Successful completion of the life cycle of Amblyomma variegatum using tick artificial membrane feeding system

Successful completion of the life cycle of Amblyomma variegatum using tick artificial membrane feeding system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Successful completion of the life cycle of Amblyomma variegatum using tick artificial membrane feeding system Naomie Pature, Mélanie Dhune, Rubikah Vimonish, Maxime Duhayon, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6867220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 4 You are reading this latest preprint version Abstract The three-host tick Amblyomma variegatum , commonly known as the tropical bont tick, poses a major threat to livestock health and productivity in tropical and subtropical regions worldwide. This tick is agressive, transmits multiple pathogens including Ehrlichia ruminantium, an intracellular obligate bacterium that causes heartwater, and directly damages the skin, and causes losses in productivity. The tropical bont tick, which belongs to the Ixodidae family, has long mouth parts and feeding behaviors are characterized by prolonged blood meals during each life stage. The inability to control the tick and prevent the diseases it transmits is partly due to the necessity of rearing the tick on animals. Thus, the goal of this study was to develop an artificial membrane feeding system to complete the life cycle of A. variegatum . All life stages of A. variegatum were fed using fresh goat blood at 38°C, and blood replacement occurred every 12 hours. Key parameters, such as humidity, temperature, and membrane thickness, were optimized to mimic natural tick feeding conditions. The attachment of ticks to the artificial membranes was induced by synthetic pheromones and host hairs. The attachment and engorgement rates for immature tick stages exceeded 80%, demonstrating high feeding success using the artificial system. The reproductive capacity of A. variegatum adult female ticks proved to be successful, with an oviposition rate of 35%. The larvae resulting from these eggs exhibited feeding patterns comparable to larvae derived from female ticks fed on goats. Collectively, these findings demonstrate the feasibility of using artificial feeding system to complete the breeding cycle of A. variegatum without the use of live hosts for tick engorgement. Consequently, this innovative approach will facilitate further research to close the knowledge gap, including understanding the tick-pathogen interactions and feeding of other tick species or hematophagous arthropods of human and veterinary importance. Biological sciences/Zoology/Entomology Biological sciences/Biological techniques/Biological models/Animal disease models hard ticks Amblyomma variegatum in vitro feeding system heartwater Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hematophagous arthropods such as ticks, mosquitoes, phlebotomine sandflies, and black flies, are of considerable interest due to their role as biological vectors for various diseases affecting humans and animals worldwide. These ectoparasites bite and take blood form large diversity of vertebrates(Cuthbert et al., 2023 ; Miyake et al., 2019 ). Of these, ticks are notable for their specialized role as obligate, ectoparasites, which have deleterious effects on animal health (Cupp, 1991 ). Unlike other hematophagous ectoparasites, ticks feed for long time periods, thus artificial feeding is challenging. According to Waladde and Rice ( 1982 ), the feeding phase of ticks consists of multiples steps, including host hunting, skin discovery and exploration, penetration and attachment, blood meal ingestion, and detachment (Waladde and Rice, 1982 ). After the on-host phase, engorged ticks undergo critical physiological processes, including ecdysis (molting) into nymphs and adults, and oviposition in the case of adult female ticks. Hard tick species living in open vegetative environments typically require three blood meals during their lifetime (Anderson, 2002 ; Sonenshine and Roe, 2013 ; Suzin et al., 2020 ). The tropical bont tick, Amblyomma variegatum (Fabricius, 1794), is the most widely distributed biological vector of heartwater in Africa (Bezuidenhout, 1987 ). The tick is also present in numerous islands in the Caribbean region. The introduction into the region is thought to have occurred through infested African zebu cattle from Senegal (Curasson, 1943 ). Amblyomma variegatum was the first African Amblyomma spp. to become established outside the African continent (Jongejan and Uilenberg, 2004 ). Climate changes has the potential to increase the abundance and survival of ticks in cooler and unaffected areas (Estrada-Pena et al., 2012 ; Ogden et al., 2004 ). For instance, the progressive increase in temperature documented in Zimbabwe in 2008 has been linked to the expansion of A. variegatum tick populations into areas that are climatically suitable for their survival (Estrada-Peña et al., 2008 ). In accordance with its spatial distribution, A. variegatum is known to vectorize Ehrlichia ruminantium , the causative agent of heartwater, and can transmit microorganisms responsible for theileriosis ( Theileria mutans, T. velifera ) and, dermatophilosis ( Dermatophilus congolensis ) (Barré, 1989 ). A. variegatum ’s biology is characterized by its three-host life cycle, and active hunting strategy. The life cycle of A. variegatum is long, necessitating a blood meal at each life stage on a living host. This phenomenon gives rise to a number of ethical and regulatory challenges for animal welfare when the tick needs to be reared in the laboratory. In accordance with the tropical environment pattern, the rearing of A. variegatum ticks necessitates climatic conditions favorable to their survival. A. variegatum has an elongated rostrum and similar to all Ixodidae, secretes cement, which serves to anchor the tick mouth parts to the host. This adaptation enables the tick to withstand host grooming (Waladde et al., 1996 ). Beyond the transmission of pathogens, ticks with long mouthparts, like A. variegatum , cause substantial skin damage, often predisposing to abscessation (Boulanger et al., 2019 ) (Ahoussou et al., 2010 ) In addition, heavy tick burdens reduce the growth and milk production of livestock (Stachurski et al., 1993 ). Throughout the years, the controlled infestation and reproduction of A. variegatum have proven to be arduous tasks. The feeding behavior of A. variegatum ticks appears to be variable and unpredictable. This is indicated by the specific attachment pattern of these ticks, which necessitates the presence of pioneer males to lead to high attachment rates. In the case of three-host ticks, larvae and nymphs feed on small mammals, while adults exhibit a host tropism associated with larger animals. This pattern differs from one-host ticks, which carry out their entire life cycle on the same host. From this host, the replete females detach to lay their eggs. The study of tick biology often requires the use of experimental animals to obtain laboratory-reared colonies of ticks. The development of artificial feeding methods allows for the reduction in the use of experimental animals (Burgdorfer, 1957 ). Laboratory feeding of ticks has been attempted using a variety of methods, including the use of capillary tubes (Chabaud, 1950 ), parafilm (Hokama et al., 1987 ), skin-derived matrix (Kemp et al., 1975 ), and silicone-based membranes (Kröber and Guerin, 2007a ; Nijhof and Tyson, 2018 ; Waladde et al., 1991 ) or even animal skin (Bonnet et al., 2007 ). There is extensive literature on the artificial feeding of soft ticks. However, hard ticks pose additional challenges due to their complex feeding behavior, which need to attach and feed for days to weeks to achieve full engorgement. The lack of research on artificial feeding of A. variegatum can be attributed to technical challenges in artificial engorgement in Longirostrata ticks, which possess larger mouthparts and require larger and more substantial blood meals. Consequently, artificial feeding methods must be optimized for each tick species. Additionally, there is limited information available regarding the artificial feeding of immature tick stages. In the present study, a previously described in vitro feeding system (Vimonish et al., 2020 ) was used to feed all the instars of A. variegatum ticks. Through a series of adjustments, including modifications to membrane thickness and environmental factors such as temperature, humidity, photoperiod, the tick attachment and engorgement rates of the ticks were improved. Thus, the lifecycle of A. variegatum was completed using the optimized in vitro feeding system, and each consecutive life stage of the tick fed successfully without the use of experimental animals. Materials and Methods Animals and Ethics statement Naïve creole goats, free of Ehrlichia ruminantium , in accordance with the experimental procedure of the project in animal experimentation approved by the ethics committee Antilles-Guyane (Tick Feeding Project Number #22432 2019100919443284 registered by Comité National de Réflexion Ethique sur l’Expérimentation Animale and authorized by Ministère de l’Enseignement Supérieur et de la Recherche) were used for tick feeding or as a source of fresh blood for in vitro feeding. Animals were confirmed negative for E. ruminantium using an indirect ELISA specific to detect anti- E. ruminantium antibodies and a Sol1-qPCR (Cangi et al., 2017). Ticks rearing The ticks used for this study came from female specimens collected on cattle in the Bellevue section field in Lamentin, Guadeloupe. The colony has been maintained since 13 years within the animal facility of CIRAD, located in the Duclos Domain in Guadeloupe. The adult ticks were fed on the flanks of the goats, while the immatures were fed on the sides of the ears in a Mölnlycke Tubifast® 2-Way Stretch® green line bandage, attached to the animal with with Kamar® adhesive (Vital Concept, Loudeac, France). Unfed and engorged ticks were maintained in acrylic humidity chambers containing a saturated solution of 9.43 mol of sodium carbonate for 500 ml of water, resulting in constant humidity > 90%. The ticks were kept in the animal facility at ambient temperature with a 12-hour dark/light cycle. Freshly molted ticks that were less than 4 months-old for adults, and less than 3 weeks-old for nymphs and larvae, were used for the artificial feeding experiments. In order to facilitate the acclimatation of the ticks to the laboratory environment, the ticks were stored in conical-bottomed polystyrene tubes (Corning-Gosselin, Borre, France) at room temperature with a relative humidity > 75% for several days prior to the experiment. Hypostome lengths measurement To measure the length of the hypostome, the distance between the base of the hypostome at the basis capitula and the apex of the hypostome was measured using a Leica M205FA fluorescence microscope equipped with Fluocombi (Leica, Wetzlar, Germany). This measurement excluded the palps. Initially, hypostome lengths were measured to adapt membrane thickness to each tick life stage. Later, the hypostome lengths of all the artificially-fed life stages of A. variegatum was measured to compare the mouthparts of the in vitro -fed ticks versus those of the naturally-fed ticks. To make this comparison, more than 10 ticks per life stages were measured. In vitro feeding system The artificial feeding system has been described by Vimonish et al in a previous study (Vimonish et al., 2020 ). In summary, the feeding chamber was composed of five principal components: a blood heating lid, a blood receptacle, a membrane frame, a connector and a tick receptacle (Fig. 1 A). A digital heating power source (Fig. 1 B) was connected to the heating lid in order to mimic the temperature of the host. In total, the digital device was capable of supporting four independent feeders simultaneously, which can be maintained at a constantly defined temperature (Fig. 1 B). Feeding membrane preparation and assembling The fabrication of artificial feeding membranes is a 2-day process that requires extended polymerization times. The silicone membranes were prepared as previously described (Vimonish et al., 2020 ), with the following modifications. First, stretch cling film was taped on a smooth surface, taking care to avoid bubbles and impurities and ensuring the plastic wrap was as smooth as possible. Subsequently, bovine intestinal Goldbeater’s skin (Talas, NY, USA) was employed as the matrix and affixed to the plastic wrap. A mixture was then prepared using Ecoflex Supersoft 00–50 components A and B (Creation Silicone, Plougasnou, France) in a 1:1 ratio. Next, a volume of hexane was added to the mixture, proportional to the life stage (600 µl for larvae and 800 µl for nymphs and adults) (Carlo Erba, Val de Reuil, France), with the aim of reducing the viscosity of the silicone. The components were blended on a piece of glass prior to the application of the mixture to the Goldbeater’s skin. A small silicone squeegee was used to ensure uniform distribution of the mixture across the Goldbeater’s skin. The thickness of the silicone membrane was determined through a series of trials, with the length of the tick mouthparts serving as the variable. This was done to ascertain the optimal thickness for feeding each life stage. The membranes were allowed to dry and polymerize overnight at room temperature. On the following day, the membranes were removed from the plastic wrap and allowed to dry evenly 24 hours. The thickness of the silicone membrane was determined using a digital caliper (Mitutoyo, USA). Membranes with a thickness of 700–750 µm, 310–350 µm and 150–200 µm were used for adult, nymph, and larval feeding, respectively. On the day of the experiment, the tackiness of the membranes was reduced, if desired, by uniform contact with the manipulator’s hands. Prior to utilization, all components of the in vitro system were autoclaved and sterilized through immersion in 70% ethanol for a period of 30 minutes. Once the components had been thoroughly dried, the silicone membranes were attached to the membrane frame using a mixture of Ecoflex 00–50 silicone components A and B (Fig. 1 A). Blood preparation and tick nutrients To artificially feed ticks in the system, 10 ml of E. ruminantium -free, fresh goat blood were collected daily in Vacutainer tubes (17 U.I/ml) (BD Medical, NJ, USA) from goats reared at the CIRAD animal facility. The blood was processed aseptically in a microbiological hood and either immediately used or stored at 4°C until the evening for the subsequent blood change. To enhance the attachment of ticks, the blood was supplemented with 0.010 mM ATP, which serves as a phagostimulant and 3 g/l glucose as an erythrocyte protectant. Prior to placement in the receptacle, the blood was warmed to 38°C in a preheated water bath, and no antibiotic or antifungal were added. A volume of 7 mL of blood per feeder was added to the blood receptacle of the feeding chamber. Artificial feeding Tick feeding was conducted at 26 ± 4°C, with a relative humidity exceeding 70% and a photoperiod day/night of 12h–12h. To prevent contamination, all procedures were conducted under a microbiological laminar hood. Following sterilization of the components, the silicone membranes were treated with pheromone extract to enhance attachment by A. variegatum ticks. Specifically, 100 µL of a synthetic mixture of attraction, attachment, and aggregation pheromones (AAA pheromone mixture) were added around the membrane, taking care not to place any in the center to prevent permeation between the silicone and the Goldbeater’s skin (Maranga et al., 2003 ; Norval et al., 1991 ; Schöni et al., 1984 ). The pheromone extract was allowed to evaporate for a minimum of 30 min, after which the membranes were tested for tightness by the addition of 5 mL of PBS 1X for 2 min. Next, the membrane frame with the silicone membrane was assembled with the blood receptacle, connector, and tick receptacle (Fig. 1 A). Goat hair cut on the same day were added on the membrane, and the ticks placed in the receptacle (Fig. 1 C). The number of ticks included in each assay was dependent on the instar. For instance, each feeder contained 5 adult males and 5 adult females, or 50 nymphs or 200–250 larvae. Next, a containment mesh was positioned within the tick receptacle to restrict the movement of the ticks and facilitate contact between the ticks and the silicone membrane (Fig. 1 A, 1 D). The feeders were then closed with paper and mesh, which were sealed with a rubber gasket to prevent the accidental escape of ticks (Fig. 1 A, 1 B). Blood was then added to the blood receptacle as described above and the feeders were installed on the digital heat source. Blood was replaced twice a day (every 12 hours ± 2 h) under a microbiological laminar hood. During each blood change, the blood receptacle was rinsed with sterile water, pre-heated 1x PBS (to remove blood cells and blood clots on the membrane), and 70% ethanol, refilled with pre-warmed goat blood, and the feeder was mounted back on the digital heat source. Monitoring of the feeding parameters During the artificial feeding studies, a number of parameters were assessed, including attachment and engorgement rates, presence of feces, mortality, and contamination. These parameters were recorded during the course of the blood change. The presence of tick feces indicates successful feeding on the artificial membrane. Given the absence of antibiotics or antifungals, it was essential to identify any contamination of the membrane, as this might compromise the survival and fitness of the feeding ticks. The number of dead ticks was recorded. They were removed from the tick receptacle and stored at -20°C to ensure their death. In the case of adult ticks, several additional criteria were considered, including the proportion of replete females, duration of feeding, pre-oviposition and oviposition periods, weight of eggs, and incubation period of eggs resulting in larvae. The proportion of fully engorged females was determined by their spontaneous detachment from the silicon membrane at the end of their blood meal. The length of tick feeding included the time between attachment with the cement cone and spontaneous detachment. For females, the pre-oviposition period included the time between spontaneous detachment from the membrane and initiation of egg laying. For nymphs and larvae, molting rate and duration were measured. Results Lengths of the A. variegatum hypostome All measurements of artificially- A. variegatum larvae, nymphs, adult males and females tick hypostome are presented in Table 1 . Table 1 Mean values for the hypostome length of A. variegatum ticks. Life stages Mean length of hypostome (µm) SD (µm) Larva 120 [110–130] 9 Nymph 361 [340–410] 32 Male 907 [860–970] 42 Female 1,050 [890–1,117] 137 Optimizing the artificial feeding of A. variegatum ticks A series of preliminary experiments were conducted to improve the efficiency of tick feeding, including membrane materials and thickness, frequency of blood changes, and hygrometry. Membrane thickness adjustment according to tick mouthparts . The first critical step in optimizing membrane-based artificial tick feeding was membrane preparation. The thickness and type of membranes played a key role in the success of artificial feeding. In our previous trials, 5 types of membranes were tested: tsetse flies’ membranes, Ecoflex 00–10 with a rigid mesh, Lens paper as a matrix, Ecoflex 00–10 alone, and Ecoflex 00–50. The utilization of tsetse flies’ membranes for adult ticks proved to be ineffective in promoting attachment. Furthermore, ticks failed to attached to membranes composed of Lens paper. Though A. variegatum attached to the silicone membrane, if the membrane was too thin, holes formed when tick hypostomes were withdrawn, resulting in perforation and leakage of the membrane. Blood temperature and frequency of blood change . The normal body temperature of a goat is 38°– 39° C. Initially, the blood was heated to 39°C in the blood receptacles which resulted in the blood “burning” upon contact with the air on the tick side. Upon exposure to the ambient air within the tick chamber, the blood exhibited a dark and hardened consistency. This resulted in blood leakage into the tick receptacle, which was associated with heavy coagulation and a putrid odor. To assess the effect of blood temperature and frequency of change on tick feeding, the same experiment was conducted with adjustments. The blood temperature, in the automated system, was set at 38°C, and the blood was refreshed twice a day. This, in conjunction with correct membrane thicknesses, led to an increased tick attachment rate with no blood leakage or contamination (Fig. 2 A, 4 A, 4 D). Environmental conditions . Maintaining the system at room temperature, rather than in an incubator, introduced additional challenges to controlling the temperature and relative humidity of the room. Initially, temperatures between 24–27°C allowed tick attachment. However, survival rates of unattached ticks declined when relative humidity was below 60%. In order to enhance the survival rate of ticks in connection with their attachment rate, we increased the humidity of the room by adding a humidifier. Subsequently, the relative humidity increased by 20% and was maintained at 80%. These optimizations, in association with adjustments to membrane thickness, blood temperature, and blood change frequency, permitted the attachment and subsequent engorgement of all instars of the tick (Fig. 2 A, 2 C, 4 A, 4 B, 4 D and 4 E). Table 2 Parameters monitored during the feeding of A. variegatum female tick in the artificial feeding system. Female tick (SD) Attachment rate (%) 100 [17/17] Feeding duration (days) 19.3 [15–24] ± 1.1 Detachment weight (mg) 550.5 [133–847] ± 249.7 Pre-oviposition (days) 19.2 [10–30] ± 9.3 Egg weight (mg) 115.7 [42–197] ± 68.4 Incubation period of eggs (days) 58 [56–60] ± 1.4 Tick behavior in the artificial feeding system Tick attachment rate . Tick behavior during artificial feeding was consistent with that observed in their natural habitat, including host-seeking behavior, skin exploration, attachment, and engorgement. Immediately after being placed in the tick receptacle, A. variegatum ticks, regardless of instar, started rapid crawling within the tick receptacle. The addition of chemical stimuli, including goat hair and the AAA pheromone mixture, along with physical stimuli such as temperature, led to the immediate interaction of all stages of the tick with the membrane. Indeed, they rapidly came into