R. appendiculatus (n = 9) and Ixodes ricinus (n = 7), with only isolated reports for soft ticks. MF studies were also dominated by hard ticks, including I. ricinus (n = 34), R. microplus (n = 12), and I. scapularis (n = 10). Among soft ticks, Ornithodoros moubata accounted for the highest number of MF studies (n = 24), while other species were represented by fewer than 10 studies each. For MF, only species with ≥3 studies are shown due to space constraints.
Figure 4 Long description
The image consists of two bar graphs labeled A and B. Graph A shows the number of studies on various tick species using capillary feeding. The x-axis is labeled 'Number of studies' and the y-axis lists tick species, including R. microplus, R. appendiculatus and I. ricinus, among others. Hard ticks are represented in green and soft ticks in magenta. Graph B displays the number of studies using membrane feeding, with the same x-axis label and a similar list of tick species. The species I. ricinus and O. moubata have the highest study counts. The legend indicates the color coding for soft and hard ticks.
Key studies on the application of capillary feeding for understanding tick physiology
Table 1 Long description
The table examines various studies on capillary feeding in ticks, focusing on species, objectives, life stages, feeding solutions, conditions, and outcomes. Key findings include Argas persicus preferring heparinised blood, while A. cajennense achieves the best egg production with 24-hour continuous feeding on citrated bovine blood. A. americanum shows enhanced feeding with KATP channel activation, and R. microplus gains maximum weight with bovine blood. The studies highlight the importance of feeding medium and conditions on tick physiology, with variations in outcomes based on species and experimental setups. The data suggest that feeding duration and blood type significantly impact tick feeding success and reproductive outcomes.
N, A, nymph, adult tick stage; b.wt., body weight; NP, not provided.
Pathogen transmission studies
CF has been used in 36 studies (35 in hard ticks; 1 in soft ticks) to investigate pathogen transmission (Table 2). In contrast to other techniques, such as the direct injection of ticks with pathogens, CF mimics the natural route of pathogen acquisition. It allows for a precise measurement of the ingested fluid volume and accurate titration of the pathogen dose. Furthermore, it facilitates the introduction of single or multiple strains or species of TBPs.
Key studies on the application of capillary feeding for understanding transmission of tick-borne pathogens
Table 2 Long description
The table provides a comprehensive overview of studies examining the transmission of tick-borne pathogens through capillary feeding. It includes data on different tick species, life stages, and the pathogens they acquire or transmit. Key findings include successful pathogen acquisition and transmission in both soft and hard ticks, with specific conditions such as temperature, humidity, and feeding duration influencing outcomes. Notable studies show successful trans-stadial and transovarial transmission, as well as pathogen maintenance in ticks. The table also highlights variations in transmission efficiency between tick life stages and species, with some studies noting enhanced transmission in adult ticks compared to nymphs. Overall, the data underscores the complexity of tick-pathogen interactions and the importance of environmental conditions in influencing transmission dynamics.
L, N, A, larva, nymph, adult tick stage; TOT, transovarial transmission; TS, transstadial transmission; MEM, minimal essential medium; SG, salivary glands; CF, capillary feeding; CFU, colony forming unit; PFU, plaque-forming unit; CN, copy number; HSP, heat shock protein; MSP, major surface protein; Omp, outer membrane protein; i.d., inner diameter.
CF was first used to investigate the transmission of Leptospira (L.) pomona by D. andersoni and Amblyomma maculatum (Burgdorfer, Reference Burgdorfer1957). Subsequently, it has been used for the transmission of other pathogens (Fig. 5; Table 2), including Th. parva by Rhipicephalus (R.) appendiculatus (Purnell, Reference Purnell1970; Purnell and Joyner, Reference Purnell and Joyner1967; Walker et al., Reference Walker, Brown, Bell and McKellar1979), Borrelia (Bo.) burgdorferi by Ixodes (I.) ricinus and I. scapularis (Korshus et al., Reference Korshus, Munderloh, Bey and Kurtti2004; Kurtenbach et al., Reference Kurtenbach, Dizij, Seitz, Margos, Moter, Kramer, Wallich, Schaible and Simon1994), Ehrlichia chaffeensis by A. americanum, Dermacentor Variabilis, and Rhipicephalus sanguineus (Rechav et al., Reference Rechav, Zyzak, Fielden and Childs1999), Anaplasma (An.) marginale by D. variabilis (Kocan et al., Reference Kocan, Yoshioka, Sonenshine, de la Fuente, Ceraul, Blouin and Almazan2005), Mycobacterium leprae by A. sculptum (Ferreira et al., Reference Ferreira, Oliveira, Santos, Ribeiro, Baêta, Teixeira, Neumann, Rosa, Pessolani, Moraes, Bechara, Oliveira, Sorgine, Suffys, Fontes, Bell-Sakyi, Fonseca and Lara2018), and Rickettsia (Ri.) parkeri by A. maculatum, and Rhipicephalus microplus (Cordeiro et al., Reference Cordeiro, de Azevedo Baêta, Cepeda, Teixeira, Ribeiro, de Almeida Valim, Pinter and Fonseca2018; Lee et al., Reference Lee, Moraru, Stokes, Benton, Wills, Nabors, Smith, Lawrence, Willeford and Varela-Stokes2019) (Fig. 5; Table 2).
In hard ticks, most studies have focused on the transmission of Bo. burgdorferi sensu lato, the causative agent of Lyme borreliosis, in I. ricinus and I. scapularis. Among animal pathogens, most studies were performed on Th. parva, the causative agent of East Coast fever, and its tick vector R. appendiculatus (Fig. 5; Table 2).
Number of studies using capillary feeding to assess tick vector competence for pathogens. The figure shows that such studies are currently concentrated on a limited number of tick–pathogen pairs, with the highest representation for Rhipicephalus appendiculatus–Theileria parva (n = 6), followed by Borrelia burgdorferi interactions with Ixodes ricinus (n = 4) and I. scapularis (n = 2). Repeated studies were also reported for Amblyomma maculatum–Rickettsia parkeri. Most other tick–pathogen combinations were represented by single studies, and soft ticks (e.g. Ornithodoros moubata) were included only rarely.
Figure 5 Long description
A grid displays interactions between various tick species and pathogens, with study counts indicated in each cell. The tick species are listed vertically on the left, including O. moubata, A. americanum, A. maculatum and others. Pathogens are listed horizontally at the top, such as Anaplasma marginale, Borrelia burgdorferi and Theileria parva. The grid cells contain numbers representing the number of studies for each tick-pathogen pair. Notable counts include 6 for R. appendiculatus with Theileria parva, 4 for I. ricinus with Borrelia burgdorferi and 2 for I. scapularis with Borrelia burgdorferi. A color gradient on the right indicates study frequency, ranging from 0 to 6.