contact with the treated side of the silicone membrane. Shortly after, ticks started to explore the artificial membrane with their first pair of legs, in order to identify an optimal attachment site. The initial attachment of ticks was observed less than 12 hours after the onset of the experiment. The nymphs and adult males, clustered at the angles between the membranes and tick receptacle (Fig. 2 A, 4 D). In contrast, larvae attached to the silicone membrane in a non-aggregated manner (Fig. 4 A). On the 4th day, the remaining mobile ticks attached to the membrane at the same area for nymphs and adults. During this period, the tick apices of the hypostomes were observed on the blood side of the membrane, and the subsequent cement cones were visible around them (Fig. 2 B). For A. variegatum adult ticks, it was necessary to add the females to the feeding receptacle 3 to 5 days after the males. This delay ensured that the male ticks have time to attach and secrete sufficient pheromones to stimulate female tick attachment and aggregation. On the 7th day of blood feeding, the attachment rates for larvae, nymphs, and adult female ticks were 85.9%, 82.9%, and 100%, respectively (Tables 1 and 2 ). Males were not included in these rates, as they often detach from the membrane in order to copulate with multiple females. Tick engorgement success . The feeding process, which was initiated when the feces of ticks were present in the tick receptacle, started 24 h after the ticks were placed in the feeding chamber. Since there, the ticks began their slow-feeding phase, as evidenced by the progressive production of their new cuticle, which permits its expansion and a rounded-like shape (Fig. 2 C, 4 B, 4 E). We observed engorgement rates of 100% and 89.7% for larvae and nymphs, respectively. Completion of adult ticks mating . As described above, A. variegatum adult males are allowed to initiate feeding 3–5 days before the females to allow for the secretion of attraction pheromones and maturation of sperm. After 7 days of feeding, A. variegatum females increase in size and their cuticle becomes greenish. Several days later, the female’ cuticles enlarged and adopted an ebony-brown color (Fig. 2 C). At this time, the males and females could copulate several times with different partners on the membrane. We observed that the males tended to detach from the artificial membrane, crawled onto the feeding females, and position themselves venter to venter with females (Fig. 3 A). The males held the females firmly with their legs and brought their hypostomes close to the female gonopore (Fig. 3 B). In addition, in the special case where ticks were manually removed from the artificial membrane, males and females A. variegatum exhibited off-membrane mating behavior (Fig. 3 A). Tick detachment rate . For the nymphs, 52 nymphs with an average weight of 34.4 ± 16.5 mg were obtained after artificial membrane feeding for 17 ± 0.4 days (Table 3 ). After a mean feeding period of 19 ± 1.1 days, fully engorged adult females that had detached from the membranes were weighed. The median engorgement weight of fed females was 550.50 ± 249.65 mg (Table 1 ). During their pre-oviposition period, the detached females exhibited the same behavior as natural female ticks. Specifically, they started walking around the collection tube looking for the right place to lay their eggs. Table 3 Parameters monitored during the feeding of A. variegatum immature stages in the artificial feeding system. Larvae 1 Larvae 2 Nymphs Attachment rate (%) 44/45 [97.8] 85/99 [85.9] 58/70 [82.9] Engorgement rate (%) 43/44 [97.7] 85/85 [100.0] 52/58 [89.7] Feeding duration (days) 15,2 [12–19] ± 1.2 19,7 [12–21] ± 2.1 17,1 [13–20] ± 0.4 Detachment weight (mg) – – 34,4 [19–52] ± 16.5 Molting rate (%) 34/43 [79.1] 77/85 [90.7] 38/52 [73.1] Molting duration (days) 25 [24–26] ± 0.4 28 [21–35] ± 5.1 28 [21–35] ± 2.3 1 Larvae from a female that was artificially fed with the system. 2 Larvae from a female that was fed on a goat. Assessing the tick life cycle after the artificial feeding Egg laying and hatching . Following the ingestion of a blood meal and subsequent detachment, the average pre-oviposition period was determined to be 19.17 ± 9.3 days. During this period, the females displayed a questing behavior, navigating the tube and extending their first pair of legs in search of a suitable location for oviposition (unpublished data). To facilitate this process, a cotton ball was added into the tube. This period was crucial for ensuring that engorged females digest their blood meal and prepare their clutch. After a mean 19-day interval, 41% (7/17) of the engorged females began egg-laying (Fig. 3 C). During the process of oviposition, A. variegatum females exhibit a distinctive physical characteristic with an orange-brown alloscutum that becomes visibly crumpled (Fig. 3 C). Of the females that oviposited, 86% successfully completed the process. However, the eggs exhibited a low hatch rate (17%). Molting success of nymphs . The molting rate of fed larvae and nymphs to nymphs and adults, respectively, was considered as an indicator of tick feeding success. A high molting rate was observed for “semi-natural” larvae, with 77 out of 85 engorged ticks undergoing molting over a duration of 28 ± 5.1 days. For nymphs, 73% successfully molted to the adult stage within a mean duration of 28 ± 2.3 days, comparable to the larvae (Table 3 ). These findings collectively indicate the high feeding success for both larvae and nymphs. Artificial feeding of the offspring of artificially fed ticks compared to “semi-natural” larvae . For the larvae from engorged female on goat, considered as “semi-natural” larvae, they fed for 19 ± 2.1 days. Due to their low weight, the fed larvae could not be weighed. A high molting rate was observed for “semi-natural” larvae, with 77 out of 85 engorged ticks undergoing molting over a duration of 28 ± 5.1 days. A batch of 45 larvae derived from a single artificially fed female was successfully fed in vitro . The outcomes of the artificial feeding of the “artificial” larvae are presented in Table 2 . In summary, 97% of the larvae were successful in attaching, with all larvae engorging on the artificial membrane. From these artificially fed larvae, 100% engorged and detached, and 79% successfully molted into nymphs. Of these molted nymphs, 34 were recovered and fed again on our artificial feeding membrane system (Fig. 5 ). Discussion For several decades, artificial feeding systems have been developed for various tick species (Asri et al., 2023 ; Bilgiç et al., 2023 ; Elati et al., 2024 ; Garcia Guizzo et al., 2023 ; Krull et al., 2017 ). These apparatuses were utilized in laboratory settings to circumvent the use of experimental animals and to better understand tick biology, including feeding behavior or pathogen transmission. Furthermore, numerous artificial feeding methods for Amblyomma species have been explored, including capillary feeding and skin membranes (Abel et al., 2016 , 2008 ; Rechav et al., 1982 ; Voigt et al., 1993 ; Young et al., 1996 ). However, the utilization of in vitro feeding membranes systems for Amblyomma spp. ticks remains under-explored (Barré et al., 1998 ; Bullard et al., 2016 ; Kuhnert et al., 1995 ; Li et al., 2019 ; Moura et al., 1997 ; Rochlin et al., 2023 ). Since the seminal work of Voigt et al in 1993, there has been limited progress in the field, particularly concerning the feeding of the three-host tick A variegatum . Previous attempts to feed consecutive artificially-fed instars in the same way have not been successful, which has made artificial rearing impossible (Voigt et al., 1993 ). In this report, we present our achievement in successfully feeding all stages of A. variegatum ticks and their offspring using an artificial feeding system. Remarkably, a high attachment rate (97%) was observed in larvae derived from artificially-fed females. These larvae fed to repletion and detached normally, displaying physical characteristics similar to those of coming from a naturally fed female, and considered as “semi-natural”. However, molting success was higher in “semi-natural” larvae (90%) than in artificially reared ones (79%). Due to delayed feeding attempts on artificially reared nymphs (over seven months after molting), high mortality and low molting success in adults were recorded. These observations underscore the importance of tick fitness and timely feeding, as the pre-feeding phase is essential for successful engorgement - typically two months for nymphs and adults, and two weeks for larvae (Khoo et al., 2022 ; Tukahirwa, 1976 ). To date, studies on artificial feeding of consecutive stages remain scarce (Kuhnert et al., 1995 ; Militzer et al., 2023 , 2021 ). The use of artificial membranes is a major breakthrough in feeding hard ticks. The components of these membranes are commercially available, ready for immediate use, and easy to make. Baudruche membranes, also known as Goldbeater’s skin, are made from the serous layer of bovine intestine, from which soluble and natural compounds have been extracted and measured 20–40 µm (Waladde et al., 1996 , 1993 , 1991 ). These artificial membranes allow clear visualization of tick attachment and subsequent engorgement due to their transparency. Despite the fact that Goldbeater’s skin membranes are thinner than Lens paper and more suitable for immature stages, we were able to successfully feed A. variegatum adult ticks when they were used as a matrix. The silicone membranes used in this study were produced at various thickness to feed all life stages of A. variegatum ticks. This is particularly important because the three tick stages have different rostrum sizes. It was therefore necessary to adapt the thickness of the silicone membranes to the size of tick mouthparts. The most challenging membrane preparation processes were for larvae and adults because the former have short hypostomes and the latter have the largest. The length of the hypostome of larval offspring of artificially fed A. variegatum larvae is approximately 120 µm, which is larger than the hypostome length of Dermacentor reticulatus larvae (72.4 µm) (Krull et al., 2017 ) or Rhipicephalus microplus larvae (78.5 µm) (Estrada-Peña et al., 2012 ). Thus, membranes with a thickness of 150–200 µm obtained for A. variegatum larvae were sufficient. This thickness ensures that the membranes would be thin enough to allow the attachment of the larval hypostome, but strong enough to prevent blood leakage into the tick receptable. For A. variegatum nymphs, the length of their hypostome was about 359 µm, which is three times longer than the nymphs of Ixodes wioyliei (Ash et al., 2017 ) and R. annulatus (Abdel-Shafy and Namaky, 2013 ). The hypostome length of “artificial” A. variegatum adults was approximately 994 µm, male and female ticks included, which is longer than Hyalomma excavatum (429–465 µm) and H. marginatum (411–503 µm) adults (Bilgiç et al., 2023 ). The hypostome length reported by Kröber and Guerin in 2007 (1,100 µm) for adult A. variegatum ticks was consistent with our findings. Baudruche silicone membranes utilized in this study, resulted in attachment rates of 85.9% for “semi-natural” larvae (from a female fed on goat) and 97.8% for “artificial” larvae (from an artificially-fed female). These rates were slightly higher than those documented for A. hebraeum (Kuhnert et al., 1995 ). Further studies by Kuhnert ( 1996 ) reported comparable results for R. microplus larvae, with attachment rates exceeding 80%. The results reported here for nymph attachment rate are significantly higher than those reported by Kuhnert ( 1996 ) (39%) but comparable to the rates obtained by Barré et al, 1998 , who observed nymph attachment rates of approximately 87%. Kuhnert’s 1995 study on A. hebraeum also reported rates exceeding 90% where he highlighted the similar attachment rates between in vivo and in vitro -reared nymphs. In contrast, Tajeri et al. failed to observe both H. anatolicum and R. bursa nymphs feeding on silicone membranes (Tajeri et al., 2016 ). These observations suggest that, in absence of inappropriate stimuli and environmental conditions, silicone membranes are not necessarily appropriate to feed all tick species. As previously demonstrated by Kuhnert (1995), A. hebraeum female ticks exhibited an attachment rate of 46% one hour after their introduction to sexually mature male ticks. Later, other observations revealed an attachment rate of 67% for A. variegatum female ticks, recorded on the fourth day following the application of unfed female ticks with attached male ticks (Kuhnert, 1996 ). In the present study, A. variegatum females were applied after a 5-day period, which resulted in an attachment rate of 94% after 2 days of engorgement and 100% rate after 10 days. This rate of attachment was higher than the rates of attachment of I. ricinus female ticks (20–60%) when fed on artificial membranes (Fourie et al., 2019 ). Voigt et al . demonstrated that blood temperatures below 35°C and above 39°C did not result in A. variegatum ticks attaching to the artificial membrane (Voigt et al., 1993 ). These results are consistent with those obtained by Waladde (1991) and Asri et al ( 2023 ) on adult R. appendiculatus ticks. In our study, all the instars of A. variegatum ticks fed successfully on blood heated at 38°C. Moreover, blood changes were done every 12 hours. The repeated blood changes did not appear to affect tick feeding as previously reported by Kuhnert (1995). In contrast, Kozisek (2024) noted that premature detachment was frequently associated with the frequency of blood changes. In consequence, they observed that blood changes occurring every 24 hours were more favorable than those performed every 12 hours. However, at the onset of our experiments, the blood was changed daily, resulting in contamination by the third or fourth day. Addressing this limitation by adding a peristaltic pump into the artificial feeding system has been previously suggested (Vimonish et al., 2020 ). This device could reduce the interval of blood changes and simulate circulation, avoiding clotting and further contamination. The addition of phagostimulants such as ATP is essential for feeding, as these substances are recognized by specific cheliceral gustatory receptors (Waladde, 1977 ). Indeed, glucose stabilizes erythrocytes, while ATP serves as a general tick stimulant (Krober and Guerin, 2007). (Galun, 1967 ). Heparin was used as the anticoagulant. Several studies have indicated the heparin as a more suitable anticoagulant agent than defibrinated blood or ethylenediaminetetraacetic acid (EDTA) for the feeding of hard ticks (Young et al., 1996 ). In comparison with mosquitoes, where the maintenance of colonies is less difficult, triggering tick feeding behavior necessitates supplementary life-like stimuli (Romano et al., 2018 ). The sensitivity of ticks to semiochemicals varies depending on the species. In the case of Amblyomma spp., such as A. variegatum , the addition of AAA pheromones to the membrane is essential to stimulate tick attachment (Norval et al., 1991 ). These pheromones facilitate the localization of infested hosts and induce cluster-like behaviors at specific locations. Schöni et al demonstrated that the combination of o -nitrophenol, methyl salicylate, and nonanoic acid with diethyl ether induces orientation and dynamic aggregation associated with mounting and clasping behavior. As observed in nature, A. variegatum female ticks encounter difficulties in attaching to cattle if pioneer male ticks have not been attached for several days (Stachurski, 2000 ). As demonstrated in our experimental study, all life A. variegatum stages successfully attached to the artificial feeding membranes within 24 hours. The elasticity and thickness of the artificial membranes were conferred by the silicone layer in contact with the ticks. As shown by previous studies (Barré et al., 1998 ; Vimonish et al., 2020 ), silicone membranes are soft but strong, and adapted to the tick mouthparts. The softness of silicone membranes permits the withdrawal of the tick mouthparts by allowing for elastic retraction of the penetration sites and facilitating reattachment elsewhere (Kröber and Guerin, 2007b ). However, in cases where artificial membranes were not thick enough, blood leakage could occur rapidly in the feeder as we experienced in our first trials. This phenomenon could be explained by the fact that ticks exert considerable pressure on the membrane as they feed in clusters, which could increase the fragility of the membrane (Asri et al., 2023 ; Militzer et al., 2021 ; Yamasaki et al., 2024 ). A previous data analysis described the critical impact of temperature and relative humidity on the success of molting in both larval and nymphal stages (Yonow, 1995 ). The mean weight of detached ticks was recorded as 34.4 ± 16.5 mg for nymphs. As described in previous studies, tick molting success is closely related to a “critical weight” (Koch, 1986 ). Thus, ticks that have consumed a partially ingested blood meal may represent a significant proportion in the wild. The critical tick weight is defined as the transition from the slow to the rapid feeding phase where ticks reach 10 times their unfed weight (Harris and Kaufman, 1984 ). Ticks must reach this “critical weight” to obtain a sufficient blood meal to continue their developmental stages (oviposition and molting). The present study successfully obtained a high molting rate of 90% and 73% for “semi-natural” larvae and nymphs, respectively. Although Yonow ( 1995 ) pointed out that the mean engorgement periods of larvae and nymphs fed in vivo did not appear to be related to temperature and relative humidity, several recent studies have shown the opposite results. When humidity was reduced below 95%, I. scapularis nymphs failed to attach to silicone membranes (Kozisek et al., 2024 ). In this study, A. variegatum nymphs were successfully engorged at a rate of 89.7% and for a feeding duration of 17 ± 0.4 days. The ambient relative humidity in our study was maintained at approximately 75–80%, and the ticks were exposed to natural light. It is important to note that the relative humidity in the tick chamber was higher, as evidenced by the presence of condensation drops within the receptacle. However, it was not possible to measure this parameter within the feeder. Previous works on I. scapularis confirmed that relative humidity is critical for large tick attachment rates (Oliver et al., 2016 ). In that study, it also highlighted the importance of light exposure, which reproduces their circadian rhythm in nature. Feeding experiments were conducted at room temperature as in Barré (1998). A recent study showed that low ambient temperatures in the artificial experiment for A. tonelliae resulted in reduced host-seeking activity (Sebastian et al., 2023 ). In 1979, Doube and Kemp observed a high rate of R. microplus larvae attachment when the temperature of the environment and the skin membrane was identical (Doube and Kemp, 1979 ). Another important parameter to be consider to improve feeding tick in artificial conditions is the use of carbon dioxide (CO 2 ) to attract tick to the silicone membrane. In this study all experiments were performed with human natural CO 2 exhaled. Prior studies indicate that elevated CO 2 levels do not improve the attachment of A. variegatum ticks to synthetic membranes (Barré et al., 1998 ; Voigt et al., 1993 ). However, in some cases, high atmospheric CO2 concentrations enhance tick-seeking behavior in tick feeding experiments involving skin membranes or animals (Perritt et al., 1993 ). This phenomenon is particularly pronounced in the case of hunting ticks, such as Amblyomma spp. and Hyalomma spp, which can travel up to 21 meters to reach their host (Sauer et al., 1974 ). The engorgement rate for larvae from a naturally-fed female was found to be 100%, which aligns with the engorgement rate of 97.7% for the larvae from an artificially-fed female tick. This outcome is particularly noteworthy in light of the fact that artificial feeding methods have been observed to engorge larvae to a lesser extent, regardless of tick species (Elati et al., 2024 ; Garcia Guizzo et al., 2023 ; Kuhnert, 1996 ). The average duration of the feeding period for “semi-natural” larvae was 19 ± 2.1 days, while for artificially-fed larvae was 15 ± 1.2 days. The mean engorgement period recorded for A. variegatum female ticks was 19 ± 1.1 days, which is consistent with those reported in vivo in Africa (Yonow, 1995 ). In a previous study with H. luitanicum female ticks, a similar feeding period on sheep was observed (Cota Guajardo, 2015 ). In a subsequent artificial membrane feeding study, an engorgement rate of 40% was observed with H. lusitanicum female ticks (González et al., 2017 ). Later, Bohme et al. (2018) documented a low engorgement rate in D. reticulatus females when utilizing both semi-automated and conventional in vitro methods (27%) (Böhme et al., 2018 ). Conversely, when I. ricinus female ticks were fed using the same artificial methods, higher engorgement rates (80%) were observed. High engorgement rates may be explained by the fact that the quality of the blood meal is one of the most critical parameters for successful tick feeding, with tick feeding efficiency depending on this variable. Given that A. variegatum is an ectoparasite that infests ruminants, the type and the temperature of the blood selected for tick feeding are of significance. Although the blood donor source in previous studies did not affect the tick feeding weight, using the blood from the tick natural host remains the optimal choice for modeling the tick life cycle in the wild (Bonnet and Liu, 2012 ). Furthermore, the quantity and quality of blood ingested from a donor source have consequences on tick weights after molting (Koch and Hair, 1975 ). Moreover, it is important to note that the collection of blood from slaughterhouses does not guarantee aseptic conditions, which are essential for preventing blood contamination. To address the non-sterility of the blood collection process, all blood changes were carried out under a microbiological hood. This process ensures the limitation of bacterial growth from ambient relative humidity in the feeding chamber. In order to prevent bacterial and fungal contamination during tick feeding. The addition of antibiotics could be considered, however, given the well-documented deleterious effects of antibiotics on the tick microbiome and interactions with pathogens, their use was not an option in this study (González et al., 2021 ; Liu et al., 2014 ; Militzer et al., 2023 ; Sebastian et al., 2023 ). During the course of the feeding experiments, A. variegatum males demonstrated the capacity to copulate multiple times with several females. However, due to the inability to mark the males and thereby monitor their movement patterns, the number of copulations per male could not be determined. In a parallel study, Kröber and Guerin (2007) documented a similar copulating behavior in A. hebraeum adults on silicone membranes. As described by Feldman-Musham and Borut, the copulating behavior of Metastriate ticks, in which males introduce their chelicerae, without the palps, into the females genital pore, was also noted (Feldman-Muhsam and Borut, 1971 ). While it is challenging to induce Ixodid male ticks to engage in copulation, observations revealed the presence of a spermatophore attached near the female gonopore following transfer (Pature et al., 2025 ). The process of mating ensures that female ticks become capable of feeding to repletion and begin their vitellogenesis to produce eggs (Sanches et al., 2012 ). In the present study, the mean detachment weight recorded for A. variegatum females was 550.5 ± 249.7 mg. Given that female A. variegatum ticks can reach a weight of 3.5 g when fully fed, it is evident that in vitro feeding had an impact on oviposition. In accordance with prior findings, the lower engorgement weight, which has been shown to result in a decrease in egg production and oviposition, is consistent with artificial feeding yields being lower than natural feeding on animal hosts. We hypothesize that the low hatch rate (17%) for eggs was probably due to a less engorgement of the females and the presence of excessively low temperatures during the egg-laying phase (< 21°C). However, the incubation period of eggs is strongly dependent on temperature, as evidenced by Barré ( 1989 ). Specifically, the hatching of eggs was impacted by temperatures approximating 16°C. Yano’s observations indicated a tendency for a progressive decline in hatch-ratio as temperatures decreased (< 20°C) (Yano et al., 1987 ). He deduced that the incubation period was correlated with temperature variations. Furthermore, the viability of larvae that hatched from eggs subjected to low temperatures or desiccation was found to be reduced (Sutherst and Bourne, 2006 ). Moreover, studies have shown that the developmental stage of the tick egg influences their response to temperature (Ajayi et al., 2024 ). Consequently, eggs in their early stages of embryogenesis exhibit a high vulnerability to both warm and cold temperatures. The placement of incubated eggs must be optimized through meticulous management of the environmental conditions, particularly the temperature and relative humidity, within an incubator for example. Taken together, all of the above factors provide a suitable environment to mimic a natural host to facilitate voluntary tick attachment and further engorgement (Waladde and Ochieng, 1992 ). The use of artificial feeding systems confers the advantage of limiting the use of experimental animals with the aim of enhancing animal welfare. Furthermore, Butler’s (1984) research indicated a considerable reduction in the economic burden associated with maintaining live hosts in animal facilities (Butler et al., 1984 ). Through artificial feeding systems, the 3R Principle, which stands for “reduction, replacement and refinement” could be applied. These methods will help to minimize the use of experimental animals for research purpose (Balls, 2010 ; Russell and Burch, 1960 ). As demonstrated in the case of D. andersoni , R. appendiculatus , and Ixodes spp., the feeding system can be adapted for a broader range of ticks species through the optimization of membrane thicknesses and associated SOPs (Asri et al., 2023 ; Vimonish et al., 2020 ; Yamasaki et al., 2024 ). Further work can be conducted to study the development cycle of a given pathogen, such as E. ruminantium , the causative agent of heartwater, within its vector. This obligate intracellular bacterium is of particular interest given its widespread distribution in some geographic regions, including sub-Saharan Africa and some Caribbean islands (Barré et al., 1987 ; Camus et al., 1988 ; Camus and Barré, 1992 ; Molia et al., 2008 ). The utilization of an artificial feeding system will facilitate the investigation of vector efficiency or competency by infecting ticks with a controlled inoculum. This approach would facilitate the exploration of the effect of the pathogen dose and the minimal infection threshold required for further infection of the vector, thereby enabling the elucidation of the pathogen transmission patterns. In addition to the more profound comprehension of tick biology, the use of artificial feeding systems will further facilitate the elucidation of tick-pathogen interactions at molecular and transcriptomic scales. This will lead to a better understanding of tick and pathogen compartments during the course of the infection. Finally, to reduce the transmission of tick-borne diseases, the implementation of in vitro methods for vector control represents a promising approach. Specifically, evaluating various acaricides using artificial membranes under standardized laboratory conditions could be highly beneficial (Kröber and Guerin, 2007a ). Declarations Conflict of interest disclosure The authors declare that they have no financial conflicts of interest in relation to the content of the article. Funding This work is supported by the United States Department of Agriculture grant 58-3022-1-018-F (Risk of Arthropod-borne diseases in the Caribbean). Author Contribution For reading convenience, N.P. is referring to Naomie Pature and N.Pg. is referring to Nonito Pages; M.D. is referring to Mélanie Dhune and M.Dy. is referring to Maxime Duhayon.N.P. conceptualized the study, developed the methodology, conducted the investigation, curated the data, prepared the figures, and wrote the original draft. M.D., V.R. and M.Dy. contributed to the investigation. N.Pg. curated the data and contributed to editing the manuscript. M.U. supervised the study, curated the data, contributed to manuscript editing, and provided resources and funding. V.R. supervised the study, curated the data, contributed to manuscript editing, and provided resources and funding. D.F.M. conceptualized the study, curated the data, contributed to figure preparation, supervised the research, reviewed and edited the manuscript, secured resources and funding, and administered the project. All authors reviewed and approved the final manuscript. Acknowledgement The authors are grateful to Rosalie Aprelon for her assistance with artificial feeding and tick rearing. In addition, the authors would like to thank Jimmy Dédy and Loïc Jacquet-Crétides from the CIRAD animal facility for their dedicated contribution with the daily collection of blood tubes for this study. The authors acknowledge Dr Susan M. Noh for editorial input. References Abdel-Shafy, S. & Namaky, A. E. Scanning Electron Microscopy of Nymphal and Larval Stages of The Cattle Tick Rhipicephalus (Boophilus) annulatus (Say) 1821 (Acari: Ixodidae). Global Vet. 10 , 1–8 (2013). Abel, I. et al. Artificial feeding of Amblyomma cajennense (Acari: Ixodidae) fasting females through capillary tube technique. Rev. Bras. Parasitol. Vet. 17 , 128–132. https://doi.org/10.1590/S1984-29612008000300002 (2008). Abel, I. et al. do Artificial feeding of partially engorged Amblyomma sculptum females through capillaries. Brazilian Journal of Veterinary Medicine 38, 113–118. (2016). Ahoussou, S. et al. Analysis of Amblyomma surveillance data in the Caribbean: Lessons for future control programmes. Vet. Parasitol. 167 , 327–335. https://doi.org/10.1016/j.vetpar.2009.09.035 (2010). Ajayi, O. M. et al. Egg hatching success is influenced by the time of thermal stress in four hard tick species. J. Med. Entomol. 61 , 110–120. https://doi.org/10.1093/jme/tjad142 (2024). Anderson, J. F. The natural history of ticks. Med. Clin. North. Am. 86 , 205–218. https://doi.org/10.1016/s0025-7125(03)00083-x (2002). Ash, A. et al. Morphological and molecular description of Ixodes woyliei n. sp. (Ixodidae) with consideration for co-extinction with its critically endangered marsupial host. Parasites Vectors . 10 , 70. https://doi.org/10.1186/s13071-017-1997-8 (2017). Asri, B. et al. Assessment of an In Vitro Tick Feeding System for the Successful Feeding of Adult Rhipicephalus appendiculatus Ticks. Parasitologia 3 , 101–108. https://doi.org/10.3390/parasitologia3020012 (2023). Balls, M. The principles of humane experimental technique: timeless insights and unheeded warnings. ALTEX - Alternatives to animal experimentation 27, 144–148. (2010). https://doi.org/10.14573/altex.2010.2.144 Barré, N. Biologie et écologie de la tique Amblyomma variegatum (Acarina: Ixodina) en Guadeloupe (Antilles Françaises) (thesis). CIRAD-IEMVT. (1989). Barré, N., Aprelon, R. & Eugéne, M. Attempts to Feed Amblyomma variegatum Ticks on Artificial Membranes. Ann. N. Y. Acad. Sci. 849 , 384–390. https://doi.org/10.1111/j.1749-6632.1998.tb11077.x (1998). Barré, N., Uilenberg, G., Morel, P. C. & Camus, E. Danger of introducing heartwater onto the American mainland: potential role of indigenous and exotic Amblyomma ticks. Onderstepoort J. Vet. Res. 54 , 405–417 (1987). Bezuidenhout, J. D. Natural transmission of heartwater. Onderstepoort J. Vet. Res. 54 , 349–351 (1987). Bilgiç, H. B. et al. In vitro feeding of Hyalomma excavatum and Hyalomma marginatum tick species. Parasitol. Res. 122 , 1641–1649. https://doi.org/10.1007/s00436-023-07867-7 (2023). Böhme, B., Krull, C., Clausen, P. H. & Nijhof, A. M. Evaluation of a semi-automated in vitro feeding system for Dermacentor reticulatus and Ixodes ricinus adults. Parasitol. Res. 117 , 565–570. https://doi.org/10.1007/s00436-017-5648-y (2018). Bonnet, S. et al. Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique. Parasitology 134 , 197–207. https://doi.org/10.1017/S0031182006001545 (2007). Bonnet, S. & Liu, X. Y. Laboratory artificial infection of hard ticks: a tool for the analysis of tick-borne pathogen transmission. Acarologia 52 , 453–464. https://doi.org/10.1051/acarologia/20122068 (2012). Boulanger, N., Boyer, P., Talagrand-Reboul, E. & Hansmann, Y. Ticks and tick-borne diseases. Méd. Mal. Infect. 49 , 87–97. https://doi.org/10.1016/j.medmal.2019.01.007 (2019). Bullard, R. et al. Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum). Ticks Tick-borne Dis. 7 , 880–892. https://doi.org/10.1016/j.ttbdis.2016.04.006 (2016). Burgdorfer, W. Artificial Feeding of Ixodid Ticks for Studies on the Transmission of Disease Agents. J. Infect. Dis. 100 , 212–214 (1957). Butler, J., Hess, W., Endris, R. & Holscher, K. In vitro feeding of Ornithodoros ticks for rearing and assessment of disease transmission. (1984). Camus, E. & Barré, N. The role of Amblyomma variegatum in the transmission of heartwater with special reference to Guadeloupe. Ann. N Y Acad. Sci. 653 , 33–41. https://doi.org/10.1111/j.1749-6632.1992.tb19627.x (1992). Camus, E., Barré, N., Martinez, D. & Uilenberg, G. Heartwater (cowdriosis). A review (OIE, 1988). Chabaud, A. G. Sur la nutrition artificielle des tiques. Ann. Parasitol. Hum. Comp. 25 , 42–47. https://doi.org/10.1051/parasite/1950251042 (1950). Cota Guajardo, S. C. Control biológico e integrado de la garrapata Hyalomma lusitanicum en explotaciones silvo-agro-cinegéticas de ecosistema mesomediterráneo http://purl.org/dc/dcmitype/Text . Control biológico e integrado de la garrapata Hyalomma lusitanicum en explotaciones silvo-agro-cinegéticas de ecosistema mesomediterráneo. Universidad Complutense de Madrid. (2015). Cupp, E. W. Biology of Ticks. Veterinary Clinics of North America. Small Anim. Pract. 21 , 1–26. https://doi.org/10.1016/S0195-5616(91)50001-2 (1991). Curasson, G. Traité de protozoologie vétérinaire et comparée. (1943). Cuthbert, R. N. et al. Invasive hematophagous arthropods and associated diseases in a changing world. Parasites Vectors . 16 , 291. https://doi.org/10.1186/s13071-023-05887-x (2023). Doube, B. M. & Kemp, D. H. The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices. Int. J. Parasitol. 9 , 449–454. https://doi.org/10.1016/0020-7519(79)90048-1 (1979). Elati, K. et al. In vitro feeding of all life stages of two-host Hyalomma excavatum and Hyalomma scupense and three-host Hyalomma dromedarii ticks. Sci Rep 14, 444. (2024). https://doi.org/10.1038/s41598-023-51052-w Estrada-Pena, A., Ayllon, N. & De La Fuente, J. Impact of Climate Trends on Tick-Borne Pathogen Transmission. Front. Physiol. 3 https://doi.org/10.3389/fphys.2012.00064 (2012). Estrada-Peña, A., Horak, I. G. & Petney, T. Climate changes and suitability for the ticks Amblyomma hebraeum and Amblyomma variegatum (Ixodidae) in Zimbabwe (1974–1999). Vet. Parasitol. 151 , 256–267. https://doi.org/10.1016/j.vetpar.2007.11.014 (2008). Estrada-Peña, A. et al. Reinstatement of Rhipicephalus (Boophilus) australis (Acari: Ixodidae) With Redescription of the Adult and Larval Stages. J. Med. Entomol. 49 , 794–802. https://doi.org/10.1603/ME11223 (2012). Feldman-Muhsam, B. & Borut, S. Copulation in Ixodid Ticks. J. Parasitol. 57 , 630. https://doi.org/10.2307/3277930 (1971). Fourie, J. J. et al. Transmission of Anaplasma phagocytophilum (Foggie, 1949) by Ixodes ricinus (Linnaeus, 1758) ticks feeding on dogs and artificial membranes. Parasites Vectors . 12 , 136. https://doi.org/10.1186/s13071-019-3396-9 (2019). Galun, R. Feeding stimuli and artificial feeding. Bull. World Health Organ. 36 , 590–593 (1967). Garcia Guizzo, M. et al. Optimizing tick artificial membrane feeding for Ixodes scapularis. Sci. Rep. 13 , 16170. https://doi.org/10.1038/s41598-023-43200-z (2023). González, J., Bickerton, M. & Toledo, A. Applications of artificial membrane feeding for ixodid ticks. Acta Trop. 215 , 105818. https://doi.org/10.1016/j.actatropica.2020.105818 (2021). González, J., Valcárcel, F., Aguilar, A. & Olmeda, A. S. In vitro feeding of Hyalomma lusitanicum ticks on artificial membranes. Exp. Appl. Acarol . 72 , 449–459. https://doi.org/10.1007/s10493-017-0167-1 (2017). Harris, R. A. & Kaufman, W. R. Neural Involvement in the Control of Salivary Gland Degeneration in the Ixodid Tick Amblyomma hebraeum. J. Exp. Biol. 109 , 281–290. https://doi.org/10.1242/jeb.109.1.281 (1984). Hokama, Y., Lane, R. S. & Howarth, J. A. Maintenance of Adult and Nymphal Ornithodoros coriaceus (Acari: Argasidae) by Artificial Feeding Through a Parafilm Membrane. J. Med. Entomol. 24 , 319–323. https://doi.org/10.1093/jmedent/24.3.319 (1987). Jongejan, F. & Uilenberg, G. The global importance of ticks. Parasitology 129 , S3–S14. https://doi.org/10.1017/S0031182004005967 (2004). Kemp, D. H., Koudstaal, D., Roberts, J. A. & Kerr, J. D. Feeding of Boophilus microplus larvae on a partially defined medium through thin slices of cattle skin. Parasitology 70 , 243–254. https://doi.org/10.1017/s0031182000049702 (1975). Khoo, B., Cull, B. & Oliver, J. D. Tick Artificial Membrane Feeding for Ixodes scapularis. J. Vis. Exp. https://doi.org/10.3791/64553 (2022). Koch, H. G. Development of the Lone Star Tick, Amblyomma americanum (Acari: Ixodidae), from Immatures of Different Engorgement Weights. J. Kansas Entomol. Soc. 59 , 309–313 (1986). Koch, H. G. & Hair, J. A. The Effect of Host Species on the Engorgement, Molting Success, and Molted Weight of the Gulf Coast Tick, Amblyomma maculatum Koch (Acarina: Ixodidae). J. Med. Entomol. 12 , 213–219. https://doi.org/10.1093/jmedent/12.2.213 (1975). Kozisek, F. et al. An optimized artificial blood feeding assay to study tick cuticle biology. Insect Biochem. Mol. Biol. 168 , 104113. https://doi.org/10.1016/j.ibmb.2024.104113 (2024). Kröber, T. & Guerin, P. M. In vitro feeding assays for hard ticks. Trends Parasitol. 23 , 445–449. https://doi.org/10.1016/j.pt.2007.07.010 (2007a). Kröber, T. & Guerin, P. M. An in vitro feeding assay to test acaricides for control of hard ticks. Pest Manag. Sci. 63 , 17–22. https://doi.org/10.1002/ps.1293 (2007b). Krull, C., Böhme, B., Clausen, P. H. & Nijhof, A. M. Optimization of an artificial tick feeding assay for Dermacentor reticulatus. Parasit. Vectors . 10 , 60. https://doi.org/10.1186/s13071-017-2000-4 (2017). Kuhnert, F. Feeding of Hard Ticks In Vitro: New Perspectives for Rearing and for the Identification of Systemic Acaricides. ALTEX 13 , 76–87 (1996). Kuhnert, F., Diehl, P. A. & Guerin, P. M. The life-cycle of the bont tick Amblyomma hebraeum in vitro. Int. J. Parasitol. 25 , 887–896. https://doi.org/10.1016/0020-7519(95)00009-Q (1995). Li, Z., Macaluso, K. R., Foil, L. D. & Swale, D. R. Inward rectifier potassium (Kir) channels mediate salivary gland function and blood feeding in the lone star tick, Amblyomma americanum . PLoS Negl. Trop. Dis. 13 , e0007153. https://doi.org/10.1371/journal.pntd.0007153 (2019). Liu, X. Y., Cote, M., Paul, R. E. L. & Bonnet, S. I. Impact of feeding system and infection status of the blood meal on Ixodes ricinus feeding. Ticks Tick. Borne Dis. 5 , 323–328. https://doi.org/10.1016/j.ttbdis.2013.12.008 (2014). Maranga, R. O., Hassanali, A., Kaaya, G. P. & Mueke, J. M. Attraction of Amblyomma variegatum (ticks) to the attraction-aggregation-attachment-pheromone with or without carbon dioxide. Exp. Appl. Acarol . 29 , 121–130. https://doi.org/10.1023/a:1024265529030 (2003). Militzer, N., Bartel, A., Clausen, P. H., Hoffmann-Köhler, P. & Nijhof, A. M. Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus. Vaccines 9 , 385. https://doi.org/10.3390/vaccines9040385 (2021). Militzer, N., Socias, P., Nijhof, S. & A.M Changes in the Ixodes ricinus microbiome associated with artificial tick feeding. Front. Microbiol. 13 https://doi.org/10.3389/fmicb.2022.1050063 (2023). Miyake, T. et al. Bloodmeal host identification with inferences to feeding habits of a fish-fed mosquito, Aedes baisasi. Sci. Rep. 9 , 4002. https://doi.org/10.1038/s41598-019-40509-6 (2019). Molia, S. et al. Amblyomma variegatum in cattle in Marie Galante, French Antilles: Prevalence, control measures, and infection by Ehrlichia ruminantium. Vet. Parasitol. 153 , 338–346. https://doi.org/10.1016/j.vetpar.2008.01.046 (2008). de Moura, S. T., Fonseca, A. H., da, Fernandes, C. G. N. & Butler, J. F. Artificial Feeding of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) through Silicone Membrane. Mem. Inst. Oswaldo Cruz . 92 , 545–548. https://doi.org/10.1590/S0074-02761997000400019 (1997). Nijhof, A. M. & Tyson, K. R. In vitro Feeding Methods for Hematophagous Arthropods and Their Application in Drug Discovery, in: (eds Meng, C. Q. & Sluder, A. E.) Ectoparasites. Wiley, 187–204. https://doi.org/10.1002/9783527802883.ch9 (2018). Norval, R. A. I., Peter, T., Yunker, C. E., Sonenshine, D. E. & Burridge, M. J. Responses of the ticks Amblyomma hebraeum and A. variegatum to known or potential components of the aggregation-attachment pheromone. I. Long-range attraction. Exp. Appl. Acarol . 13 , 11–18 (1991). Ogden, N. H. et al. Investigation of Relationships Between Temperature and Developmental Rates of Tick Ixodes scapularis (Acari: Ixodidae) in the Laboratory and Field. J. Med. Entomol. 41 , 622–633. https://doi.org/10.1603/0022-2585-41.4.622 (2004). Oliver, J. D. et al. Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding. J. Med. Entomol. 53 , 409–415. https://doi.org/10.1093/jme/tjv241 (2016). Pature, N., Pages, N., Rodrigues, V. & Meyer, D. F. Dissection and Internal Anatomy of the Giant Tropical Bont Tick Amblyomma Variegatum. (2025). https://doi.org/10.2139/ssrn.5173455 Perritt, D. W., Couger, G. & Barker, R. W. Computer-controlled olfactometer system for studying behavioral responses of ticks to carbon dioxide. J. Med. Entomol. 30 , 571–578. https://doi.org/10.1093/jmedent/30.3.571 (1993). Rechav, Y., Norval, R. A. I. & Oliver, J. H. Interspecific Mating of Amblyomma Hebraeum and Amblyomma Variegatum (Acari: Ixodidae). J. Med. Entomol. 19 , 139–142. https://doi.org/10.1093/jmedent/19.2.139 (1982). Rochlin, I. et al. Optimization of artificial membrane feeding system for lone star ticks, Amblyomma americanum (Acari: Ixodidae), and experimental infection with Rickettsia amblyommatis (Rickettsiales: Rickettsiaceae). Journal of Medical Entomology tjad158. (2023). https://doi.org/10.1093/jme/tjad158 Romano, D., Stefanini, C., Canale, A. & Benelli, G. Artificial blood feeders for mosquitoes and ticks—Where from, where to? Acta Trop. 183 , 43–56. https://doi.org/10.1016/j.actatropica.2018.04.009 (2018). Russell, W. & Burch, R. The Principles of Humane Experimental Technique. Med. J. Aust. 1 , 500–500. https://doi.org/10.5694/j.1326-5377.1960.tb73127.x (1960). Sanches, G. S. et al. Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). J. Insect. Physiol. 58 , 1020–1027. https://doi.org/10.1016/j.jinsphys.2012.05.006 (2012). Sauer, J. R., Hair, J. A. & Houts, M. S. Chemo-Attraction in the Lone Star Tick (Acarina: Ixodidae). 2. Responses to Various Concentrations of Cco21. Ann. Entomol. Soc. Am. 67 , 150–152. https://doi.org/10.1093/aesa/67.1.150 (1974). Schöni, R., Hess, E., Blum, W. & Ramstein, K. The aggregation-attachment pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari, Ixodidae): Isolation, identification and action of its components. J. Insect. Physiol. 30 , 613–618. https://doi.org/10.1016/0022-1910(84)90045-3 (1984). Sebastian, P. S. et al. Preliminary Study on Artificial versus Animal-Based Feeding Systems for Amblyomma Ticks (Acari: Ixodidae). Microorganisms 11 , 1107. https://doi.org/10.3390/microorganisms11051107 (2023). Sonenshine, E. & Roe, D. E. (eds) byR.M., Biology of Ticks Volume 2, Second Edition, New to this Edition:, Second Edition, New to this Edition: ed. Oxford University Press, Oxford, New York. (2013). Stachurski, F. Invasion of West African cattle by the tick Amblyomma variegatum. Med. Vet. Entomol. 14 , 391–399. https://doi.org/10.1046/j.1365-2915.2000.00246.x (2000). Stachurski, F., Musonge, E. N., Achu-kwi, M. D. & Saliki, J. T. Impact of natural infestation of Amblyomma variegatum on the liveweight gain of male Gudali cattle in Adamawa (Cameroon). Vet. Parasitol. 49 , 299–311. https://doi.org/10.1016/0304-4017(93)90128-A (1993). Sutherst, R. W. & Bourne, A. S. The effect of desiccation and low temperature on the viability of eggs and emerging larvae of the tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Ixodidae). Int. J. Parasitol. 36 , 193–200. https://doi.org/10.1016/j.ijpara.2005.09.007 (2006). Suzin, A. et al. Free-living ticks (Acari: Ixodidae) in the Iguaçu National Park, Brazil: Temporal dynamics and questing behavior on vegetation. Ticks Tick-borne Dis. 11 , 101471. https://doi.org/10.1016/j.ttbdis.2020.101471 (2020). Tajeri, S., Razmi, G. & Haghparast, A. Establishment of an Artificial Tick Feeding System to Study Theileria lestoquardi Infection. PLOS ONE . 11 , e0169053. https://doi.org/10.1371/journal.pone.0169053 (2016). Tukahirwa, E. M. The feeding behaviour of larvae, nymphs and adults of Rhipicephalus appendiculatus. Parasitology 72 , 65–74. https://doi.org/10.1017/S0031182000058479 (1976). Vimonish, R. et al. Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro. Sci. Rep. 10 , 470. https://doi.org/10.1038/s41598-019-57390-y (2020). Voigt, W. P. et al. In vitro feeding of instars of the ixodid tick Amblyomma variegatum on skin membranes and its application to the transmission of Theileria mutans and Cowdria ruminantium. Parasitology 107 , 257–263. https://doi.org/10.1017/S0031182000079233 (1993). Waladde, S. M., THE SENSORY NERVOUS SYSTEM OF & THE ADULT CATTLE TICK BOOPHILUS MICROPLUS (CANESTRINI) IXODIDAE. PART I. LIGHT MICROSCOPY. Australian J. Entomol. 15 , 379–387. https://doi.org/10.1111/j.1440-6055.1976.tb01720.x (1977). Waladde, S. M. & Ochieng, S. A. Advances in the Artificial Feeding of Ticks. Int. J. Trop. Insect Sci. 13 , 579–583. https://doi.org/10.1017/S1742758400016167 (1992). Waladde, S. M., Ochieng’, S. A. & Gichuhi, P. M. Artificial-membrane feeding of the ixodid tick, Rhipicephalus appendiculatus, to repletion. Exp. Appl. Acarol . 11 , 297–306. https://doi.org/10.1007/BF01202876 (1991). Waladde, S. M. & Rice, M. J. The Sensory Basis of Tick Feeding Behaviour pp. 71–118 (Elsevier, 1982). https://doi.org/10.1016/B978-0-08-024937-7.50008-1 Waladde, S. M., Young, A. S. & Morzaria, S. P. Artificial feeding of ixodid ticks. Parasitol. Today . 12 , 272–278. https://doi.org/10.1016/0169-4758(96)10027-2 (1996). Waladde, S. M., Young, A. S., Ochieng’, S. A., Mwaura, S. N. & Mwakima, F. N. Transmission of Theileria parva to cattle by Rhipicephalus appendiculatus adults fed as nymphae in vitro on infected blood through an artificial membrane. Parasitology 107 , 249–256. https://doi.org/10.1017/S0031182000079221 (1993). Yamasaki, Y., Singh, P., Vimonish, R., Ueti, M. & Bankhead, T. Development and Application of an In Vitro Tick Feeding System to Identify Ixodes Tick Environment-Induced Genes of the Lyme Disease Agent, Borrelia burgdorferi. Pathogens 13 , 487. https://doi.org/10.3390/pathogens13060487 (2024). Yano, Y., Shiraishi, S. & Uchida, T. A. Effects of temperature on development and growth in the tick, Haemaphysalis longicornis . Exp. Appl. Acarol . 3 , 73–78. https://doi.org/10.1007/BF01200415 (1987). Yonow, T. The life-cycle of Amblyomma variegatum (Acari: Ixodidae): a literature synthesis with a view to modelling. Int. J. Parasitol. 25 , 1023–1060. https://doi.org/10.1016/0020-7519(95)00020-3 (1995). Young, A. S., Waladde, S. M. & Morzaria, S. P. Artificial Feeding Systems for Ixodid Ticks as a Tool for Study of Pathogen Transmission. Ann. N. Y. Acad. Sci. 791 , 211–218. https://doi.org/10.1111/j.1749-6632.1996.tb53527.x (1996). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Jun, 2025 Editor assigned by journal 14 Jun, 2025 Submission checks completed at journal 12 Jun, 2025 First submitted to journal 10 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6867220","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469518626,"identity":"27fb2179-edee-4e61-ada5-01eb6c163a80","order_by":0,"name":"Naomie Pature","email":"","orcid":"","institution":"CIRAD - French Agricultural Research Centre for International Development","correspondingAuthor":false,"prefix":"","firstName":"Naomie","middleName":"","lastName":"Pature","suffix":""},{"id":469518627,"identity":"790c7862-9364-4a8c-8c73-27a078a9026c","order_by":1,"name":"Mélanie Dhune","email":"","orcid":"","institution":"CIRAD - French Agricultural Research Centre for International Development","correspondingAuthor":false,"prefix":"","firstName":"Mélanie","middleName":"","lastName":"Dhune","suffix":""},{"id":469518628,"identity":"1d6bee30-a16d-4f4d-8cb4-e51a80c94a5e","order_by":2,"name":"Rubikah Vimonish","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Rubikah","middleName":"","lastName":"Vimonish","suffix":""},{"id":469518629,"identity":"7a2e8c46-5a58-427d-96b2-86172484d740","order_by":3,"name":"Maxime Duhayon","email":"","orcid":"","institution":"ASTRE, CIRAD, INRAE, Univ Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Maxime","middleName":"","lastName":"Duhayon","suffix":""},{"id":469518630,"identity":"8491b5ef-8438-48bc-b819-87e6978ad32a","order_by":4,"name":"Nonito Pages","email":"","orcid":"","institution":"CIRAD - French Agricultural Research Centre for International Development","correspondingAuthor":false,"prefix":"","firstName":"Nonito","middleName":"","lastName":"Pages","suffix":""},{"id":469518632,"identity":"13f3c8c9-4cad-44ec-a9d8-0029e7bbc909","order_by":5,"name":"Massaro W. Ueti","email":"","orcid":"","institution":"Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Massaro","middleName":"W.","lastName":"Ueti","suffix":""},{"id":469518633,"identity":"3e47bf9b-a3b4-4588-996f-eae0aac2e0e8","order_by":6,"name":"Valerie Rodrigues","email":"","orcid":"","institution":"CIRAD - French Agricultural Research Centre for International Development","correspondingAuthor":false,"prefix":"","firstName":"Valerie","middleName":"","lastName":"Rodrigues","suffix":""},{"id":469518634,"identity":"e48e4332-9908-4da0-84c2-3a11da597bc3","order_by":7,"name":"Damien F. MEYER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDCCA2AEBR8YJIBkApAmVgvjDIgWIE1ACxww84ApAlr4jp9OPPBxB0M0v0Tysc82NRb5/O0JjM0VeLRInsndcHDmGYbcmTPSkmfnHJOwnHHmAWPjGTxaDA7kbjjM28aQu+FGjjFzboOEAcONBPaHDfi0nH+74fBfoJb9N/I/M1sCtcjfSGBsxKvlBtAWRpAtEjnMzIxALQaEtEjeeLvhYG+bRO6MM8+MGXuOSRgYnnnYiFcL3/nczR9+ttnk9rcnP2b4UVNnIHc8+SBeLVAAjEGBBBiHkQgNYMB/gEiFo2AUjIJRMOIAAExXV/TsZlPYAAAAAElFTkSuQmCC","orcid":"","institution":"CIRAD - French Agricultural Research Centre for International Development","correspondingAuthor":true,"prefix":"","firstName":"Damien","middleName":"F.","lastName":"MEYER","suffix":""}],"badges":[],"createdAt":"2025-06-11 02:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6867220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6867220/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-23801-6","type":"published","date":"2025-11-17T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84435004,"identity":"e6ff3b15-0713-43ce-a42e-2ad265172cf2","added_by":"auto","created_at":"2025-06-12 02:05:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215121,"visible":true,"origin":"","legend":"\u003cp\u003eThe\u003cem\u003e in vitro \u003c/em\u003etick feeding system used for this study. \u003cstrong\u003e(A)\u003c/strong\u003e Diagram of all components of the \u003cstrong\u003e(a) \u003c/strong\u003eunassembled and \u003cstrong\u003e(b) \u003c/strong\u003eassembled feeder adapted for \u003cem\u003eA. variegatum\u003c/em\u003e tick feeding (adapted from Yamasaki \u003cem\u003eet al\u003c/em\u003e, 2024). \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eA. variegatum\u003c/em\u003emale and female adult ticks in the tick receptacle before placing the containment membrane. \u003cstrong\u003e(C)\u003c/strong\u003e Tick receptacle with adult ticks after the installation of the containment membrane. \u003cstrong\u003e(D) \u003c/strong\u003eThe entire feeding system with a digital heating power source with four tick feeding units.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/0f88dadbc8767e55ecaed644.jpeg"},{"id":84435005,"identity":"b9895245-8456-4b6d-8b83-325962e0a15b","added_by":"auto","created_at":"2025-06-12 02:05:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179598,"visible":true,"origin":"","legend":"\u003cp\u003eThe different stages of feeding experiments with \u003cem\u003eA. variegatum\u003c/em\u003e adults. \u003cstrong\u003e(A)\u003c/strong\u003e Male and female ticks attached to the artificial membrane. Tick excreta and goat hairs are visible. The males were fed for 7 days, while the females were fed for 2 days, because the males had to secrete pheromones first to stimulate the females’ fixation. \u003cstrong\u003e(B)\u003c/strong\u003eCement cones seen from the inside of the artificial membrane (indicated by yellow arrows). Membrane damages resembling to feeding lesions are visible around the cement cones. \u003cstrong\u003e(C)\u003c/strong\u003e Cluster of partially engorged \u003cem\u003eA. variegatum\u003c/em\u003e adult ticks after 15 days of feeding. Several copulating couples are observed (indicated by white arrows). \u003cstrong\u003e(D)\u003c/strong\u003e Engorged ticks on the silicon membrane after 21 days of feeding. Fully repleted females that detached from the artificial membrane, were removed from the tick receptacle.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/ea96d528f0c678999e0e0271.jpeg"},{"id":84434993,"identity":"0a91d487-da02-42d4-b805-22425ecf39ba","added_by":"auto","created_at":"2025-06-12 02:05:16","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118786,"visible":true,"origin":"","legend":"\u003cp\u003eThe complete reproduction cycle of adult ticks. \u003cstrong\u003e(A) \u003c/strong\u003eMicroscopic view of copulating \u003cem\u003eA. variegatum \u003c/em\u003eticks with the male inserting its hypostome near the female gonopore. The female’s hypostome was left in the membrane, sealed by the cement cone during removal. \u003cstrong\u003e(B) \u003c/strong\u003eMated female with male spermatophore (pink triangle) attached near gonopore (blue triangle), viewed under microscope. \u003cstrong\u003e(C) \u003c/strong\u003eArtificially fed female laying eggs.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/f3d4f61ee7781e7b58f1a059.jpeg"},{"id":84434998,"identity":"6ca87cef-8285-43b2-af67-4389a5ff4abe","added_by":"auto","created_at":"2025-06-12 02:05:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292057,"visible":true,"origin":"","legend":"\u003cp\u003eAttachment and engorgement of the immature stages of \u003cem\u003eA. variegatum\u003c/em\u003e ticks \u003cem\u003ein vitro\u003c/em\u003e. Larvae ticks attached to the artificial silicon membrane \u003cstrong\u003e(A)\u003c/strong\u003e 4 days after the onset of the engorgement, \u003cstrong\u003e(B)\u003c/strong\u003e 12 days after the onset of the engorgement and \u003cstrong\u003e(C)\u003c/strong\u003e17 days after the onset of the engorgement. Nymph ticks attached to the artificial silicon membrane \u003cstrong\u003e(D)\u003c/strong\u003e 4 days after the onset of the engorgement, \u003cstrong\u003e(E)\u003c/strong\u003e 12 days after the onset of the engorgement and \u003cstrong\u003e(F)\u003c/strong\u003e17 days after the onset of the engorgement.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/366c285112a20fdc23348fa3.jpeg"},{"id":84435600,"identity":"e5114da6-35ad-422d-a973-a0340c4d72d2","added_by":"auto","created_at":"2025-06-12 02:13:16","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172339,"visible":true,"origin":"","legend":"\u003cp\u003eThe complete life cycle of \u003cem\u003eA. variegatum \u003c/em\u003eticks under artificial conditions. The initial step involved engorgement of “semi-natural” adult ticks in the artificial feeding system until repletion and further oviposition of females. From these eggs, freshly molted “artificial” larvae were fed again with the system and left to molt. After ecdysis, newly molted “artificial” nymphs were permitted to feed on silicone membranes prior to constituting the new generation of artificial adult ticks (adapted from Kuhnert \u003cem\u003eet al\u003c/em\u003e, 1995) (created with Biorender).\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/82a709c09b7a9ec284909e0d.jpeg"},{"id":96650079,"identity":"214822da-4cd0-4c6c-85c3-415c3f9ecc83","added_by":"auto","created_at":"2025-11-24 16:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2087695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6867220/v1/73c27aee-961b-4afe-a8fd-723291efa029.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSuccessful completion of the life cycle of \u003cem\u003eAmblyomma variegatum\u003c/em\u003e using tick artificial membrane feeding system\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHematophagous arthropods such as ticks, mosquitoes, phlebotomine sandflies, and black flies, are of considerable interest due to their role as biological vectors for various diseases affecting humans and animals worldwide. These ectoparasites bite and take blood form large diversity of vertebrates(Cuthbert et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Miyake et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Of these, ticks are notable for their specialized role as obligate, ectoparasites, which have deleterious effects on animal health (Cupp, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Unlike other hematophagous ectoparasites, ticks feed for long time periods, thus artificial feeding is challenging. According to Waladde and Rice (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), the feeding phase of ticks consists of multiples steps, including host hunting, skin discovery and exploration, penetration and attachment, blood meal ingestion, and detachment (Waladde and Rice, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). After the on-host phase, engorged ticks undergo critical physiological processes, including ecdysis (molting) into nymphs and adults, and oviposition in the case of adult female ticks. Hard tick species living in open vegetative environments typically require three blood meals during their lifetime (Anderson, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sonenshine and Roe, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Suzin et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe tropical bont tick, \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (Fabricius, 1794), is the most widely distributed biological vector of heartwater in Africa (Bezuidenhout, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The tick is also present in numerous islands in the Caribbean region. The introduction into the region is thought to have occurred through infested African zebu cattle from Senegal (Curasson, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1943\u003c/span\u003e). \u003cem\u003eAmblyomma variegatum\u003c/em\u003e was the first African \u003cem\u003eAmblyomma\u003c/em\u003e spp. to become established outside the African continent (Jongejan and Uilenberg, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Climate changes has the potential to increase the abundance and survival of ticks in cooler and unaffected areas (Estrada-Pena et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ogden et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For instance, the progressive increase in temperature documented in Zimbabwe in 2008 has been linked to the expansion of \u003cem\u003eA. variegatum\u003c/em\u003e tick populations into areas that are climatically suitable for their survival (Estrada-Pe\u0026ntilde;a et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In accordance with its spatial distribution, \u003cem\u003eA. variegatum\u003c/em\u003e is known to vectorize \u003cem\u003eEhrlichia ruminantium\u003c/em\u003e, the causative agent of heartwater, and can transmit microorganisms responsible for theileriosis (\u003cem\u003eTheileria mutans, T. velifera\u003c/em\u003e) and, dermatophilosis (\u003cem\u003eDermatophilus congolensis\u003c/em\u003e) (Barr\u0026eacute;, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. variegatum\u003c/em\u003e\u0026rsquo;s biology is characterized by its three-host life cycle, and active hunting strategy. The life cycle of \u003cem\u003eA. variegatum\u003c/em\u003e is long, necessitating a blood meal at each life stage on a living host. This phenomenon gives rise to a number of ethical and regulatory challenges for animal welfare when the tick needs to be reared in the laboratory. In accordance with the tropical environment pattern, the rearing of \u003cem\u003eA. variegatum\u003c/em\u003e ticks necessitates climatic conditions favorable to their survival. \u003cem\u003eA. variegatum\u003c/em\u003e has an elongated rostrum and similar to all Ixodidae, secretes cement, which serves to anchor the tick mouth parts to the host. This adaptation enables the tick to withstand host grooming (Waladde et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Beyond the transmission of pathogens, ticks with long mouthparts, like \u003cem\u003eA. variegatum\u003c/em\u003e, cause substantial skin damage, often predisposing to abscessation (Boulanger et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Ahoussou et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) In addition, heavy tick burdens reduce the growth and milk production of livestock (Stachurski et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Throughout the years, the controlled infestation and reproduction of \u003cem\u003eA. variegatum\u003c/em\u003e have proven to be arduous tasks. The feeding behavior of \u003cem\u003eA. variegatum\u003c/em\u003e ticks appears to be variable and unpredictable. This is indicated by the specific attachment pattern of these ticks, which necessitates the presence of pioneer males to lead to high attachment rates. In the case of three-host ticks, larvae and nymphs feed on small mammals, while adults exhibit a host tropism associated with larger animals. This pattern differs from one-host ticks, which carry out their entire life cycle on the same host. From this host, the replete females detach to lay their eggs.\u003c/p\u003e \u003cp\u003eThe study of tick biology often requires the use of experimental animals to obtain laboratory-reared colonies of ticks. The development of artificial feeding methods allows for the reduction in the use of experimental animals (Burgdorfer, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). Laboratory feeding of ticks has been attempted using a variety of methods, including the use of capillary tubes (Chabaud, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1950\u003c/span\u003e), parafilm (Hokama et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), skin-derived matrix (Kemp et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), and silicone-based membranes (Kr\u0026ouml;ber and Guerin, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007a\u003c/span\u003e; Nijhof and Tyson, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Waladde et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) or even animal skin (Bonnet et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). There is extensive literature on the artificial feeding of soft ticks. However, hard ticks pose additional challenges due to their complex feeding behavior, which need to attach and feed for days to weeks to achieve full engorgement. The lack of research on artificial feeding of \u003cem\u003eA. variegatum\u003c/em\u003e can be attributed to technical challenges in artificial engorgement in Longirostrata ticks, which possess larger mouthparts and require larger and more substantial blood meals. Consequently, artificial feeding methods must be optimized for each tick species. Additionally, there is limited information available regarding the artificial feeding of immature tick stages.\u003c/p\u003e \u003cp\u003eIn the present study, a previously described \u003cem\u003ein vitro\u003c/em\u003e feeding system (Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was used to feed all the instars of \u003cem\u003eA. variegatum\u003c/em\u003e ticks. Through a series of adjustments, including modifications to membrane thickness and environmental factors such as temperature, humidity, photoperiod, the tick attachment and engorgement rates of the ticks were improved. Thus, the lifecycle of \u003cem\u003eA. variegatum\u003c/em\u003e was completed using the optimized \u003cem\u003ein vitro\u003c/em\u003e feeding system, and each consecutive life stage of the tick fed successfully without the use of experimental animals.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Ethics statement\u003c/h2\u003e \u003cp\u003eNa\u0026iuml;ve creole goats, free of \u003cem\u003eEhrlichia ruminantium\u003c/em\u003e, in accordance with the experimental procedure of the project in animal experimentation approved by the ethics committee Antilles-Guyane (Tick Feeding Project Number #22432 2019100919443284 registered by Comit\u0026eacute; National de R\u0026eacute;flexion Ethique sur l\u0026rsquo;Exp\u0026eacute;rimentation Animale and authorized by Minist\u0026egrave;re de l\u0026rsquo;Enseignement Sup\u0026eacute;rieur et de la Recherche) were used for tick feeding or as a source of fresh blood for \u003cem\u003ein vitro\u003c/em\u003e feeding. Animals were confirmed negative for \u003cem\u003eE. ruminantium\u003c/em\u003e using an indirect ELISA specific to detect anti- \u003cem\u003eE. ruminantium\u003c/em\u003e antibodies and a Sol1-qPCR (Cangi et al., 2017).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTicks rearing\u003c/h3\u003e\n\u003cp\u003eThe ticks used for this study came from female specimens collected on cattle in the Bellevue section field in Lamentin, Guadeloupe. The colony has been maintained since 13 years within the animal facility of CIRAD, located in the Duclos Domain in Guadeloupe. The adult ticks were fed on the flanks of the goats, while the immatures were fed on the sides of the ears in a M\u0026ouml;lnlycke Tubifast\u0026reg; 2-Way Stretch\u0026reg; green line bandage, attached to the animal with with Kamar\u0026reg; adhesive (Vital Concept, Loudeac, France). Unfed and engorged ticks were maintained in acrylic humidity chambers containing a saturated solution of 9.43 mol of sodium carbonate for 500 ml of water, resulting in constant humidity\u0026thinsp;\u0026gt;\u0026thinsp;90%. The ticks were kept in the animal facility at ambient temperature with a 12-hour dark/light cycle. Freshly molted ticks that were less than 4 months-old for adults, and less than 3 weeks-old for nymphs and larvae, were used for the artificial feeding experiments. In order to facilitate the acclimatation of the ticks to the laboratory environment, the ticks were stored in conical-bottomed polystyrene tubes (Corning-Gosselin, Borre, France) at room temperature with a relative humidity\u0026thinsp;\u0026gt;\u0026thinsp;75% for several days prior to the experiment.