Evaluation of drugs and vaccines
To date, 16 studies have applied CF for drug and vaccine efficacy testing in hard ticks (Table 2). Tested substances include antibodies against antigens such as the Boophilus microplus 86 kDa protein (Bm86) from R. microplus, B. microplus 2 protein from a Brazilian R. microplus strain (BrBm2), salivary gland Kunitz-type inhibitor protein (SILK), subolesin (SUB), tick receptor for OspA (TROSPA), and R. microplus phosphatase (RmPAP), as well as the acaricide fluazuron, tested against R. microplus (Antunes et al., Reference Antunes, Merino, Mosqueda, Moreno-Cid, Bell-Sakyi, Fragkoudis, Weisheit, Pérez de la Lastra, Alberdi, Domingos and de la Fuente2014; Gonsioroski et al., Reference Gonsioroski, Bezerra, Utiumi, Driemeier, Farias, Vaz and Masuda2012; Lew-Tabor et al., Reference Lew-Tabor, Bruyeres, Zhang and Valle2014; Lu et al., Reference Lu, Parizi, Torquato, Vaz Junior and Tanaka2019; Reck et al., Reference Reck, Klafke, Webster, Dall’Agnol, Scheffer, Souza, Corassini, Vargas, Santos and Martins2014). These findings underscore the value of CF as a tool for drug screening and vaccine development targeting ticks (Table 3).
Key studies on the application of capillary feeding to assess the efficacy of drugs, vaccines and antisera against ticks and tick-borne pathogens
Table 3 Long description
The table evaluates the efficacy of drugs, vaccines, and antisera against various tick species and tick-borne pathogens. Key findings include the reduction in tick oviposition and weight when treated with specific antibodies and vaccines, such as the 83% efficacy of IgG against Bm86 in R. microplus. Comparisons reveal that some treatments, like anti-calreticulin serum, show no significant effect, while others, like fluazuron, indicate drug resistance in certain strains. The studies span different tick life stages, primarily focusing on adult ticks, and highlight the importance of specific antigens and inhibitors in controlling tick populations. The data suggests that while some treatments are effective, others require further investigation to understand their impact on tick physiology and resistance mechanisms.
L, N, A, larva, nymph, and adult stages of tick; PBS, phosphate buffered saline; IgG, immunoglobulin G; dsRNA, double stranded RNA; ppm, parts per million; GSK-3, glycogen synthase kinase-3; Ab, antibodies; RmSI-7, Rhipicephalus microplus subtilisin inhibitor 7; TIL, trypsin inhibitory like; BmTI-A, B. microplus trypsin inhibitor A; NP, not provided.
Limitations
CF often fails to achieve engorgement, requiring pre- or post-feeding on live animals (Burgdorfer, Reference Burgdorfer1957; Purnell and Joyner, Reference Purnell and Joyner1967; Rau and Hannoun, Reference Rau and Hannoun1968). Although extended feeding durations or targeting the rapid engorgement phase can increase the volume ingested, this approach is labour-intensive, requiring frequent blood replacement to prevent blood coagulation (Purnell and Joyner, Reference Purnell and Joyner1967; Willadsen et al., Reference Willadsen, Kemp and McKenna1984). Moreover, CF is technically challenging for juvenile ticks due to their smaller and shorter mouthparts and limited scalability. As a result, MF systems can offer more practical alternatives for larger-scale studies.
Membrane feeding
System components
MF was first used to study the sensory perceptions of ticks (Hindle and Merriman, Reference Hindle and Merriman1912; Lees, Reference Lees1948; Totze, Reference Totze1933), and has since been adopted for tick rearing, transmission of TBPs and testing of acaricides and vaccines (González et al., Reference González, Bickerton and Toledo2021). Unlike CF, this method uses membranes, derived from animal skin or synthetic components, such as parafilm or silicone, for feeding ticks. Most studies that used MF systems contained the key components, including (i) a vessel for the containment of ticks, (ii) a feeding medium, typically blood, (iii) a membrane that mimics host skin, separating the ticks from the feeding medium, and (iv) a temperature control system (Voigt et al., Reference Voigt, Young, Mwaura, Nyaga, Njihia, Mwakima and Morzaria1993). In most MF systems, ticks are placed above the feeding medium. However, in some techniques, the feeding medium reservoir is placed above the ticks to facilitate the access of blood cells to the ticks even after their sedimentation (Bonnet et al., Reference Bonnet, Jouglin, Malandrin, Becker, Agoulon, L’Hostis and Chauvin2007; Burkot et al., Reference Burkot, Happ, Dolan and Maupin2001; Vimonish et al., Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1995). The latter system might be advantageous for the feeding of ticks that exhibit negative geotaxis. However, a higher degree of membrane stability is needed to avoid blood leaking into the tick compartment placed below.
Types of membranes used
To date, a total of 138 studies have used MF, employing animal tissues/skin (n = 37), synthetic (n = 97), or both types of membranes (n = 4). Animal tissues used included the skin of mouse and rabbits (Tarshis, Reference Tarshis1958), chickens (Rogers and Howell, Reference Rogers and Howell1971), cattle (Kemp et al., Reference Kemp, Koudstaal, Roberts and Kerr1975), mice (Umemiya-Shirafuji et al., Reference Umemiya-Shirafuji, Hatta, Okubo, Sato, Maeda, Kume, Yokoyama, Igarashi, Tsuji, Fujisaki, Inoue and Suzuki2017; Wechtaisong et al., Reference Wechtaisong, Bonnet, Chomel, Lien, Chuang and Tsai2021, Reference Wechtaisong, Bonnet, Lien, Chuang and Tsai2020), rabbits (Howarth and Hokama, Reference Howarth and Hokama1983), pigs and chickens (Osborne and Mellor, Reference Osborne and Mellor1985), pigeons (Abbassy et al., Reference Abbassy, Stein and Osman1994), and gerbils (Bonnet et al., Reference Bonnet, Jouglin, Malandrin, Becker, Agoulon, L’Hostis and Chauvin2007; Migné et al., Reference Migné, Braga de Seixas, Heckmann, Galon, Mohd Jaafar, Monsion, Attoui and Moutailler2022a), as well as muscles derived from the diaphragm of rabbits and guinea pigs (Lees, Reference Lees1948; Totze, Reference Totze1933). In addition, animal-derived membranes, such as goldbeater’s skin (Baudruche membrane; bovine intestine-derived), have also been used in tick feeding studies (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). Most studies sourced membranes from cattle, mice and rabbits, primarily due to availability. Fresh animal skin was used in the majority of studies, although frozen skin has also been reported. In general, feeding success was higher with fresh animal skin (Kemp et al., Reference Kemp, Koudstaal, Roberts and Kerr1975).
Unlike the synthetic membranes, animal skin does not have a fixed thickness; this varies between species and anatomical regions, therefore requiring careful selection of skin types for the tick species and life stage under investigation. A key advantage of animal skin is the presence of natural chemical and physical cues that enhance tick attachment and feeding. However, using membranes from animal tissues/skin is prone to degradation and contamination, can introduce bacteria or fungi into the blood meal despite antibiotic use, may be difficult to source consistently, and can raise ethical concerns.
To circumvent these challenges, various synthetic membranes have been developed that can be produced under hygienic conditions, with a tailored thickness to match the hypostome length of different tick species and life stages. Synthetic materials that have been used for MF include cellophane (Totze, Reference Totze1933), latex (Ben-Yakir and Galun, Reference Ben-Yakir and Galun1993), parafilm (Galun and Kindler, Reference Galun and Kindler1965), and silicone (Böhme et al., Reference Böhme, Krull, Clausen and Nijhof2018; Kröber and Guerin, Reference Kröber and Guerin2007a; Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995), with parafilm and silicone being more commonly used.
Parafilm is a stretchable polyethylene foil mainly used to feed soft ticks. It is relatively inexpensive, easy to handle and has been used in several studies (Buysse et al., Reference Buysse, Duhayon, Cantet, Bonazzi and Duron2021; Hokama et al., Reference Hokama, Lane and Howarth1987; Lewis et al., Reference Lewis, Bartholomay and Blanchong2020; Schäfer et al., Reference Schäfer, Pfaff, Höper and Silaghi2022; Stewart et al., Reference Stewart, Raffel, Gherardini and Bloom2022). However, it was observed that parafilm perforates rapidly when pierced by the mouthparts of ticks, limiting its use to short-term feeding of soft ticks. Silicone membranes have been the most successful for feeding hard ticks (Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995). This material was first used for the artificial feeding of I. holocyclus (Stone et al., Reference Stone, Commins and Kemp1983) and has since been used across various hard tick species and life stages (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021; Ribeiro et al., Reference Ribeiro, Cordeiro, Cepeda, Valim, Fonseca and Baêta2023; Sebastian et al., Reference Sebastian, Król, Novoa, Nijhof, Pfeffer, Nava and Obiegala2023). A landmark achievement was the first in vitro completion of the life cycle of Amblyomma (A.) hebraeum using a silicone-based MF system (Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995). Kröber and Guerin further demonstrated its utility in acaricidal testing (Kröber and Guerin, Reference Kröber and Guerin2007b). Recently, the artificial feeding of all life stages of I. ricinus, Hyalomma (Hy.) scupense, Hy. excavatum, and Hy. dromedarii using silicone membranes was reported (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). However, it is important to note that all these tick species are either two- or three-host ticks and the successful completion of the life cycle of one-host ticks using ATFS has not yet been reported (Table 4).
Key studies on the application of membrane feeding for understanding tick physiology
Table 4 Long description
The table examines various studies on membrane feeding to understand tick physiology, focusing on different tick species, life stages, membrane types, and feeding mediums. Key findings include the efficiency of whole blood in promoting tick moulting and oviposition, and the role of reduced glutathione (GSH) in enhancing blood intake. Synthetic membranes like silicone and parafilm are often used for feeding experiments. Environmental conditions such as temperature and humidity are crucial for successful feeding and development. The studies highlight the variability in feeding success and physiological responses across different tick species and experimental setups.
L, N, A, larva, nymph and adult stages of tick; GSH, reduced glutathione; ATP, adenosine triphosphate; RBC’s, red blood cells; FBS, foetal bovine serum; FCS, foetal calf serum; MEM, minimum essential medium; ACD, acid citrate dextrose; PBS, phosphate buffered saline; Glu., glucose; pen/strep, penicillin/streptomycin; PS, phagostimulants for ticks; P/K, pheromone/kairomone for ticks; ATFS, artificial tick feeding system; TOT, transovarial transmission; Ampho., amphotericin B; Nyst., nystatin; TCM, tissue culture medium; SG, salivary glands; h, hours; Genta., gentamicin; b.wt., body weight.
Environmental requirements
Successful MF requires precise control of environmental parameters. Most successful studies reported maintenance of the blood temperature at approximately 37°C (35–39°C). Studies have used various sources to warm the blood for ticks, including hot plates (Paine et al., Reference Paine, Kemp and Allen1983), water baths (Waladde et al., Reference Waladde, Young, Ochieng, Mwaura and Mwakima1993), and incubators (Kemp et al., Reference Kemp, Koudstaal, Roberts and Kerr1975). Relative humidity is typically maintained between 70 and 95%. Additionally, a CO2 concentration of 0.05–10% has been used, and a higher CO2 concentration has been shown to enhance tick feeding and engorgement (Böhme et al., Reference Böhme, Krull, Clausen and Nijhof2018; Habedank and Hiepe, Reference Habedank and Hiepe1993; Krull et al., Reference Krull, Bohme, Clausen and Nijhof2017; Voigt et al., Reference Voigt, Young, Mwaura, Nyaga, Njihia, Mwakima and Morzaria1993; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1995). To stimulate tick attachment, tactile and olfactory stimuli such as cattle hair or hair extracts have also been used (Böhme et al., Reference Böhme, Krull, Clausen and Nijhof2018; Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Kröber and Guerin, Reference Kröber and Guerin2007a; Krull et al., Reference Krull, Bohme, Clausen and Nijhof2017; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021; Trentelman et al., Reference Trentelman, Kleuskens, van de Crommert and Schetters2017).
To ensure a fresh nutrient supply and reduce contamination risk for hard ticks, blood needs to be changed twice daily in most studies (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024), although some replaced blood three to six times per day (Vimonish et al., Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020; Waladde et al., Reference Waladde, Ochieng and Gichuhi1991, Reference Waladde, Young, Ochieng, Mwaura and Mwakima1993). To address this challenge, semi-automated MF systems (Table 5) have been developed that facilitate the routine addition and removal of blood or feeding medium (Asri et al., Reference Asri, Tahir, Evans, Meyer, Rhalem, Bouslikhane, Ueti and Madder2023; Böhme et al., Reference Böhme, Krull, Clausen and Nijhof2018; de la Vega et al., Reference de la Vega, Camejo and Fonseca2004; Kemp et al., Reference Kemp, Koudstaal, Roberts and Kerr1975; Kuhnert et al., Reference Kuhnert, Issmer and Grunewald1998; Stone et al., Reference Stone, Commins and Kemp1983; Vimonish et al., Reference Vimonish, Dinkel, Fry, Johnson, Capelli-Peixoto, Bastos, Scoles, Knowles, Madder, Chaka and Ueti2021, Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020). However, these systems have demonstrated variable success across studies. For example, Kuhnert et al. (Reference Kuhnert, Issmer and Grunewald1998) showed that a semi-automated system for A. hebraeum produced feeding and reproductive parameters (body mass, egg conversion factor, and larval hatch rate) comparable to those observed in animal-fed ticks. Similarly, Kemp et al. (Reference Kemp, Koudstaal, Roberts and Kerr1975) reported successful larval engorgement and moulting in R. microplus using fresh animal skin. More recently, Asri et al. (Reference Asri, Tahir, Evans, Meyer, Rhalem, Bouslikhane, Ueti and Madder2023) achieved high attachment rates (≥70%) and successful feeding of adult R. appendiculatus to repletion under in vitro conditions, further supporting the utility of these systems.
Key studies on the application of partially automated membrane feeding systems for artificial tick feeding
Table 5 Long description
The table examines the effectiveness of semi-automated membrane feeding systems for artificial tick feeding across various tick species and life stages. Key findings include increased tick size in A. cajennense after 5 to 7 days, comparable tick parameters to animal feeding in R. microplus and A. hebraeum, and better system performance in D. reticulatus as compared to I. ricinus. The systems utilize silicone membranes with different feeding mediums, such as heparinised blood and tissue culture mediums, under controlled environmental conditions. Notable trends include successful pathogen transmission in R. appendiculatus and improved engorgement and moulting rates with fresh cattle skin in R. microplus larvae. The table highlights the potential of these systems in enhancing tick feeding outcomes and pathogen studies.