\u003c/p\u003e\n\u003ch3\u003eHypostome lengths measurement\u003c/h3\u003e\n\u003cp\u003eTo measure the length of the hypostome, the distance between the base of the hypostome at the basis capitula and the apex of the hypostome was measured using a Leica M205FA fluorescence microscope equipped with Fluocombi (Leica, Wetzlar, Germany). This measurement excluded the palps. Initially, hypostome lengths were measured to adapt membrane thickness to each tick life stage. Later, the hypostome lengths of all the artificially-fed life stages of \u003cem\u003eA. variegatum\u003c/em\u003e was measured to compare the mouthparts of the \u003cem\u003ein vitro\u003c/em\u003e-fed ticks versus those of the naturally-fed ticks. To make this comparison, more than 10 ticks per life stages were measured.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003efeeding system\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe artificial feeding system has been described by Vimonish \u003cem\u003eet al\u003c/em\u003e in a previous study (Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In summary, the feeding chamber was composed of five principal components: a blood heating lid, a blood receptacle, a membrane frame, a connector and a tick receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A digital heating power source (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) was connected to the heating lid in order to mimic the temperature of the host. In total, the digital device was capable of supporting four independent feeders simultaneously, which can be maintained at a constantly defined temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003eFeeding membrane preparation and assembling\u003c/h3\u003e\n\u003cp\u003eThe fabrication of artificial feeding membranes is a 2-day process that requires extended polymerization times. The silicone membranes were prepared as previously described (Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with the following modifications. First, stretch cling film was taped on a smooth surface, taking care to avoid bubbles and impurities and ensuring the plastic wrap was as smooth as possible. Subsequently, bovine intestinal Goldbeater\u0026rsquo;s skin (Talas, NY, USA) was employed as the matrix and affixed to the plastic wrap. A mixture was then prepared using Ecoflex Supersoft 00\u0026ndash;50 components A and B (Creation Silicone, Plougasnou, France) in a 1:1 ratio. Next, a volume of hexane was added to the mixture, proportional to the life stage (600 \u0026micro;l for larvae and 800 \u0026micro;l for nymphs and adults) (Carlo Erba, Val de Reuil, France), with the aim of reducing the viscosity of the silicone. The components were blended on a piece of glass prior to the application of the mixture to the Goldbeater\u0026rsquo;s skin. A small silicone squeegee was used to ensure uniform distribution of the mixture across the Goldbeater\u0026rsquo;s skin. The thickness of the silicone membrane was determined through a series of trials, with the length of the tick mouthparts serving as the variable. This was done to ascertain the optimal thickness for feeding each life stage. The membranes were allowed to dry and polymerize overnight at room temperature. On the following day, the membranes were removed from the plastic wrap and allowed to dry evenly 24 hours. The thickness of the silicone membrane was determined using a digital caliper (Mitutoyo, USA). Membranes with a thickness of 700\u0026ndash;750 \u0026micro;m, 310\u0026ndash;350 \u0026micro;m and 150\u0026ndash;200 \u0026micro;m were used for adult, nymph, and larval feeding, respectively. On the day of the experiment, the tackiness of the membranes was reduced, if desired, by uniform contact with the manipulator\u0026rsquo;s hands. Prior to utilization, all components of the \u003cem\u003ein vitro\u003c/em\u003e system were autoclaved and sterilized through immersion in 70% ethanol for a period of 30 minutes. Once the components had been thoroughly dried, the silicone membranes were attached to the membrane frame using a mixture of Ecoflex 00\u0026ndash;50 silicone components A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eBlood preparation and tick nutrients\u003c/h3\u003e\n\u003cp\u003eTo artificially feed ticks in the system, 10 ml of \u003cem\u003eE. ruminantium\u003c/em\u003e-free, fresh goat blood were collected daily in Vacutainer tubes (17 U.I/ml) (BD Medical, NJ, USA) from goats reared at the CIRAD animal facility. The blood was processed aseptically in a microbiological hood and either immediately used or stored at 4\u0026deg;C until the evening for the subsequent blood change. To enhance the attachment of ticks, the blood was supplemented with 0.010 mM ATP, which serves as a phagostimulant and 3 g/l glucose as an erythrocyte protectant. Prior to placement in the receptacle, the blood was warmed to 38\u0026deg;C in a preheated water bath, and no antibiotic or antifungal were added. A volume of 7 mL of blood per feeder was added to the blood receptacle of the feeding chamber.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eArtificial feeding\u003c/h2\u003e \u003cp\u003eTick feeding was conducted at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u0026deg;C, with a relative humidity exceeding 70% and a photoperiod day/night of 12h\u0026ndash;12h. To prevent contamination, all procedures were conducted under a microbiological laminar hood. Following sterilization of the components, the silicone membranes were treated with pheromone extract to enhance attachment by \u003cem\u003eA. variegatum\u003c/em\u003e ticks. Specifically, 100 \u0026micro;L of a synthetic mixture of attraction, attachment, and aggregation pheromones (AAA pheromone mixture) were added around the membrane, taking care not to place any in the center to prevent permeation between the silicone and the Goldbeater\u0026rsquo;s skin (Maranga et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Norval et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sch\u0026ouml;ni et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). The pheromone extract was allowed to evaporate for a minimum of 30 min, after which the membranes were tested for tightness by the addition of 5 mL of PBS 1X for 2 min. Next, the membrane frame with the silicone membrane was assembled with the blood receptacle, connector, and tick receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Goat hair cut on the same day were added on the membrane, and the ticks placed in the receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The number of ticks included in each assay was dependent on the instar. For instance, each feeder contained 5 adult males and 5 adult females, or 50 nymphs or 200\u0026ndash;250 larvae. Next, a containment mesh was positioned within the tick receptacle to restrict the movement of the ticks and facilitate contact between the ticks and the silicone membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The feeders were then closed with paper and mesh, which were sealed with a rubber gasket to prevent the accidental escape of ticks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Blood was then added to the blood receptacle as described above and the feeders were installed on the digital heat source. Blood was replaced twice a day (every 12 hours\u0026thinsp;\u0026plusmn;\u0026thinsp;2 h) under a microbiological laminar hood. During each blood change, the blood receptacle was rinsed with sterile water, pre-heated 1x PBS (to remove blood cells and blood clots on the membrane), and 70% ethanol, refilled with pre-warmed goat blood, and the feeder was mounted back on the digital heat source.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMonitoring of the feeding parameters\u003c/h3\u003e\n\u003cp\u003eDuring the artificial feeding studies, a number of parameters were assessed, including attachment and engorgement rates, presence of feces, mortality, and contamination. These parameters were recorded during the course of the blood change. The presence of tick feces indicates successful feeding on the artificial membrane. Given the absence of antibiotics or antifungals, it was essential to identify any contamination of the membrane, as this might compromise the survival and fitness of the feeding ticks. The number of dead ticks was recorded. They were removed from the tick receptacle and stored at -20\u0026deg;C to ensure their death. In the case of adult ticks, several additional criteria were considered, including the proportion of replete females, duration of feeding, pre-oviposition and oviposition periods, weight of eggs, and incubation period of eggs resulting in larvae. The proportion of fully engorged females was determined by their spontaneous detachment from the silicon membrane at the end of their blood meal. The length of tick feeding included the time between attachment with the cement cone and spontaneous detachment. For females, the pre-oviposition period included the time between spontaneous detachment from the membrane and initiation of egg laying. For nymphs and larvae, molting rate and duration were measured.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLengths of the\u003c/b\u003e \u003cb\u003eA. variegatum\u003c/b\u003e \u003cb\u003ehypostome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll measurements of artificially-\u003cem\u003eA. variegatum\u003c/em\u003e larvae, nymphs, adult males and females tick hypostome are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean values for the hypostome length of \u003cem\u003eA. variegatum\u003c/em\u003e ticks.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLife stages\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean length of hypostome (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120 [110\u0026ndash;130]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNymph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e361 [340\u0026ndash;410]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e907 [860\u0026ndash;970]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,050 [890\u0026ndash;1,117]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimizing the artificial feeding of\u003c/b\u003e \u003cb\u003eA. variegatum\u003c/b\u003e \u003cb\u003eticks\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA series of preliminary experiments were conducted to improve the efficiency of tick feeding, including membrane materials and thickness, frequency of blood changes, and hygrometry.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMembrane thickness adjustment according to tick mouthparts\u003c/em\u003e. The first critical step in optimizing membrane-based artificial tick feeding was membrane preparation. The thickness and type of membranes played a key role in the success of artificial feeding. In our previous trials, 5 types of membranes were tested: tsetse flies\u0026rsquo; membranes, Ecoflex 00\u0026ndash;10 with a rigid mesh, Lens paper as a matrix, Ecoflex 00\u0026ndash;10 alone, and Ecoflex 00\u0026ndash;50. The utilization of tsetse flies\u0026rsquo; membranes for adult ticks proved to be ineffective in promoting attachment. Furthermore, ticks failed to attached to membranes composed of Lens paper. Though \u003cem\u003eA. variegatum\u003c/em\u003e attached to the silicone membrane, if the membrane was too thin, holes formed when tick hypostomes were withdrawn, resulting in perforation and leakage of the membrane.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBlood temperature and frequency of blood change\u003c/em\u003e. The normal body temperature of a goat is 38\u0026deg;\u0026ndash; 39\u0026deg; C. Initially, the blood was heated to 39\u0026deg;C in the blood receptacles which resulted in the blood \u0026ldquo;burning\u0026rdquo; upon contact with the air on the tick side. Upon exposure to the ambient air within the tick chamber, the blood exhibited a dark and hardened consistency. This resulted in blood leakage into the tick receptacle, which was associated with heavy coagulation and a putrid odor. To assess the effect of blood temperature and frequency of change on tick feeding, the same experiment was conducted with adjustments. The blood temperature, in the automated system, was set at 38\u0026deg;C, and the blood was refreshed twice a day. This, in conjunction with correct membrane thicknesses, led to an increased tick attachment rate with no blood leakage or contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnvironmental conditions\u003c/em\u003e. Maintaining the system at room temperature, rather than in an incubator, introduced additional challenges to controlling the temperature and relative humidity of the room. Initially, temperatures between 24\u0026ndash;27\u0026deg;C allowed tick attachment. However, survival rates of unattached ticks declined when relative humidity was below 60%. In order to enhance the survival rate of ticks in connection with their attachment rate, we increased the humidity of the room by adding a humidifier. Subsequently, the relative humidity increased by 20% and was maintained at 80%. These optimizations, in association with adjustments to membrane thickness, blood temperature, and blood change frequency, permitted the attachment and subsequent engorgement of all instars of the tick (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters monitored during the feeding of \u003cem\u003eA. variegatum\u003c/em\u003e female tick in the artificial feeding system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale tick\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttachment rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e100 [17/17]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeeding duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e19.3 [15\u0026ndash;24]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetachment weight (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e550.5 [133\u0026ndash;847]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;249.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-oviposition (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e19.2 [10\u0026ndash;30]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgg weight (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e115.7 [42\u0026ndash;197]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;68.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncubation period of eggs (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e58 [56\u0026ndash;60]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTick behavior in the artificial feeding system\u003c/h2\u003e \u003cp\u003e \u003cem\u003eTick attachment rate\u003c/em\u003e. Tick behavior during artificial feeding was consistent with that observed in their natural habitat, including host-seeking behavior, skin exploration, attachment, and engorgement. Immediately after being placed in the tick receptacle, \u003cem\u003eA. variegatum\u003c/em\u003e ticks, regardless of instar, started rapid crawling within the tick receptacle. The addition of chemical stimuli, including goat hair and the AAA pheromone mixture, along with physical stimuli such as temperature, led to the immediate interaction of all stages of the tick with the membrane. Indeed, they rapidly came into contact with the treated side of the silicone membrane. Shortly after, ticks started to explore the artificial membrane with their first pair of legs, in order to identify an optimal attachment site. The initial attachment of ticks was observed less than 12 hours after the onset of the experiment. The nymphs and adult males, clustered at the angles between the membranes and tick receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, larvae attached to the silicone membrane in a non-aggregated manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). On the 4th day, the remaining mobile ticks attached to the membrane at the same area for nymphs and adults. During this period, the tick apices of the hypostomes were observed on the blood side of the membrane, and the subsequent cement cones were visible around them (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). For \u003cem\u003eA. variegatum\u003c/em\u003e adult ticks, it was necessary to add the females to the feeding receptacle 3 to 5 days after the males. This delay ensured that the male ticks have time to attach and secrete sufficient pheromones to stimulate female tick attachment and aggregation. On the 7th day of blood feeding, the attachment rates for larvae, nymphs, and adult female ticks were 85.9%, 82.9%, and 100%, respectively (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Males were not included in these rates, as they often detach from the membrane in order to copulate with multiple females.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTick engorgement success\u003c/em\u003e. The feeding process, which was initiated when the feces of ticks were present in the tick receptacle, started 24 h after the ticks were placed in the feeding chamber. Since there, the ticks began their slow-feeding phase, as evidenced by the progressive production of their new cuticle, which permits its expansion and a rounded-like shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We observed engorgement rates of 100% and 89.7% for larvae and nymphs, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCompletion of adult ticks mating\u003c/em\u003e. As described above, \u003cem\u003eA. variegatum\u003c/em\u003e adult males are allowed to initiate feeding 3\u0026ndash;5 days before the females to allow for the secretion of attraction pheromones and maturation of sperm. After 7 days of feeding, \u003cem\u003eA. variegatum\u003c/em\u003e females increase in size and their cuticle becomes greenish. Several days later, the female\u0026rsquo; cuticles enlarged and adopted an ebony-brown color (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At this time, the males and females could copulate several times with different partners on the membrane. We observed that the males tended to detach from the artificial membrane, crawled onto the feeding females, and position themselves venter to venter with females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The males held the females firmly with their legs and brought their hypostomes close to the female gonopore (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, in the special case where ticks were manually removed from the artificial membrane, males and females \u003cem\u003eA. variegatum\u003c/em\u003e exhibited off-membrane mating behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTick detachment rate\u003c/em\u003e. For the nymphs, 52 nymphs with an average weight of 34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5 mg were obtained after artificial membrane feeding for 17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 days (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter a mean feeding period of 19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 days, fully engorged adult females that had detached from the membranes were weighed. The median engorgement weight of fed females was 550.50\u0026thinsp;\u0026plusmn;\u0026thinsp;249.65 mg (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). During their pre-oviposition period, the detached females exhibited the same behavior as natural female ticks. Specifically, they started walking around the collection tube looking for the right place to lay their eggs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters monitored during the feeding of \u003cem\u003eA. variegatum\u003c/em\u003e immature stages in the artificial feeding system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarvae\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLarvae\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNymphs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttachment rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44/45 [97.8]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85/99 [85.9]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58/70 [82.9]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEngorgement rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43/44 [97.7]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85/85 [100.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52/58 [89.7]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeeding duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,2 [12\u0026ndash;19]\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19,7 [12\u0026ndash;21]\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,1 [13\u0026ndash;20]\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetachment weight (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34,4 [19\u0026ndash;52]\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolting rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34/43 [79.1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77/85 [90.7]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38/52 [73.1]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolting duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 [24\u0026ndash;26]\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 [21\u0026ndash;35]\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 [21\u0026ndash;35]\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eLarvae from a female that was artificially fed with the system. \u003csup\u003e2\u003c/sup\u003eLarvae from a female that was fed on a goat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessing the tick life cycle after the artificial feeding\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEgg laying and hatching\u003c/em\u003e. Following the ingestion of a blood meal and subsequent detachment, the average pre-oviposition period was determined to be 19.17\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 days. During this period, the females displayed a questing behavior, navigating the tube and extending their first pair of legs in search of a suitable location for oviposition (unpublished data). To facilitate this process, a cotton ball was added into the tube. This period was crucial for ensuring that engorged females digest their blood meal and prepare their clutch. After a mean 19-day interval, 41% (7/17) of the engorged females began egg-laying (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). During the process of oviposition, \u003cem\u003eA. variegatum\u003c/em\u003e females exhibit a distinctive physical characteristic with an orange-brown alloscutum that becomes visibly crumpled (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Of the females that oviposited, 86% successfully completed the process. However, the eggs exhibited a low hatch rate (17%).