L, A, larva and adult stages of tick; ATP, adenosine triphosphate; FCS, foetal calf serum; Genta., gentamicin; Glu., glucose; h, hours; Nyst., nystatin; pen/strep., penicillin, streptomycin; P/K, pheromone/kairomone for ticks of tick; PS, phagostimulants for ticks; SG, salivary glands; TCM, tissue culture medium.
In contrast, less favourable outcomes have also been reported. For example, Krull et al. (Reference Krull, Bohme, Clausen and Nijhof2017) observed satisfactory results for D. reticulatus, but suboptimal performance in Ixodes ricinus, largely due to severe fungal contamination. In pathogen transmission studies, semi-automated systems have successfully supported transmission of An. marginale and Th. parva in D. andersoni and R. appendiculatus, respectively (Vimonish et al., Reference Vimonish, Dinkel, Fry, Johnson, Capelli-Peixoto, Bastos, Scoles, Knowles, Madder, Chaka and Ueti2021, Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020).
Collectively, these findings indicate that while semi-automated feeding systems are valuable tools, their performance is highly species-dependent and requires careful optimisation of feeding conditions and stringent control of experimental variables, particularly contamination.
Applications of MF systems
Physiological studies
MF has been used in 78 studies to investigate soft (n = 31) and hard (n = 47) tick physiology under controlled conditions. Early optimisation studies focused on identifying suitable membranes, attractants (pheromones/kairomones) and feeding stimuli such as adenosine triphosphate (ATP), glutathione, and CO2 that enhanced artificial feeding success (Galun and Kindler, Reference Galun and Kindler1965; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1979). A major breakthrough was achieved by Kuhnert and colleagues, who reported the successful artificial feeding of all life stages of A. hebraeum (Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995). The key to this success was the use of different combinations of stage-specific stimuli, tailoring the silicone membrane thickness to match the hypostome length of each tick stage and controlling contamination, highlighting the need to adapt feeding conditions for different species and life stages.
Despite the successful artificial feeding of A. hebraeum, only a few studies have achieved multistage feeding across all stages of hard ticks, including Hy. scupense, Hy. excavatum, Hy. dromedarii, and I. ricinus (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). Additionally, MF has also been used for artificial feeding of various life stages of soft ticks, including Ar. hermanni, Ar. persicus, Ar. arboreus, O. moubata, O. turicata, O. tartakovskyi, O. rostratus, and O. tholozani (Fig. 4B).
To feed ticks in vitro, most studies have used defibrinated or heparinised blood from various host species. However, blood alone is often insufficient for in vitro tick feeding. For instance, early experiments showed that supplementation with ATP and glutathione enhanced feeding rates of soft ticks (O. moubata, O. tholozani) (Ben-Yakir and Galun, Reference Ben-Yakir and Galun1993; Galun, Reference Galun1978; Galun and Kindler, Reference Galun and Kindler1965, Reference Galun and Kindler1968; Mango and Galun, Reference Mango and Galun1977). This enhancement is based on the principle that when a tick bites the host's skin, host cell lysis releases glutathione and ATP, signals that may be interpreted by ticks as an indication of nutrient-rich blood. In ATFS, the concentrations of these compounds in the provided blood may be insufficient to be effective and supplementation of the blood containing glucose (1 mg/ml) with ATP (10−3 M) or glutathione (10−3–10−4 M) has been shown to improve feeding success (Ben-Yakir and Galun, Reference Ben-Yakir and Galun1993; Galun and Kindler, Reference Galun and Kindler1965, Reference Galun and Kindler1968). Similarly, haem, a component of host blood haemoglobin, has been found to be critical in tick feeding and reproduction. For example, the feeding I. ricinus on haem-depleted meal (serum) resulted in the production of eggs with poor embryogenesis and larval viability. Haemoglobin supplementation of serum mitigated these effects and improved egg viability (Perner et al., Reference Perner, Sobotka, Sima, Konvickova, Sojka, de Oliveira, Hajdusek and Kopáček2016b). Subsequently, similar findings were observed for O. moubata when fed twice on a haem-free blood meal (Hatalová et al., Reference Hatalová, Erhart, Kopáček and Perner2023).
Anatomical and morphological studies
MF can also facilitate detailed morphological studies of tick feeding structures under controlled conditions. For example, many tick species secrete a cement-like substance that anchors the mouthparts and forms a protective barrier at the feeding site. Analysing the composition and architecture of cement cones in vivo is technically challenging, whereas MF enables cleaner and more accessible isolation of these structures.
Using this approach, Bullard et al. (Reference Bullard, Allen, Chao, Douglas, Das, Morgan, Ching and Karim2016) showed that cement cones from animal-fed A. americanum were composed of sheets and fibrils with a highly textured surface, whereas cones from membrane-fed ticks were smoother and lacked distinct structural features. Although both cone types contained β-sheet structures, animal-fed cones also exhibited helical protein configurations, indicating important structural differences. These findings suggest that host-derived factors, such as extracellular matrix components and immune responses, may influence cement cone formation in vivo, and that MF may capture only part of this complexity.
Further targeted comparative studies are therefore needed to better understand how host factors shape cement cone structure and function, and to refine MF systems for studying tick-feeding biology.
Number of studies using membrane feeding (MF) to assess tick vector competence for pathogens. In hard ticks, the highest study counts were reported for Rhipicephalus appendiculatus–Theileria parva (n = 4), followed by Ixodes ricinus–tick-borne encephalitis virus (TBEV) (n = 3) and I. scapularis–Borrelia burgdorferi (n = 3). Among hard ticks, I. ricinus showed the widest pathogen coverage, with several additional pathogens represented by one or two studies each. Among soft ticks, Ornithodoros moubata was most frequently studied, including O. moubata–Borrelia hermsii (n = 2) and several single-study observations. Most remaining tick–pathogen combinations were reported only once or twice.
Figure 6 Long description
The heatmap displays interactions between various tick species and pathogens, with study counts indicated by numbers within the grid. The tick species are listed vertically on the left, including Ar. persicus, O. brasiliensis, O. fonsecai, O. hermsi, O. moubata, O. savignyi, O. turicata, A. americanum, A. variegatum, D. andersoni, D. marginatus, D. reticulatus, Hae. longicornis, Hy. anatolicum, Hy. lusitanicum, I. ricinus, I. scapularis, I. persulcatus, R. appendiculatus, R. bursa and R. sanguineus. Pathogens are listed horizontally at the top, including ALSV, An. phagocytophilum, ASFV, Ba. ovata, B. anthracis, B. subtilis, Bar. henselae, Bluetongue virus, Bo. burgdorferi, Bo. duttonii, Bo. hermsii, Bo. turicatae, C. burnetii, D. viteae, E. canis, E. muris-like agent, En. faecalis, KEMV, L. icterohaemorrhagiae, N. mikurensis, Ri. raoultii, Ri. massiliae, R. monacensis, Th. lestoquardi, Th. mutans, Th. parva, TBEV, WEE virus and WNV. The study counts are represented by numbers within the grid, ranging from 1 to 6, with a color gradient indicating the frequency of studies. The highest count is 4 for R. appendiculatus with Th. parva and 3 for I. scapularis with Bo. burgdorferi and I. ricinus with TBEV.
Pathogen transmission and vector competence
To date, both soft (n = 11) and hard (n = 35) ticks have been studied using MF for studying pathogen transmission and vector competence (Fig. 6; Table 6). In hard ticks, it enabled controlled studies on the transmission of protozoal (e.g., Babesia (Ba.) divergens, Ba. ovata, Th. mutans, and Th. parva in I. ricinus, Haemaphysalis (Hae.) longicornis, A. variegatum, and R. appendiculatus, respectively (Bonnet et al., Reference Bonnet, Jouglin, Malandrin, Becker, Agoulon, L’Hostis and Chauvin2007; Maeda et al., Reference Maeda, Hatta, Alim, Tsubokawa, Mikami, Matsubayashi, Miyoshi, Umemiya-Shirafuji, Kawazu, Igarashi, Mochizuki, Tsuji and Tanaka2016; Musyoki et al., Reference Musyoki, Osir, Kiara and Kokwaro2004; Umemiya-Shirafuji et al., Reference Umemiya-Shirafuji, Hatta, Okubo, Sato, Maeda, Kume, Yokoyama, Igarashi, Tsuji, Fujisaki, Inoue and Suzuki2017; Voigt et al., Reference Voigt, Young, Mwaura, Nyaga, Njihia, Mwakima and Morzaria1993), and viral pathogens, e.g., TBEV, West Nile virus, and Kemerovo virus by I. ricinus (Liebig et al., Reference Liebig, Boelke, Grund, Schicht, Bestehorn-Willmann, Chitimia-Dobler, Dobler, Jung and Becker2021; Migné et al., Reference Migné, Braga de Seixas, Heckmann, Galon, Mohd Jaafar, Monsion, Attoui and Moutailler2022a, Reference Migné, Hönig, Bonnet, Palus, Rakotobe, Galon, Heckmann, Vyletova, Devillers and Attoui2022b; Răileanu et al., Reference Răileanu, Tauchmann, Vasic, Neumann, Tews and Silaghi2020). In addition, MF also helped to elucidate whether several European ticks could acquire and support infection (and possible onward transmission) of African swine fever virus (ASFV). No evidence of ASFV replication was found in I. ricinus and D. reticulatus, whereas it occurred in O. moubata, the confirmed vector (de Carvalho Ferreira et al., Reference de Carvalho Ferreira, Tudela Zuquete, Wijnveld, Weesendorp, Jongejan, Stegeman and Loeffen2014). Similarly, several hard and soft tick species were infected with bluetongue virus (serotype-8) using MF or CF, and O. savignyi demonstrated viral infection and RNA detection in various tissues; however, viral presence was not found in tick progeny (Bouwknegt et al., Reference Bouwknegt, van Rijn, Schipper, Hölzel, Boonstra, Nijhof, van Rooij and Jongejan2010). MF has also contributed to studying the transmission of various bacterial pathogens (An. marginale, An. phagocytophilum, Bo. burgdorferi, Bartonella (Bar.) henselae, Ehrlichia (E.) canis, and Rickettsia spp. in various ticks and also unravelling the role of various proteins (BBA52, Lp6.6, and lmp1 of Bo. burgdorferi) in the survival, persistence, and transmission of pathogens (Cotté et al., Reference Cotté, Bonnet, Le Rhun, Le Naour, Chauvin, Boulouis, Lecuelle, Lilin and Vayssier-Taussat2008; Fourie et al., Reference Fourie, Evans, Labuschagne, Crafford, Madder, Pollmeier and Schunack2019, Reference Fourie, Stanneck, Luus, Beugnet, Wijnveld and Jongejan2013; Hart et al., Reference Hart, Yang, Pal and Lin2018; Koci et al., Reference Koci, Bernard, Yang and Pal2018; Król et al., Reference Król, Militzer, Stobe, Nijhof, Pfeffer, Kempf and Obiegala2021; Olivieri et al., Reference Olivieri, Wijnveld, Bonga, Berger, Manfredi, Veronesi and Jongejan2018; Vimonish et al., Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020).
Key studies on the application of membrane feeding for understanding transmission of tick-borne pathogens
Table 6 Long description
This table presents research findings on the use of membrane feeding to study the transmission of tick-borne pathogens. It includes data on different tick species, life stages, types of membranes used, pathogens studied, and feeding solutions. Key findings indicate successful pathogen acquisition and transmission in both soft and hard ticks, with variations in feeding conditions such as temperature, humidity, and blood composition. Notable studies include successful trans-stadial transmission and pathogen maintenance across tick life stages. The table also highlights differences in vector competence and pathogen detection in tick organs, emphasizing the role of membrane type and feeding conditions in experimental outcomes.
L, N, A, larva, nymph, adult tick stage; ATP, adenosine triphosphate; CFU, colony-forming unit; Genta., gentamicin; Glu., glucose; PFU, plaque forming unit; MEM, minimal essential medium; Nyst., nystatin; TOT, transovarial transmission; TS, Trans-stadial; SG, salivary glands; EDTA, ethylenediamine tetraacetic acid; ACD, acid-citrate-dextrose; RBC, red blood cell; PCV, packed cell volume.
In soft ticks, MF has facilitated studies on the transmission of various bacterial pathogens, including L. pomona, Bo. turicatae, and Bo. hermsii in O. turicata and O. hermsi (Burgdorfer, Reference Burgdorfer1957; Neelakanta et al., Reference Neelakanta, Sultana, Sonenshine and Andersen2018; Stewart et al., Reference Stewart, Raffel, Gherardini and Bloom2022). Unsuccessful transmission of Coxiella (C.) burnetii by O. moubata has also been reported (Buysse et al., Reference Buysse, Duhayon, Cantet, Bonazzi and Duron2021). Together, these studies elucidate the pivotal role of MF in enhancing our mechanistic understanding of tick-pathogen interactions and molecular determinants of vectorial capacity of ticks for various pathogens.