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMolting success of nymphs\u003c/em\u003e. The molting rate of fed larvae and nymphs to nymphs and adults, respectively, was considered as an indicator of tick feeding success. A high molting rate was observed for \u0026ldquo;semi-natural\u0026rdquo; larvae, with 77 out of 85 engorged ticks undergoing molting over a duration of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 days. For nymphs, 73% successfully molted to the adult stage within a mean duration of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 days, comparable to the larvae (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings collectively indicate the high feeding success for both larvae and nymphs.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArtificial feeding of the offspring of artificially fed ticks compared to \u0026ldquo;semi-natural\u0026rdquo; larvae\u003c/em\u003e. For the larvae from engorged female on goat, considered as \u0026ldquo;semi-natural\u0026rdquo; larvae, they fed for 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 days. Due to their low weight, the fed larvae could not be weighed. A high molting rate was observed for \u0026ldquo;semi-natural\u0026rdquo; larvae, with 77 out of 85 engorged ticks undergoing molting over a duration of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 days. A batch of 45 larvae derived from a single artificially fed female was successfully fed \u003cem\u003ein vitro\u003c/em\u003e. The outcomes of the artificial feeding of the \u0026ldquo;artificial\u0026rdquo; larvae are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In summary, 97% of the larvae were successful in attaching, with all larvae engorging on the artificial membrane. From these artificially fed larvae, 100% engorged and detached, and 79% successfully molted into nymphs. Of these molted nymphs, 34 were recovered and fed again on our artificial feeding membrane system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor several decades, artificial feeding systems have been developed for various tick species (Asri et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bilgi\u0026ccedil; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Elati et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Garcia Guizzo et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Krull et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These apparatuses were utilized in laboratory settings to circumvent the use of experimental animals and to better understand tick biology, including feeding behavior or pathogen transmission. Furthermore, numerous artificial feeding methods for \u003cem\u003eAmblyomma\u003c/em\u003e species have been explored, including capillary feeding and skin membranes (Abel et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rechav et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Voigt et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Young et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). However, the utilization of \u003cem\u003ein vitro\u003c/em\u003e feeding membranes systems for \u003cem\u003eAmblyomma\u003c/em\u003e spp. ticks remains under-explored (Barr\u0026eacute; et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Bullard et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kuhnert et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Moura et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Rochlin et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since the seminal work of Voigt \u003cem\u003eet al\u003c/em\u003e in 1993, there has been limited progress in the field, particularly concerning the feeding of the three-host tick \u003cem\u003eA variegatum\u003c/em\u003e. Previous attempts to feed consecutive artificially-fed instars in the same way have not been successful, which has made artificial rearing impossible (Voigt et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In this report, we present our achievement in successfully feeding all stages of \u003cem\u003eA. variegatum\u003c/em\u003e ticks and their offspring using an artificial feeding system. Remarkably, a high attachment rate (97%) was observed in larvae derived from artificially-fed females. These larvae fed to repletion and detached normally, displaying physical characteristics similar to those of coming from a naturally fed female, and considered as \u0026ldquo;semi-natural\u0026rdquo;. However, molting success was higher in \u0026ldquo;semi-natural\u0026rdquo; larvae (90%) than in artificially reared ones (79%). Due to delayed feeding attempts on artificially reared nymphs (over seven months after molting), high mortality and low molting success in adults were recorded. These observations underscore the importance of tick fitness and timely feeding, as the pre-feeding phase is essential for successful engorgement - typically two months for nymphs and adults, and two weeks for larvae (Khoo et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tukahirwa, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). To date, studies on artificial feeding of consecutive stages remain scarce (Kuhnert et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Militzer et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of artificial membranes is a major breakthrough in feeding hard ticks. The components of these membranes are commercially available, ready for immediate use, and easy to make. Baudruche membranes, also known as Goldbeater\u0026rsquo;s skin, are made from the serous layer of bovine intestine, from which soluble and natural compounds have been extracted and measured 20\u0026ndash;40 \u0026micro;m (Waladde et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). These artificial membranes allow clear visualization of tick attachment and subsequent engorgement due to their transparency. Despite the fact that Goldbeater\u0026rsquo;s skin membranes are thinner than Lens paper and more suitable for immature stages, we were able to successfully feed \u003cem\u003eA. variegatum\u003c/em\u003e adult ticks when they were used as a matrix. The silicone membranes used in this study were produced at various thickness to feed all life stages of \u003cem\u003eA. variegatum\u003c/em\u003e ticks. This is particularly important because the three tick stages have different rostrum sizes. It was therefore necessary to adapt the thickness of the silicone membranes to the size of tick mouthparts. The most challenging membrane preparation processes were for larvae and adults because the former have short hypostomes and the latter have the largest. The length of the hypostome of larval offspring of artificially fed \u003cem\u003eA. variegatum\u003c/em\u003e larvae is approximately 120 \u0026micro;m, which is larger than the hypostome length of \u003cem\u003eDermacentor reticulatus\u003c/em\u003e larvae (72.4 \u0026micro;m) (Krull et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) or \u003cem\u003eRhipicephalus microplus\u003c/em\u003e larvae (78.5 \u0026micro;m) (Estrada-Pe\u0026ntilde;a et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, membranes with a thickness of 150\u0026ndash;200 \u0026micro;m obtained for \u003cem\u003eA. variegatum\u003c/em\u003e larvae were sufficient. This thickness ensures that the membranes would be thin enough to allow the attachment of the larval hypostome, but strong enough to prevent blood leakage into the tick receptable. For \u003cem\u003eA. variegatum\u003c/em\u003e nymphs, the length of their hypostome was about 359 \u0026micro;m, which is three times longer than the nymphs of \u003cem\u003eIxodes wioyliei\u003c/em\u003e (Ash et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003eR. annulatus\u003c/em\u003e (Abdel-Shafy and Namaky, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The hypostome length of \u0026ldquo;artificial\u0026rdquo; \u003cem\u003eA. variegatum\u003c/em\u003e adults was approximately 994 \u0026micro;m, male and female ticks included, which is longer than \u003cem\u003eHyalomma excavatum\u003c/em\u003e (429\u0026ndash;465 \u0026micro;m) and \u003cem\u003eH. marginatum\u003c/em\u003e (411\u0026ndash;503 \u0026micro;m) adults (Bilgi\u0026ccedil; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The hypostome length reported by Kr\u0026ouml;ber and Guerin in 2007 (1,100 \u0026micro;m) for adult \u003cem\u003eA. variegatum\u003c/em\u003e ticks was consistent with our findings. Baudruche silicone membranes utilized in this study, resulted in attachment rates of 85.9% for \u0026ldquo;semi-natural\u0026rdquo; larvae (from a female fed on goat) and 97.8% for \u0026ldquo;artificial\u0026rdquo; larvae (from an artificially-fed female). These rates were slightly higher than those documented for \u003cem\u003eA. hebraeum\u003c/em\u003e (Kuhnert et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Further studies by Kuhnert (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) reported comparable results for \u003cem\u003eR. microplus\u003c/em\u003e larvae, with attachment rates exceeding 80%. The results reported here for nymph attachment rate are significantly higher than those reported by Kuhnert (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) (39%) but comparable to the rates obtained by Barr\u0026eacute; et al, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, who observed nymph attachment rates of approximately 87%. Kuhnert\u0026rsquo;s 1995 study on \u003cem\u003eA. hebraeum\u003c/em\u003e also reported rates exceeding 90% where he highlighted the similar attachment rates between \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e-reared nymphs. In contrast, Tajeri et al. failed to observe both \u003cem\u003eH. anatolicum\u003c/em\u003e and \u003cem\u003eR. bursa\u003c/em\u003e nymphs feeding on silicone membranes (Tajeri et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These observations suggest that, in absence of inappropriate stimuli and environmental conditions, silicone membranes are not necessarily appropriate to feed all tick species. As previously demonstrated by Kuhnert (1995), \u003cem\u003eA. hebraeum\u003c/em\u003e female ticks exhibited an attachment rate of 46% one hour after their introduction to sexually mature male ticks. Later, other observations revealed an attachment rate of 67% for \u003cem\u003eA. variegatum\u003c/em\u003e female ticks, recorded on the fourth day following the application of unfed female ticks with attached male ticks (Kuhnert, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). In the present study, \u003cem\u003eA. variegatum\u003c/em\u003e females were applied after a 5-day period, which resulted in an attachment rate of 94% after 2 days of engorgement and 100% rate after 10 days. This rate of attachment was higher than the rates of attachment of \u003cem\u003eI. ricinus\u003c/em\u003e female ticks (20\u0026ndash;60%) when fed on artificial membranes (Fourie et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVoigt \u003cem\u003eet al\u003c/em\u003e. demonstrated that blood temperatures below 35\u0026deg;C and above 39\u0026deg;C did not result in \u003cem\u003eA. variegatum\u003c/em\u003e ticks attaching to the artificial membrane (Voigt et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). These results are consistent with those obtained by Waladde (1991) and Asri et al (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) on adult \u003cem\u003eR. appendiculatus\u003c/em\u003e ticks. In our study, all the instars of \u003cem\u003eA. variegatum\u003c/em\u003e ticks fed successfully on blood heated at 38\u0026deg;C. Moreover, blood changes were done every 12 hours. The repeated blood changes did not appear to affect tick feeding as previously reported by Kuhnert (1995). In contrast, Kozisek (2024) noted that premature detachment was frequently associated with the frequency of blood changes. In consequence, they observed that blood changes occurring every 24 hours were more favorable than those performed every 12 hours. However, at the onset of our experiments, the blood was changed daily, resulting in contamination by the third or fourth day. Addressing this limitation by adding a peristaltic pump into the artificial feeding system has been previously suggested (Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This device could reduce the interval of blood changes and simulate circulation, avoiding clotting and further contamination. The addition of phagostimulants such as ATP is essential for feeding, as these substances are recognized by specific cheliceral gustatory receptors (Waladde, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Indeed, glucose stabilizes erythrocytes, while ATP serves as a general tick stimulant (Krober and Guerin, 2007). (Galun, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). Heparin was used as the anticoagulant. Several studies have indicated the heparin as a more suitable anticoagulant agent than defibrinated blood or ethylenediaminetetraacetic acid (EDTA) for the feeding of hard ticks (Young et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn comparison with mosquitoes, where the maintenance of colonies is less difficult, triggering tick feeding behavior necessitates supplementary life-like stimuli (Romano et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The sensitivity of ticks to semiochemicals varies depending on the species. In the case of \u003cem\u003eAmblyomma\u003c/em\u003e spp., such as \u003cem\u003eA. variegatum\u003c/em\u003e, the addition of AAA pheromones to the membrane is essential to stimulate tick attachment (Norval et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). These pheromones facilitate the localization of infested hosts and induce cluster-like behaviors at specific locations. Sch\u0026ouml;ni \u003cem\u003eet al\u003c/em\u003e demonstrated that the combination of \u003cem\u003eo\u003c/em\u003e-nitrophenol, methyl salicylate, and nonanoic acid with diethyl ether induces orientation and dynamic aggregation associated with mounting and clasping behavior. As observed in nature, \u003cem\u003eA. variegatum\u003c/em\u003e female ticks encounter difficulties in attaching to cattle if pioneer male ticks have not been attached for several days (Stachurski, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). As demonstrated in our experimental study, all life \u003cem\u003eA. variegatum\u003c/em\u003e stages successfully attached to the artificial feeding membranes within 24 hours. The elasticity and thickness of the artificial membranes were conferred by the silicone layer in contact with the ticks. As shown by previous studies (Barr\u0026eacute; et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), silicone membranes are soft but strong, and adapted to the tick mouthparts. The softness of silicone membranes permits the withdrawal of the tick mouthparts by allowing for elastic retraction of the penetration sites and facilitating reattachment elsewhere (Kr\u0026ouml;ber and Guerin, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e). However, in cases where artificial membranes were not thick enough, blood leakage could occur rapidly in the feeder as we experienced in our first trials. This phenomenon could be explained by the fact that ticks exert considerable pressure on the membrane as they feed in clusters, which could increase the fragility of the membrane (Asri et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Militzer et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yamasaki et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA previous data analysis described the critical impact of temperature and relative humidity on the success of molting in both larval and nymphal stages (Yonow, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The mean weight of detached ticks was recorded as 34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5 mg for nymphs. As described in previous studies, tick molting success is closely related to a \u0026ldquo;critical weight\u0026rdquo; (Koch, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Thus, ticks that have consumed a partially ingested blood meal may represent a significant proportion in the wild. The critical tick weight is defined as the transition from the slow to the rapid feeding phase where ticks reach 10 times their unfed weight (Harris and Kaufman, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Ticks must reach this \u0026ldquo;critical weight\u0026rdquo; to obtain a sufficient blood meal to continue their developmental stages (oviposition and molting). The present study successfully obtained a high molting rate of 90% and 73% for \u0026ldquo;semi-natural\u0026rdquo; larvae and nymphs, respectively. Although Yonow (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) pointed out that the mean engorgement periods of larvae and nymphs fed \u003cem\u003ein vivo\u003c/em\u003e did not appear to be related to temperature and relative humidity, several recent studies have shown the opposite results. When humidity was reduced below 95%, \u003cem\u003eI. scapularis\u003c/em\u003e nymphs failed to attach to silicone membranes (Kozisek et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, \u003cem\u003eA. variegatum\u003c/em\u003e nymphs were successfully engorged at a rate of 89.7% and for a feeding duration of 17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 days. The ambient relative humidity in our study was maintained at approximately 75\u0026ndash;80%, and the ticks were exposed to natural light. It is important to note that the relative humidity in the tick chamber was higher, as evidenced by the presence of condensation drops within the receptacle. However, it was not possible to measure this parameter within the feeder. Previous works on \u003cem\u003eI. scapularis\u003c/em\u003e confirmed that relative humidity is critical for large tick attachment rates (Oliver et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In that study, it also highlighted the importance of light exposure, which reproduces their circadian rhythm in nature. Feeding experiments were conducted at room temperature as in Barr\u0026eacute; (1998). A recent study showed that low ambient temperatures in the artificial experiment for \u003cem\u003eA. tonelliae\u003c/em\u003e resulted in reduced host-seeking activity (Sebastian et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In 1979, Doube and Kemp observed a high rate of \u003cem\u003eR. microplus\u003c/em\u003e larvae attachment when the temperature of the environment and the skin membrane was identical (Doube and Kemp, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Another important parameter to be consider to improve feeding tick in artificial conditions is the use of carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) to attract tick to the silicone membrane. In this study all experiments were performed with human natural CO\u003csub\u003e2\u003c/sub\u003e exhaled. Prior studies indicate that elevated CO\u003csub\u003e2\u003c/sub\u003e levels do not improve the attachment of \u003cem\u003eA. variegatum\u003c/em\u003e ticks to synthetic membranes (Barr\u0026eacute; et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Voigt et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, in some cases, high atmospheric CO2 concentrations enhance tick-seeking behavior in tick feeding experiments involving skin membranes or animals (Perritt et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). This phenomenon is particularly pronounced in the case of hunting ticks, such as \u003cem\u003eAmblyomma\u003c/em\u003e spp. and \u003cem\u003eHyalomma\u003c/em\u003e spp, which can travel up to 21 meters to reach their host (Sauer et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1974\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe engorgement rate for larvae from a naturally-fed female was found to be 100%, which aligns with the engorgement rate of 97.7% for the larvae from an artificially-fed female tick. This outcome is particularly noteworthy in light of the fact that artificial feeding methods have been observed to engorge larvae to a lesser extent, regardless of tick species (Elati et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Garcia Guizzo et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kuhnert, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The average duration of the feeding period for \u0026ldquo;semi-natural\u0026rdquo; larvae was 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 days, while for artificially-fed larvae was 15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 days. The mean engorgement period recorded for \u003cem\u003eA. variegatum\u003c/em\u003e female ticks was 19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 days, which is consistent with those reported \u003cem\u003ein vivo\u003c/em\u003e in Africa (Yonow, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In a previous study with \u003cem\u003eH. luitanicum\u003c/em\u003e female ticks, a similar feeding period on sheep was observed (Cota Guajardo, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In a subsequent artificial membrane feeding study, an engorgement rate of 40% was observed with \u003cem\u003eH. lusitanicum\u003c/em\u003e female ticks (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Later, Bohme et al. (2018) documented a low engorgement rate in \u003cem\u003eD. reticulatus\u003c/em\u003e females when utilizing both semi-automated and conventional \u003cem\u003ein vitro\u003c/em\u003e methods (27%) (B\u0026ouml;hme et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Conversely, when \u003cem\u003eI. ricinus\u003c/em\u003e female ticks were fed using the same artificial methods, higher engorgement rates (80%) were observed. High engorgement rates may be explained by the fact that the quality of the blood meal is one of the most critical parameters for successful tick feeding, with tick feeding efficiency depending on this variable. Given that \u003cem\u003eA. variegatum\u003c/em\u003e is an ectoparasite that infests ruminants, the type and the temperature of the blood selected for tick feeding are of significance. Although the blood donor source in previous studies did not affect the tick feeding weight, using the blood from the tick natural host remains the optimal choice for modeling the tick life cycle in the wild (Bonnet and Liu, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, the quantity and quality of blood ingested from a donor source have consequences on tick weights after molting (Koch and Hair, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). Moreover, it is important to note that the collection of blood from slaughterhouses does not guarantee aseptic conditions, which are essential for preventing blood contamination. To