Drug and vaccine evaluation
A principal advantage of MF is that only small quantities of test compounds are required, and their activity can be assessed under controlled conditions with reduced experimental variability. This approach provides a scalable and precise platform for the preliminary screening of acaricides, including the evaluation of multiple concentrations to determine dose-response relationships. In addition, MF systems enable the evaluation of vaccine candidates and other immunological interventions targeting tick infestation and TBD transmission, for example, by incorporating immune sera or specific antibodies into the blood meal. A total of 16 studies (hard ticks: n = 11; soft ticks: n = 5) have used MF to evaluate the efficacy of acaricides and immunological interventions (Table 7). For example, the efficacy of drugs such as ivermectin, fipronil, and fluralaner was tested against A. hebraeum, I. ricinus, O. moubata, and O. erraticus (Kröber and Guerin, Reference Kröber and Guerin2007b; Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995; Pérez-Sánchez and Oleaga, Reference Pérez-Sánchez and Oleaga2017; Williams et al., Reference Williams, Zoller, Roepke, Zschiesche and Heckeroth2015). Animals vaccinated with the B. microplus female extract showed gut damage in infested ticks, and ticks fed via MF with plasma from vaccinated animals showed similar effects (Kemp et al., Reference Kemp, Agbede, Johnston and Gough1986). Importantly, this study demonstrated that host-derived antibodies ingested during feeding can induce pathological effects in ticks, providing strong support for the concept of hidden antigens as viable vaccine targets. These findings were subsequently validated in in vivo animal trials and contributed to the identification and development of the Bm86 antigen. This work ultimately led to the commercialisation of anti-tick vaccines, including TickGARD and TickGARD Plus in Australia and Gavac in Cuba, highlighting the translational potential of ATFS in anti-tick vaccine discovery (Andreotti, Reference Andreotti2006; Boué et al., Reference Boué, Redondo, Montero, Rodriguez and De La Fuente1999; de la Fuente et al., Reference de la Fuente, Rodriguez, Redondo, Montero, García-García, Méndez, Serrano, Valdés, Enriquez, Canales, Ramos, Boué, Machado, Lleonart, de Armas, Rey, Rodríguez, Artiles and García1998; Jonsson et al., Reference Jonsson, Matschoss, Pepper, Green, Albrecht, Hungerford and Ansell2000; Rand et al., Reference Rand, Moore, Sriskantha, Spring, Tellam, Willadsen and Cobon1989; Rodríguez Valle et al., Reference Rodríguez Valle, Méndez, Valdez, Redondo, Espinosa, Vargas, Cruz, Barrios, Seoane, Ramírez, Boué, Vigil, Machado, Nordelo and Piñeiro2004; Willadsen et al., Reference Willadsen, Bird, Cobon and Hungerford1995, Reference Willadsen, Riding, McKenna, Kemp, Tellam, Nielsen and Gough1989). Recently, the efficacy of Bm86 vaccines was also tested against R. australis (Trentelman et al., Reference Trentelman, Kleuskens, van de Crommert and Schetters2017, Reference Trentelman, Teunissen, Kleuskens, van de Crommert, de la Fuente, Hovius and Schetters2019). Furthermore, MF was used to test monoclonal antibodies targeting omHC31 and om21 against O. moubata (Matsuo et al., Reference Matsuo, Inoue, Ruheta, Taylor and Fujisaki2004, Reference Matsuo, Tsukamoto, Inoue and Fujisaki2003).
Key studies on the application of membrane feeding to assess the efficacy of drugs, vaccines and antisera against ticks and tick-borne pathogens
Table 7 Long description
The table evaluates the efficacy of various drugs, vaccines, and antisera against different tick species and life stages. Fluralaner consistently demonstrates anti-tick effects in O. moubata, R. sanguineus, and other species. DPP shows superior anti-tick feeding efficacy and blocks pathogen acquisition in I. ricinus and I. scapularis. Ivermectin and fipronil are compared, with fipronil being more effective in adult I. ricinus. Histamine causes tick detachment in R. microplus larvae, while feeding blood from vaccinated animals increases tick mortality in R. microplus adults. The effectiveness of monoclonal antibodies and antisera varies, with some combinations reducing larval feeding significantly. The table highlights the importance of life stage and species in determining treatment efficacy.
L, N, A, larva, nymph and adult stages of tick; mAb, monoclonal antibody; TPEs, tick protein extracts; SUB, Subolesin; DPP, dinotefuran, pyriproxyfen, and permethrin.
MF can also help to understand the mechanisms of vaccine-induced protection. For instance, calves immunised with homogenised tick extracts hosted fewer ticks, yet sera or blood from these animals failed to reproduce protective effects in vitro (Knorr et al., Reference Knorr, Anguita, Cortazar, Hajdusek, Kopáček, Trentelman, Kershaw, Hovius and Nijhof2018). This also highlights that the complex interactions of the intact host immune system, such as cellular responses, tissue-associated factors, and systemic immune interactions, are largely absent under in vitro conditions, underscoring that MF systems cannot fully replicate the complexity of in vivo immune responses.
Limitations
Unlike natural host feeding, where multiple behavioural and physiological cues, such as skin and hair odours and preferred attachment sites, are present, MF systems often achieve lower attachment and engorgement rates. Reproductive performance of hard ticks is frequently reduced, and protocols must be tailored to specific tick species and life stages, further limiting scalability.
Contamination by bacteria and fungi is also common and can result in failure to feed ticks to engorgement, necessitating strict hygiene and aseptic handling throughout feeding. Although antibiotics and antimycotics are frequently added to control microbial growth, their effects on the tick’s native microbiome and long-term colony maintenance, particularly in hard ticks, remain poorly understood. Indeed, to date, no studies have reported the successful use of ATFS for the sustained maintenance of hard tick colonies beyond a single generation. Finally, MF systems are labour-intensive, highlighting the need for further development of automated or semi-automated ATFS platforms to improve efficiency, reproducibility, and scalability (Böhme et al., Reference Böhme, Krull, Clausen and Nijhof2018; Guizzo et al., Reference Guizzo, Meneses, Amado Cecilio, Hessab Alvarenga, Sonenshine and Ribeiro2023; Khoo et al., Reference Khoo, Cull and Oliver2022; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021, Reference Militzer, Pinecki Socias and Nijhof2023).
Discussion
This review synthesises a growing body of literature to comprehensively evaluate and compare ATF techniques, with a focus on MF and CF systems across soft and hard tick species and their life stages and identifies promising adaptations to overcome the biological and technical challenges associated with in vitro tick feeding.
ATFS provide a powerful alternative to animal feeding and have been used under a range of experimental conditions, including evaluation of anti-tick vaccines and drugs, studies on tick-pathogen interactions and vector competence assessments (Bonnet and Liu, Reference Bonnet and Liu2012; Kröber and Guerin, Reference Kröber and Guerin2007b; Tajeri et al., Reference Tajeri, Razmi and Haghparast2016; Vimonish et al., Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020). MF more closely resembles natural skin feeding and is often preferred over CF due to its resemblance to in vivo conditions. It also improves safety by minimising tick escape, as ticks are confined within chambers and secured using stoppers (Sojka et al., Reference Sojka, Pytelková, Perner, Horn, Konvičková, Schrenková, Mareš and Kopáček2016; Trentelman et al., Reference Trentelman, Kleuskens, van de Crommert and Schetters2017).
Although ATFS have strong potential to support the 3Rs in animal research, initial optimisations can be technically challenging for particular tick species/stage and their performance varies substantially among tick groups. Soft ticks, which typically have shorter feeding durations and less complex pre-feeding behaviours than hard ticks, appear more amenable to MF. For example, Osborne and Mellor (Reference Osborne and Mellor1985) reported that O. moubata adults fed via membranes showed rapid engorgement and higher egg production than animal-fed ticks. Similarly, Schwan et al. (Reference Schwan, Hutton, Shields and Townson1991) found that O. moubata fed through parafilm on different blood sources showed no reduction in reproductive performance. Consequently, routine colony maintenance using MF currently seems more feasible for some soft tick species. In contrast, although multi-stage feeding and life-cycle completion have been demonstrated in certain hard ticks, sustained colony maintenance using ATFS alone has not yet been reliably achieved, owing to the biological and technical limitations discussed below.
A major challenge is the need for a large and continuous supply of ticks, particularly during extended optimisation trials, which may be challenging under some circumstances (Goodrich et al., Reference Goodrich, Murray and Holmes1978). In addition, ticks fed via MF often exhibit reduced attachment rates and longer feeding duration than those fed on animals (Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). These differences may be influenced by frequent blood changes, ineffective tactile and olfactory cues (e.g., animal hair and kairomones), and the mechanical properties of the membranes used.
Membrane thickness plays a critical role in feeding success. Thinner membranes improve attachment rates but are more prone to tearing, while thicker membranes offer structural integrity but reduce tick-feeding efficiency (Khoo et al., Reference Khoo, Cull and Oliver2022; Tajeri et al., Reference Tajeri, Razmi and Haghparast2016). The use of silicone membranes overlaid with goldbeater’s skin has proven effective in striking a balance between mechanical strength and flexibility (Krull et al., Reference Krull, Bohme, Clausen and Nijhof2017; Trentelman et al., Reference Trentelman, Kleuskens, van de Crommert and Schetters2017; Vimonish et al., Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020).
Species- and stage-specific differences in tick feeding behaviour require tailored approaches to feeding system design. I. scapularis larvae, for example, show optimal feeding success 2 weeks after hatching, while nymphs and adults perform better 10 weeks post-moult (Khoo et al., Reference Khoo, Cull and Oliver2022). Adult tick feeding is comparatively challenging as overcrowding of adult ticks (they tend to aggregate during feeding) can rupture membranes and reduce feeding success, whereas larvae and nymphs exhibit shorter feeding times and higher engorgement rates (Khoo et al., Reference Khoo, Cull and Oliver2022). A possible solution for adult tick feeding may be to reduce their numbers in a feeding chamber.
To improve feeding efficiency, researchers have used a variety of chemical and biological phagostimulants, such as animal hair, tick faeces, tick pheromones (synthetic), ATP (1–2 × 10−3 mol/L), and glutathione (10−3 mol/L) (Khoo et al., Reference Khoo, Cull and Oliver2022; Kuhnert et al., Reference Kuhnert, Diehl and Guerin1995; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1979). However, the effects of these compounds are highly species- and stage-dependent. Additionally, the use of raw animal hair in high-humidity conditions can promote fungal growth and tick mortality (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024).
Blood from a variety of animal species, including rabbit, cattle, pig, sheep, rat, and chicken, has been used for ATF (Barré et al., Reference Barré, Aprelon and Eugène1998; de Carvalho Ferreira et al., Reference de Carvalho Ferreira, Tudela Zuquete, Wijnveld, Weesendorp, Jongejan, Stegeman and Loeffen2014; González et al., Reference González, Valcarcel, Aguilar and Olmeda2017; Hart et al., Reference Hart, Yang, Pal and Lin2018; Liu et al., Reference Liu, Cote, Paul and Bonnet2014; Mango and Galun, Reference Mango and Galun1977; Migné et al., Reference Migné, Hönig, Bonnet, Palus, Rakotobe, Galon, Heckmann, Vyletova, Devillers and Attoui2022b; Schwan et al., Reference Schwan, Hutton, Shields and Townson1991). However, bovine blood remains the most commonly employed source, largely due to its ready availability in larger quantities from abattoirs. As a result, many studies rely on blood from non-natural hosts, which may influence tick feeding success and experimental outcomes. For experiments aimed at tick rearing or pathogen transmission, blood should be free of TBPs and acaricidal residues, as both can compromise tick fitness and confound transmission outcomes. For this reason, some studies have used sterile or screened blood to minimise contamination and experimental bias (Knorr et al., Reference Knorr, Anguita, Cortazar, Hajdusek, Kopáček, Trentelman, Kershaw, Hovius and Nijhof2018; Körner et al., Reference Körner, Makert, Mertens-Scholz, Henning, Pfeffer, Starke, Nijhof and Ulbert2020; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1995). Although whole blood is the most widely used feeding medium, other alternatives such as tissue culture medium (TCM-199) supplemented with glucose or serum have shown promise in certain species (Kemp et al., Reference Kemp, Koudstaal, Roberts and Kerr1975; Stone et al., Reference Stone, Commins and Kemp1983). More recently, packed bovine red blood cells (RBCs) have been used for artificial feeding of D. andersoni and R. appendiculatus (Vimonish et al., Reference Vimonish, Dinkel, Fry, Johnson, Capelli-Peixoto, Bastos, Scoles, Knowles, Madder, Chaka and Ueti2021, Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020). Defrosted blood extends the usability window of feeding media but may reduce engorgement success and increase the risk of contamination (Barré et al., Reference Barré, Aprelon and Eugène1998; González et al., Reference González, Valcarcel, Aguilar and Olmeda2017; Krull et al., Reference Krull, Bohme, Clausen and Nijhof2017).
Adding anticoagulants is necessary to prevent clotting during ATF, but the choice of anticoagulant can also influence feeding success. Heparinised blood has generally produced the most consistent results, followed by defibrinated blood (Barré et al., Reference Barré, Aprelon and Eugène1998; Waladde et al., Reference Waladde, Young, Mwaura, Njihia and Mwakima1979, Reference Waladde, Young, Mwaura, Njihia and Mwakima1995, Reference Waladde, Young, Ochieng, Mwaura and Mwakima1993). In contrast, citrate and ethylenediaminetetraacetic acid have been associated with reduced attachment, engorgement or survival rates, and are therefore not recommended for most ATF applications (Asri et al., Reference Asri, Tahir, Evans, Meyer, Rhalem, Bouslikhane, Ueti and Madder2023; Voigt et al., Reference Voigt, Young, Mwaura, Nyaga, Njihia, Mwakima and Morzaria1993; Waladde et al., Reference Waladde, Young, Ochieng, Mwaura and Mwakima1993).
Blood, being nutrient-rich, is susceptible to bacterial and fungal contamination, particularly under high-humidity conditions (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Krull et al., Reference Krull, Bohme, Clausen and Nijhof2017). To address this, several antibiotics and antimycotics are frequently added to the blood (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Khoo et al., Reference Khoo, Cull and Oliver2022). However, these agents can have unintended effects on the tick microbiome and may compromise tick health and experimental validity (Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017; Militzer et al., Reference Militzer, Pinecki Socias and Nijhof2023).
Several studies have shown that antibiotic exposure negatively affects tick fitness by altering microbiome composition and reducing the density of endosymbionts such as Rickettsia spp. in I. scapularis (Oliver et al., Reference Oliver, Price, Burkhardt, Heu, Khoo, Thorpe, Kurtti and Munderloh2021). These alterations are associated with reduced oviposition, poor larval viability, and impaired development (Duron et al., Reference Duron, Morel, Noel, Buysse, Binetruy, Lancelot, Loire, Menard, Bouchez, Vavre and Vial2018; Zhang et al., Reference Zhang, Li, Zhang, Qiu, Li, Li and Liu2017). In some cases, larvae produced from artificially fed adults failed to attach or feed in subsequent experiments, though they could still feed successfully on live hosts (Bilgiç et al., Reference Bilgiç, Hacilarlioğlu, Pekağirbaş, Karagenç, Eren and Bakirci2023; Tajeri et al., Reference Tajeri, Razmi and Haghparast2016). These discrepancies highlight the need for longitudinal, standardised studies to assess transgenerational impacts of ATFS.
Interestingly, vitamin B supplementation during artificial feeding was found to mitigate some of the adverse effects of antibiotics, partially restoring engorgement weight and fecundity (Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). This suggests that antibiotic-induced dysbiosis may impair vitamin biosynthesis pathways essential for tick physiology (Duron et al., Reference Duron, Morel, Noel, Buysse, Binetruy, Lancelot, Loire, Menard, Bouchez, Vavre and Vial2018). However, this hypothesis requires further investigation across different tick species and life stages.
Another important limitation of in vitro feeding systems is that certain host responses present during natural feeding may not be fully reproduced. At the attachment site, for example, tick saliva modulates vascular permeability, haemostasis, inflammation and extracellular matrix remodelling, processes that are difficult to replicate in ATF systems (Chmelar et al., Reference Chmelar, Calvo, Pedra, Francischetti and Kotsyfakis2012). Similarly, studying paralysis-inducing ticks such as I. holocyclus and D. andersoni is challenging because toxin activity depends on interactions at the host neuromuscular junction (Hall‐Mendelin et al., Reference Hall‐Mendelin, Craig, Hall, O’donoghue, Atwell, Tulsiani and Graham2011; Lysyk, Reference Lysyk2014). In such cases, however, ATF can still facilitate toxin collection, which may be difficult to achieve in vivo (Stone et al., Reference Stone, Commins and Kemp1983).
Host specificity is another constraint, as ATF cannot always reproduce natural host cues, potentially altering gene expression profiles. For example, I. ricinus fed through ATF showed reduced gene expression compared with animal-fed ticks. However, the same study demonstrated differential expression between blood- and serum-fed ticks, comparisons that are not feasible under in vivo conditions (Perner et al., Reference Perner, Provaznik, Schrenková, Urbanová, Ribeiro and Kopáček2016a). Similarly, animal-fed ticks expressed additional salivary gland proteins, including metalloproteases and anti-complement proteins, during early feeding compared with MF ticks fed on leukocyte-depleted blood (Perner et al., Reference Perner, Kropáčková, Kopáček and Ribeiro2018b), highlighting the importance of host immune components in establishing feeding. Finally, in the context of vaccine candidate discovery, it is important to recognise that ATFS do not fully capture the complexity of natural host immune responses. Critical processes such as local skin inflammation at the tick attachment site, cellular immune responses (including recruitment of neutrophils, macrophages and lymphocytes), cytokine signalling and complement-mediated effects are not adequately replicated under in vitro conditions. Consequently, ATFS cannot fully recapitulate the dynamic and multifaceted nature of in vivo host–tick interactions. This limitation should be carefully considered when interpreting findings and underscores the need to validate promising vaccine candidates in appropriate in vivo models. Although these findings indicate that ATF may under-represent certain biological processes, controlled MF studies can still identify key molecules and candidate vaccine targets when interpreted cautiously. Thus, ATF should be viewed as a complementary approach that, alongside animal feeding, can help to elucidate tick biology and guide vaccine discovery.
Despite significant advances in ATFS methodology, particularly over the past two decades, many tick species have not yet been successfully fed using these systems. Feeding success continues to vary depending on species, life stage, and age (Elati et al., Reference Elati, Benyedem, Fukatsu, Hoffmann-Köhler, Mhadhbi, Bakirci, Bilgiç, Karagenç, Darghouth and Nijhof2024; Khoo et al., Reference Khoo, Cull and Oliver2022; Militzer et al., Reference Militzer, Bartel, Clausen, Hoffmann-Köhler and Nijhof2021). Although ATFS aligns with the 3Rs principles (Replacement, Reduction, and Refinement) of animal use in research, its application is still limited by labour intensity, technical complexity, and high contamination risk (Vimonish et al., Reference Vimonish, Dinkel, Fry, Johnson, Capelli-Peixoto, Bastos, Scoles, Knowles, Madder, Chaka and Ueti2021, Reference Vimonish, Johnson, Mousel, Brayton, Scoles, Noh and Ueti2020). These issues are especially pronounced in long-term experiments or trans-stadial studies requiring consistent blood changes and stringent hygiene protocols.
Conclusion and future recommendations
Since its first description more than a century ago, ATFS have progressed substantially, particularly over the past two decades. ATFS now play an important role as tools in advancing research on tick biology and physiology, host–pathogen interactions, vector competence, and the discovery of anti-tick drugs and vaccine targets.
Despite these advances, several practical and biological challenges continue to limit the broader application of ATFS in research on TBDs. Based on evidence synthesised from more than 100 years of studies included in this review, we highlight the following priorities for future ATFS development:
(i) Broaden species coverage: Optimise ATFS protocols for a wider range of tick species and life stages of veterinary and medical importance.
(ii) Improve scalability and automation: Develop semi- or fully automated feeding systems to reduce labour intensity, improve reproducibility, and enable larger experimental throughput.
(iii) Address contamination risks: Improve system design, sterility protocols, and blood preparation methods to minimise bacterial and fungal contamination without disrupting the tick microbiome.
(iv) Develop improved feeding media: Explore synthetic or blood-free nutritional formulations that support feeding, development, and reproduction without compromising microbiome integrity or overall tick fitness.
(v) Enhance feeding success and colony sustainability: Design experiments to improve attachment, engorgement, reproductive performance, and long-term colony maintenance – especially for hard ticks.
(vi) Expand applications in drug and vaccine evaluation: Use ATFS alongside animal models to screen vaccine candidates, acaricides, and transmission-blocking interventions under controlled conditions.
As these challenges are addressed through methodological innovation and interdisciplinary collaboration, ATFS are likely to become an increasingly powerful and ethical tool for studying tick biology and controlling TBDs of medical and veterinary importance. Importantly, ATFS should be viewed not as a complete replacement for animal-based studies at present, but as a complementary platform that enables controlled experimentation, reduces animal use, and accelerates discovery in TBD research.
Acknowledgements
Bahar E. Mustafa is a PhD student at the University of Melbourne and is grateful to the University of Melbourne for providing him with a Melbourne Research Scholarship (MRS) for his PhD studies.
Author contributions
Conceptualisation: A.J., A.N., and A.G. Data curation: B.E.M., A.G., and G.A. Methodology: B.E.M., A.G., and G.A. Project administration: A.J., A.N., I.B., and A.G. Software: B.E.M. and A.G. Supervision: A.J., A.N., I.B., and A.G. Validation: A.J., A.N., and A.G. Writing – original draft: B.E.M. Writing – review and editing: V.D., G.A., C.G., S.A., A.C.-C., M.U., A.J., A.N., I.B., and A.G.
Funding statement
A.J. and A.N. are grateful to the Berlin University Alliance and the University of Melbourne for funding, which resulted in this review article.
Competing interests
The authors declare none.
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