address the non-sterility of the blood collection process, all blood changes were carried out under a microbiological hood. This process ensures the limitation of bacterial growth from ambient relative humidity in the feeding chamber. In order to prevent bacterial and fungal contamination during tick feeding. The addition of antibiotics could be considered, however, given the well-documented deleterious effects of antibiotics on the tick microbiome and interactions with pathogens, their use was not an option in this study (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Militzer et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sebastian et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the course of the feeding experiments, \u003cem\u003eA. variegatum\u003c/em\u003e males demonstrated the capacity to copulate multiple times with several females. However, due to the inability to mark the males and thereby monitor their movement patterns, the number of copulations per male could not be determined. In a parallel study, Kr\u0026ouml;ber and Guerin (2007) documented a similar copulating behavior in \u003cem\u003eA. hebraeum\u003c/em\u003e adults on silicone membranes. As described by Feldman-Musham and Borut, the copulating behavior of Metastriate ticks, in which males introduce their chelicerae, without the palps, into the females genital pore, was also noted (Feldman-Muhsam and Borut, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). While it is challenging to induce Ixodid male ticks to engage in copulation, observations revealed the presence of a spermatophore attached near the female gonopore following transfer (Pature et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The process of mating ensures that female ticks become capable of feeding to repletion and begin their vitellogenesis to produce eggs (Sanches et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the present study, the mean detachment weight recorded for \u003cem\u003eA. variegatum\u003c/em\u003e females was 550.5\u0026thinsp;\u0026plusmn;\u0026thinsp;249.7 mg. Given that female \u003cem\u003eA. variegatum\u003c/em\u003e ticks can reach a weight of 3.5 g when fully fed, it is evident that \u003cem\u003ein vitro\u003c/em\u003e feeding had an impact on oviposition. In accordance with prior findings, the lower engorgement weight, which has been shown to result in a decrease in egg production and oviposition, is consistent with artificial feeding yields being lower than natural feeding on animal hosts. We hypothesize that the low hatch rate (17%) for eggs was probably due to a less engorgement of the females and the presence of excessively low temperatures during the egg-laying phase (\u0026lt;\u0026thinsp;21\u0026deg;C). However, the incubation period of eggs is strongly dependent on temperature, as evidenced by Barr\u0026eacute; (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Specifically, the hatching of eggs was impacted by temperatures approximating 16\u0026deg;C. Yano\u0026rsquo;s observations indicated a tendency for a progressive decline in hatch-ratio as temperatures decreased (\u0026lt;\u0026thinsp;20\u0026deg;C) (Yano et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). He deduced that the incubation period was correlated with temperature variations. Furthermore, the viability of larvae that hatched from eggs subjected to low temperatures or desiccation was found to be reduced (Sutherst and Bourne, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Moreover, studies have shown that the developmental stage of the tick egg influences their response to temperature (Ajayi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, eggs in their early stages of embryogenesis exhibit a high vulnerability to both warm and cold temperatures. The placement of incubated eggs must be optimized through meticulous management of the environmental conditions, particularly the temperature and relative humidity, within an incubator for example. Taken together, all of the above factors provide a suitable environment to mimic a natural host to facilitate voluntary tick attachment and further engorgement (Waladde and Ochieng, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of artificial feeding systems confers the advantage of limiting the use of experimental animals with the aim of enhancing animal welfare. Furthermore, Butler\u0026rsquo;s (1984) research indicated a considerable reduction in the economic burden associated with maintaining live hosts in animal facilities (Butler et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Through artificial feeding systems, the 3R Principle, which stands for \u0026ldquo;reduction, replacement and refinement\u0026rdquo; could be applied. These methods will help to minimize the use of experimental animals for research purpose (Balls, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Russell and Burch, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). As demonstrated in the case of \u003cem\u003eD. andersoni\u003c/em\u003e, \u003cem\u003eR. appendiculatus\u003c/em\u003e, and \u003cem\u003eIxodes\u003c/em\u003e spp., the feeding system can be adapted for a broader range of ticks species through the optimization of membrane thicknesses and associated SOPs (Asri et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Vimonish et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yamasaki et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Further work can be conducted to study the development cycle of a given pathogen, such as \u003cem\u003eE. ruminantium\u003c/em\u003e, the causative agent of heartwater, within its vector. This obligate intracellular bacterium is of particular interest given its widespread distribution in some geographic regions, including sub-Saharan Africa and some Caribbean islands (Barr\u0026eacute; et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Camus et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Camus and Barr\u0026eacute;, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Molia et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The utilization of an artificial feeding system will facilitate the investigation of vector efficiency or competency by infecting ticks with a controlled inoculum. This approach would facilitate the exploration of the effect of the pathogen dose and the minimal infection threshold required for further infection of the vector, thereby enabling the elucidation of the pathogen transmission patterns. In addition to the more profound comprehension of tick biology, the use of artificial feeding systems will further facilitate the elucidation of tick-pathogen interactions at molecular and transcriptomic scales. This will lead to a better understanding of tick and pathogen compartments during the course of the infection. Finally, to reduce the transmission of tick-borne diseases, the implementation of \u003cem\u003ein vitro\u003c/em\u003e methods for vector control represents a promising approach. Specifically, evaluating various acaricides using artificial membranes under standardized laboratory conditions could be highly beneficial (Kr\u0026ouml;ber and Guerin, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007a\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest disclosure\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no financial conflicts of interest in relation to the content of the article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work is supported by the United States Department of Agriculture grant 58-3022-1-018-F (Risk of Arthropod-borne diseases in the Caribbean).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFor reading convenience, N.P. is referring to Naomie Pature and N.Pg. is referring to Nonito Pages; M.D. is referring to M\u0026eacute;lanie Dhune and M.Dy. is referring to Maxime Duhayon.N.P. conceptualized the study, developed the methodology, conducted the investigation, curated the data, prepared the figures, and wrote the original draft. M.D., V.R. and M.Dy. contributed to the investigation. N.Pg. curated the data and contributed to editing the manuscript. M.U. supervised the study, curated the data, contributed to manuscript editing, and provided resources and funding. V.R. supervised the study, curated the data, contributed to manuscript editing, and provided resources and funding. D.F.M. conceptualized the study, curated the data, contributed to figure preparation, supervised the research, reviewed and edited the manuscript, secured resources and funding, and administered the project. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Rosalie Aprelon for her assistance with artificial feeding and tick rearing. In addition, the authors would like to thank Jimmy D\u0026eacute;dy and Lo\u0026iuml;c Jacquet-Cr\u0026eacute;tides from the CIRAD animal facility for their dedicated contribution with the daily collection of blood tubes for this study. The authors acknowledge Dr Susan M. Noh for editorial input.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Shafy, S. \u0026amp; Namaky, A. E. Scanning Electron Microscopy of Nymphal and Larval Stages of The Cattle Tick Rhipicephalus (Boophilus) annulatus (Say) 1821 (Acari: Ixodidae). \u003cem\u003eGlobal Vet.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;8 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbel, I. et al. Artificial feeding of Amblyomma cajennense (Acari: Ixodidae) fasting females through capillary tube technique. \u003cem\u003eRev. Bras. Parasitol. Vet.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 128\u0026ndash;132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S1984-29612008000300002\u003c/span\u003e\u003cspan address=\"10.1590/S1984-29612008000300002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbel, I. et al. do Artificial feeding of partially engorged Amblyomma sculptum females through capillaries. Brazilian Journal of Veterinary Medicine 38, 113\u0026ndash;118. (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhoussou, S. et al. Analysis of Amblyomma surveillance data in the Caribbean: Lessons for future control programmes. \u003cem\u003eVet. Parasitol.\u003c/em\u003e \u003cb\u003e167\u003c/b\u003e, 327\u0026ndash;335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetpar.2009.09.035\u003c/span\u003e\u003cspan address=\"10.1016/j.vetpar.2009.09.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjayi, O. M. et al. Egg hatching success is influenced by the time of thermal stress in four hard tick species. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 110\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jme/tjad142\u003c/span\u003e\u003cspan address=\"10.1093/jme/tjad142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson, J. F. The natural history of ticks. \u003cem\u003eMed. Clin. North. Am.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, 205\u0026ndash;218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0025-7125(03)00083-x\u003c/span\u003e\u003cspan address=\"10.1016/s0025-7125(03)00083-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsh, A. et al. Morphological and molecular description of Ixodes woyliei n. sp. (Ixodidae) with consideration for co-extinction with its critically endangered marsupial host. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13071-017-1997-8\u003c/span\u003e\u003cspan address=\"10.1186/s13071-017-1997-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsri, B. et al. Assessment of an In Vitro Tick Feeding System for the Successful Feeding of Adult Rhipicephalus appendiculatus Ticks. \u003cem\u003eParasitologia\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 101\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/parasitologia3020012\u003c/span\u003e\u003cspan address=\"10.3390/parasitologia3020012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalls, M. The principles of humane experimental technique: timeless insights and unheeded warnings. ALTEX - Alternatives to animal experimentation 27, 144\u0026ndash;148. (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14573/altex.2010.2.144\u003c/span\u003e\u003cspan address=\"10.14573/altex.2010.2.144\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarr\u0026eacute;, N. Biologie et \u0026eacute;cologie de la tique \u003cem\u003eAmblyomma variegatum\u003c/em\u003e (Acarina: Ixodina) en Guadeloupe (Antilles Fran\u0026ccedil;aises) (thesis). CIRAD-IEMVT. (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarr\u0026eacute;, N., Aprelon, R. \u0026amp; Eug\u0026eacute;ne, M. Attempts to Feed Amblyomma variegatum Ticks on Artificial Membranes. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e \u003cb\u003e849\u003c/b\u003e, 384\u0026ndash;390. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1749-6632.1998.tb11077.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1749-6632.1998.tb11077.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarr\u0026eacute;, N., Uilenberg, G., Morel, P. C. \u0026amp; Camus, E. Danger of introducing heartwater onto the American mainland: potential role of indigenous and exotic Amblyomma ticks. \u003cem\u003eOnderstepoort J. Vet. Res.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 405\u0026ndash;417 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBezuidenhout, J. D. Natural transmission of heartwater. \u003cem\u003eOnderstepoort J. Vet. Res.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 349\u0026ndash;351 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBilgi\u0026ccedil;, H. B. et al. In vitro feeding of Hyalomma excavatum and Hyalomma marginatum tick species. \u003cem\u003eParasitol. Res.\u003c/em\u003e \u003cb\u003e122\u003c/b\u003e, 1641\u0026ndash;1649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00436-023-07867-7\u003c/span\u003e\u003cspan address=\"10.1007/s00436-023-07867-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026ouml;hme, B., Krull, C., Clausen, P. H. \u0026amp; Nijhof, A. M. Evaluation of a semi-automated \u003cem\u003ein vitro\u003c/em\u003e feeding system for \u003cem\u003eDermacentor reticulatus\u003c/em\u003e and \u003cem\u003eIxodes ricinus\u003c/em\u003e adults. \u003cem\u003eParasitol. Res.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, 565\u0026ndash;570. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00436-017-5648-y\u003c/span\u003e\u003cspan address=\"10.1007/s00436-017-5648-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnet, S. et al. Transstadial and transovarial persistence of \u003cem\u003eBabesia divergens\u003c/em\u003e DNA in \u003cem\u003eIxodes ricinus\u003c/em\u003e ticks fed on infected blood in a new skin-feeding technique. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e134\u003c/b\u003e, 197\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0031182006001545\u003c/span\u003e\u003cspan address=\"10.1017/S0031182006001545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnet, S. \u0026amp; Liu, X. Y. Laboratory artificial infection of hard ticks: a tool for the analysis of tick-borne pathogen transmission. \u003cem\u003eAcarologia\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, 453\u0026ndash;464. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/acarologia/20122068\u003c/span\u003e\u003cspan address=\"10.1051/acarologia/20122068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoulanger, N., Boyer, P., Talagrand-Reboul, E. \u0026amp; Hansmann, Y. Ticks and tick-borne diseases. \u003cem\u003eM\u0026eacute;d. Mal. Infect.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 87\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.medmal.2019.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.medmal.2019.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBullard, R. et al. Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum). \u003cem\u003eTicks Tick-borne Dis.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 880\u0026ndash;892. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ttbdis.2016.04.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ttbdis.2016.04.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurgdorfer, W. Artificial Feeding of Ixodid Ticks for Studies on the Transmission of Disease Agents. \u003cem\u003eJ. Infect. Dis.\u003c/em\u003e \u003cb\u003e100\u003c/b\u003e, 212\u0026ndash;214 (1957).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButler, J., Hess, W., Endris, R. \u0026amp; Holscher, K. In vitro feeding of Ornithodoros ticks for rearing and assessment of disease transmission. (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamus, E. \u0026amp; Barr\u0026eacute;, N. The role of Amblyomma variegatum in the transmission of heartwater with special reference to Guadeloupe. \u003cem\u003eAnn. N Y Acad. Sci.\u003c/em\u003e \u003cb\u003e653\u003c/b\u003e, 33\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1749-6632.1992.tb19627.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1749-6632.1992.tb19627.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamus, E., Barr\u0026eacute;, N., Martinez, D. \u0026amp; Uilenberg, G. \u003cem\u003eHeartwater (cowdriosis). A review\u003c/em\u003e (OIE, 1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChabaud, A. G. Sur la nutrition artificielle des tiques. \u003cem\u003eAnn. Parasitol. Hum. Comp.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 42\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/parasite/1950251042\u003c/span\u003e\u003cspan address=\"10.1051/parasite/1950251042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1950).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCota Guajardo, S. C. Control biol\u0026oacute;gico e integrado de la garrapata Hyalomma lusitanicum en explotaciones silvo-agro-cineg\u0026eacute;ticas de ecosistema mesomediterr\u0026aacute;neo \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://purl.org/dc/dcmitype/Text\u003c/span\u003e\u003cspan address=\"http://purl.org/dc/dcmitype/Text\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Control biol\u0026oacute;gico e integrado de la garrapata Hyalomma lusitanicum en explotaciones silvo-agro-cineg\u0026eacute;ticas de ecosistema mesomediterr\u0026aacute;neo. Universidad Complutense de Madrid. (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCupp, E. W. Biology of Ticks. Veterinary Clinics of North America. \u003cem\u003eSmall Anim. Pract.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 1\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0195-5616(91)50001-2\u003c/span\u003e\u003cspan address=\"10.1016/S0195-5616(91)50001-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurasson, G. Trait\u0026eacute; de protozoologie v\u0026eacute;t\u0026eacute;rinaire et compar\u0026eacute;e. (1943).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuthbert, R. N. et al. Invasive hematophagous arthropods and associated diseases in a changing world. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 291. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13071-023-05887-x\u003c/span\u003e\u003cspan address=\"10.1186/s13071-023-05887-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoube, B. M. \u0026amp; Kemp, D. H. The influence of temperature, relative humidity and host factors on the attachment and survival of \u003cem\u003eBoophilus microplus\u003c/em\u003e (Canestrini) larvae to skin slices. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 449\u0026ndash;454. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0020-7519(79)90048-1\u003c/span\u003e\u003cspan address=\"10.1016/0020-7519(79)90048-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1979).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElati, K. et al. \u003cem\u003eIn vitro\u003c/em\u003e feeding of all life stages of two-host \u003cem\u003eHyalomma excavatum\u003c/em\u003e and \u003cem\u003eHyalomma scupense\u003c/em\u003e and three-host \u003cem\u003eHyalomma dromedarii\u003c/em\u003e ticks. Sci Rep 14, 444. (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-51052-w\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-51052-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrada-Pena, A., Ayllon, N. \u0026amp; De La Fuente, J. Impact of Climate Trends on Tick-Borne Pathogen Transmission. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2012.00064\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2012.00064\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrada-Pe\u0026ntilde;a, A., Horak, I. G. \u0026amp; Petney, T. Climate changes and suitability for the ticks Amblyomma hebraeum and Amblyomma variegatum (Ixodidae) in Zimbabwe (1974\u0026ndash;1999). \u003cem\u003eVet. Parasitol.\u003c/em\u003e \u003cb\u003e151\u003c/b\u003e, 256\u0026ndash;267. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetpar.2007.11.014\u003c/span\u003e\u003cspan address=\"10.1016/j.vetpar.2007.11.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrada-Pe\u0026ntilde;a, A. et al. Reinstatement of Rhipicephalus (Boophilus) australis (Acari: Ixodidae) With Redescription of the Adult and Larval Stages. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 794\u0026ndash;802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1603/ME11223\u003c/span\u003e\u003cspan address=\"10.1603/ME11223\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeldman-Muhsam, B. \u0026amp; Borut, S. Copulation in Ixodid Ticks. \u003cem\u003eJ. Parasitol.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/3277930\u003c/span\u003e\u003cspan address=\"10.2307/3277930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFourie, J. J. et al. Transmission of Anaplasma phagocytophilum (Foggie, 1949) by Ixodes ricinus (Linnaeus, 1758) ticks feeding on dogs and artificial membranes. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13071-019-3396-9\u003c/span\u003e\u003cspan address=\"10.1186/s13071-019-3396-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalun, R. Feeding stimuli and artificial feeding. \u003cem\u003eBull. World Health Organ.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 590\u0026ndash;593 (1967).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia Guizzo, M. et al. Optimizing tick artificial membrane feeding for Ixodes scapularis. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 16170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-43200-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-43200-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez, J., Bickerton, M. \u0026amp; Toledo, A. Applications of artificial membrane feeding for ixodid ticks. \u003cem\u003eActa Trop.\u003c/em\u003e \u003cb\u003e215\u003c/b\u003e, 105818. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actatropica.2020.105818\u003c/span\u003e\u003cspan address=\"10.1016/j.actatropica.2020.105818\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez, J., Valc\u0026aacute;rcel, F., Aguilar, A. \u0026amp; Olmeda, A. S. In vitro feeding of Hyalomma lusitanicum ticks on artificial membranes. \u003cem\u003eExp. Appl. Acarol\u003c/em\u003e. \u003cb\u003e72\u003c/b\u003e, 449\u0026ndash;459. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10493-017-0167-1\u003c/span\u003e\u003cspan address=\"10.1007/s10493-017-0167-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris, R. A. \u0026amp; Kaufman, W. R. Neural Involvement in the Control of Salivary Gland Degeneration in the Ixodid Tick Amblyomma hebraeum. \u003cem\u003eJ. Exp. Biol.\u003c/em\u003e \u003cb\u003e109\u003c/b\u003e, 281\u0026ndash;290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.109.1.281\u003c/span\u003e\u003cspan address=\"10.1242/jeb.109.1.281\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHokama, Y., Lane, R. S. \u0026amp; Howarth, J. A. Maintenance of Adult and Nymphal Ornithodoros coriaceus (Acari: Argasidae) by Artificial Feeding Through a Parafilm Membrane. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 319\u0026ndash;323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jmedent/24.3.319\u003c/span\u003e\u003cspan address=\"10.1093/jmedent/24.3.319\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJongejan, F. \u0026amp; Uilenberg, G. The global importance of ticks. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e129\u003c/b\u003e, S3\u0026ndash;S14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0031182004005967\u003c/span\u003e\u003cspan address=\"10.1017/S0031182004005967\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKemp, D. H., Koudstaal, D., Roberts, J. A. \u0026amp; Kerr, J. D. Feeding of Boophilus microplus larvae on a partially defined medium through thin slices of cattle skin. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e, 243\u0026ndash;254. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/s0031182000049702\u003c/span\u003e\u003cspan address=\"10.1017/s0031182000049702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1975).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhoo, B., Cull, B. \u0026amp; Oliver, J. D. Tick Artificial Membrane Feeding for Ixodes scapularis. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/64553\u003c/span\u003e\u003cspan address=\"10.3791/64553\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoch, H. G. Development of the Lone Star Tick, Amblyomma americanum (Acari: Ixodidae), from Immatures of Different Engorgement Weights. \u003cem\u003eJ. Kansas Entomol. Soc.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e, 309\u0026ndash;313 (1986).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoch, H. G. \u0026amp; Hair, J. A. The Effect of Host Species on the Engorgement, Molting Success, and Molted Weight of the Gulf Coast Tick, Amblyomma maculatum Koch (Acarina: Ixodidae). \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 213\u0026ndash;219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jmedent/12.2.213\u003c/span\u003e\u003cspan address=\"10.1093/jmedent/12.2.213\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1975).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozisek, F. et al. An optimized artificial blood feeding assay to study tick cuticle biology. \u003cem\u003eInsect Biochem. Mol. Biol.\u003c/em\u003e \u003cb\u003e168\u003c/b\u003e, 104113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ibmb.2024.104113\u003c/span\u003e\u003cspan address=\"10.1016/j.ibmb.2024.104113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026ouml;ber, T. \u0026amp; Guerin, P. M. In vitro feeding assays for hard ticks. \u003cem\u003eTrends Parasitol.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 445\u0026ndash;449. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pt.2007.07.010\u003c/span\u003e\u003cspan address=\"10.1016/j.pt.2007.07.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007a).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026ouml;ber, T. \u0026amp; Guerin, P. M. An in vitro feeding assay to test acaricides for control of hard ticks. \u003cem\u003ePest Manag. Sci.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e, 17\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.1293\u003c/span\u003e\u003cspan address=\"10.1002/ps.1293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007b).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrull, C., B\u0026ouml;hme, B., Clausen, P. H. \u0026amp; Nijhof, A. M. Optimization of an artificial tick feeding assay for Dermacentor reticulatus. \u003cem\u003eParasit. Vectors\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13071-017-2000-4\u003c/span\u003e\u003cspan address=\"10.1186/s13071-017-2000-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuhnert, F. Feeding of Hard Ticks In Vitro: New Perspectives for Rearing and for the Identification of Systemic Acaricides. \u003cem\u003eALTEX\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 76\u0026ndash;87 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuhnert, F., Diehl, P. A. \u0026amp; Guerin, P. M. The life-cycle of the bont tick Amblyomma hebraeum in vitro. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 887\u0026ndash;896. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0020-7519(95)00009-Q\u003c/span\u003e\u003cspan address=\"10.1016/0020-7519(95)00009-Q\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Z., Macaluso, K. R., Foil, L. D. \u0026amp; Swale, D. R. Inward rectifier potassium (Kir) channels mediate salivary gland function and blood feeding in the lone star tick, \u003cem\u003eAmblyomma americanum\u003c/em\u003e. \u003cem\u003ePLoS Negl. Trop. Dis.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, e0007153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0007153\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0007153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, X. Y., Cote, M., Paul, R. E. L. \u0026amp; Bonnet, S. I. Impact of feeding system and infection status of the blood meal on \u003cem\u003eIxodes ricinus\u003c/em\u003e feeding. \u003cem\u003eTicks Tick. Borne Dis.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 323\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ttbdis.2013.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.ttbdis.2013.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaranga, R. O., Hassanali, A., Kaaya, G. P. \u0026amp; Mueke, J. M. Attraction of Amblyomma variegatum (ticks) to the attraction-aggregation-attachment-pheromone with or without carbon dioxide. \u003cem\u003eExp. Appl. Acarol\u003c/em\u003e. \u003cb\u003e29\u003c/b\u003e, 121\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/a:1024265529030\u003c/span\u003e\u003cspan address=\"10.1023/a:1024265529030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilitzer, N., Bartel, A., Clausen, P. H., Hoffmann-K\u0026ouml;hler, P. \u0026amp; Nijhof, A. M. Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus. \u003cem\u003eVaccines\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/vaccines9040385\u003c/span\u003e\u003cspan address=\"10.3390/vaccines9040385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilitzer, N., Socias, P., Nijhof, S. \u0026amp; A.M Changes in the Ixodes ricinus microbiome associated with artificial tick feeding. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.1050063\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.1050063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyake, T. et al. Bloodmeal host identification with inferences to feeding habits of a fish-fed mosquito, Aedes baisasi. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 4002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-40509-6\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-40509-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolia, S. et al. Amblyomma variegatum in cattle in Marie Galante, French Antilles: Prevalence, control measures, and infection by Ehrlichia ruminantium. \u003cem\u003eVet. Parasitol.\u003c/em\u003e \u003cb\u003e153\u003c/b\u003e, 338\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetpar.2008.01.046\u003c/span\u003e\u003cspan address=\"10.1016/j.vetpar.2008.01.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Moura, S. T., Fonseca, A. H., da, Fernandes, C. G. N. \u0026amp; Butler, J. F. Artificial Feeding of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) through Silicone Membrane. \u003cem\u003eMem. Inst. Oswaldo Cruz\u003c/em\u003e. \u003cb\u003e92\u003c/b\u003e, 545\u0026ndash;548. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0074-02761997000400019\u003c/span\u003e\u003cspan address=\"10.1590/S0074-02761997000400019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNijhof, A. M. \u0026amp; Tyson, K. R. In vitro Feeding Methods for Hematophagous Arthropods and Their Application in Drug Discovery, in: (eds Meng, C. Q. \u0026amp; Sluder, A. E.) Ectoparasites. Wiley, 187\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/9783527802883.ch9\u003c/span\u003e\u003cspan address=\"10.1002/9783527802883.ch9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorval, R. A. I., Peter, T., Yunker, C. E., Sonenshine, D. E. \u0026amp; Burridge, M. J. Responses of the ticks Amblyomma hebraeum and A. variegatum to known or potential components of the aggregation-attachment pheromone. I. Long-range attraction. \u003cem\u003eExp. Appl. Acarol\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e, 11\u0026ndash;18 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgden, N. H. et al. Investigation of Relationships Between Temperature and Developmental Rates of Tick Ixodes scapularis (Acari: Ixodidae) in the Laboratory and Field. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 622\u0026ndash;633. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1603/0022-2585-41.4.622\u003c/span\u003e\u003cspan address=\"10.1603/0022-2585-41.4.622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliver, J. D. et al. Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 409\u0026ndash;415. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jme/tjv241\u003c/span\u003e\u003cspan address=\"10.1093/jme/tjv241\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePature, N., Pages, N., Rodrigues, V. \u0026amp; Meyer, D. F. Dissection and Internal Anatomy of the Giant Tropical Bont Tick Amblyomma Variegatum. (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2139/ssrn.5173455\u003c/span\u003e\u003cspan address=\"10.2139/ssrn.5173455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerritt, D. W., Couger, G. \u0026amp; Barker, R. W. Computer-controlled olfactometer system for studying behavioral responses of ticks to carbon dioxide. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 571\u0026ndash;578. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jmedent/30.3.571\u003c/span\u003e\u003cspan address=\"10.1093/jmedent/30.3.571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechav, Y., Norval, R. A. I. \u0026amp; Oliver, J. H. Interspecific Mating of Amblyomma Hebraeum and Amblyomma Variegatum (Acari: Ixodidae). \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 139\u0026ndash;142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jmedent/19.2.139\u003c/span\u003e\u003cspan address=\"10.1093/jmedent/19.2.139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1982).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRochlin, I. et al. Optimization of artificial membrane feeding system for lone star ticks, Amblyomma americanum (Acari: Ixodidae), and experimental infection with Rickettsia amblyommatis (Rickettsiales: Rickettsiaceae). Journal of Medical Entomology tjad158. (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jme/tjad158\u003c/span\u003e\u003cspan address=\"10.1093/jme/tjad158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomano, D., Stefanini, C., Canale, A. \u0026amp; Benelli, G. Artificial blood feeders for mosquitoes and ticks\u0026mdash;Where from, where to? \u003cem\u003eActa Trop.\u003c/em\u003e \u003cb\u003e183\u003c/b\u003e, 43\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actatropica.2018.04.009\u003c/span\u003e\u003cspan address=\"10.1016/j.actatropica.2018.04.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRussell, W. \u0026amp; Burch, R. The Principles of Humane Experimental Technique. \u003cem\u003eMed. J. Aust.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 500\u0026ndash;500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5694/j.1326-5377.1960.tb73127.x\u003c/span\u003e\u003cspan address=\"10.5694/j.1326-5377.1960.tb73127.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1960).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanches, G. S. et al. Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). \u003cem\u003eJ. Insect. Physiol.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, 1020\u0026ndash;1027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2012.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2012.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSauer, J. R., Hair, J. A. \u0026amp; Houts, M. S. Chemo-Attraction in the Lone Star Tick (Acarina: Ixodidae). 2. Responses to Various Concentrations of Cco21. \u003cem\u003eAnn. Entomol. Soc. Am.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 150\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aesa/67.1.150\u003c/span\u003e\u003cspan address=\"10.1093/aesa/67.1.150\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1974).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026ouml;ni, R., Hess, E., Blum, W. \u0026amp; Ramstein, K. The aggregation-attachment pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari, Ixodidae): Isolation, identification and action of its components. \u003cem\u003eJ. Insect. Physiol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 613\u0026ndash;618. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-1910(84)90045-3\u003c/span\u003e\u003cspan address=\"10.1016/0022-1910(84)90045-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebastian, P. S. et al. Preliminary Study on Artificial versus Animal-Based Feeding Systems for Amblyomma Ticks (Acari: Ixodidae). \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 1107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms11051107\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms11051107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonenshine, E. \u0026amp; Roe, D. E. (eds) byR.M., Biology of Ticks Volume 2, Second Edition, New to this Edition:, Second Edition, New to this Edition: ed. Oxford University Press, Oxford, New York. (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStachurski, F. Invasion of West African cattle by the tick Amblyomma variegatum. \u003cem\u003eMed. Vet. Entomol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 391\u0026ndash;399. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-2915.2000.00246.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2915.2000.00246.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStachurski, F., Musonge, E. N., Achu-kwi, M. D. \u0026amp; Saliki, J. T. Impact of natural infestation of Amblyomma variegatum on the liveweight gain of male Gudali cattle in Adamawa (Cameroon). \u003cem\u003eVet. Parasitol.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 299\u0026ndash;311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0304-4017(93)90128-A\u003c/span\u003e\u003cspan address=\"10.1016/0304-4017(93)90128-A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutherst, R. W. \u0026amp; Bourne, A. S. The effect of desiccation and low temperature on the viability of eggs and emerging larvae of the tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Ixodidae). \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 193\u0026ndash;200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijpara.2005.09.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ijpara.2005.09.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzin, A. et al. Free-living ticks (Acari: Ixodidae) in the Igua\u0026ccedil;u National Park, Brazil: Temporal dynamics and questing behavior on vegetation. \u003cem\u003eTicks Tick-borne Dis.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 101471. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ttbdis.2020.101471\u003c/span\u003e\u003cspan address=\"10.1016/j.ttbdis.2020.101471\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTajeri, S., Razmi, G. \u0026amp; Haghparast, A. Establishment of an Artificial Tick Feeding System to Study Theileria lestoquardi Infection. \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, e0169053. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0169053\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0169053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTukahirwa, E. M. The feeding behaviour of larvae, nymphs and adults of Rhipicephalus appendiculatus. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, 65\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0031182000058479\u003c/span\u003e\u003cspan address=\"10.1017/S0031182000058479\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1976).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVimonish, R. et al. Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 470. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-57390-y\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-57390-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoigt, W. P. et al. In vitro feeding of instars of the ixodid tick Amblyomma variegatum on skin membranes and its application to the transmission of Theileria mutans and Cowdria ruminantium. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 257\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0031182000079233\u003c/span\u003e\u003cspan address=\"10.1017/S0031182000079233\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M., THE SENSORY NERVOUS SYSTEM OF \u0026amp; THE ADULT CATTLE TICK BOOPHILUS MICROPLUS (CANESTRINI) IXODIDAE. PART I. LIGHT MICROSCOPY. \u003cem\u003eAustralian J. Entomol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 379\u0026ndash;387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1440-6055.1976.tb01720.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1440-6055.1976.tb01720.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M. \u0026amp; Ochieng, S. A. Advances in the Artificial Feeding of Ticks. \u003cem\u003eInt. J. Trop. Insect Sci.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 579\u0026ndash;583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S1742758400016167\u003c/span\u003e\u003cspan address=\"10.1017/S1742758400016167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M., Ochieng\u0026rsquo;, S. A. \u0026amp; Gichuhi, P. M. Artificial-membrane feeding of the ixodid tick, Rhipicephalus appendiculatus, to repletion. \u003cem\u003eExp. Appl. Acarol\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, 297\u0026ndash;306. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01202876\u003c/span\u003e\u003cspan address=\"10.1007/BF01202876\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M. \u0026amp; Rice, M. J. \u003cem\u003eThe Sensory Basis of Tick Feeding Behaviour\u003c/em\u003epp. 71\u0026ndash;118 (Elsevier, 1982). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-08-024937-7.50008-1\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-08-024937-7.50008-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M., Young, A. S. \u0026amp; Morzaria, S. P. Artificial feeding of ixodid ticks. \u003cem\u003eParasitol. Today\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 272\u0026ndash;278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0169-4758(96)10027-2\u003c/span\u003e\u003cspan address=\"10.1016/0169-4758(96)10027-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaladde, S. M., Young, A. S., Ochieng\u0026rsquo;, S. A., Mwaura, S. N. \u0026amp; Mwakima, F. N. Transmission of Theileria parva to cattle by Rhipicephalus appendiculatus adults fed as nymphae in vitro on infected blood through an artificial membrane. \u003cem\u003eParasitology\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 249\u0026ndash;256. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0031182000079221\u003c/span\u003e\u003cspan address=\"10.1017/S0031182000079221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamasaki, Y., Singh, P., Vimonish, R., Ueti, M. \u0026amp; Bankhead, T. Development and Application of an In Vitro Tick Feeding System to Identify Ixodes Tick Environment-Induced Genes of the Lyme Disease Agent, Borrelia burgdorferi. \u003cem\u003ePathogens\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens13060487\u003c/span\u003e\u003cspan address=\"10.3390/pathogens13060487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYano, Y., Shiraishi, S. \u0026amp; Uchida, T. A. Effects of temperature on development and growth in the tick, \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e. \u003cem\u003eExp. Appl. Acarol\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e, 73\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01200415\u003c/span\u003e\u003cspan address=\"10.1007/BF01200415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYonow, T. The life-cycle of Amblyomma variegatum (Acari: Ixodidae): a literature synthesis with a view to modelling. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 1023\u0026ndash;1060. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0020-7519(95)00020-3\u003c/span\u003e\u003cspan address=\"10.1016/0020-7519(95)00020-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung, A. S., Waladde, S. M. \u0026amp; Morzaria, S. P. Artificial Feeding Systems for Ixodid Ticks as a Tool for Study of Pathogen Transmission. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e \u003cb\u003e791\u003c/b\u003e, 211\u0026ndash;218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1749-6632.1996.tb53527.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1749-6632.1996.tb53527.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1996).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hard ticks, Amblyomma variegatum, in vitro feeding system, heartwater","lastPublishedDoi":"10.21203/rs.3.rs-6867220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6867220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe three-host tick \u003cem\u003eAmblyomma variegatum\u003c/em\u003e, commonly known as the tropical bont tick, poses a major threat to livestock health and productivity in tropical and subtropical regions worldwide. This tick is agressive, transmits multiple pathogens including \u003cem\u003eEhrlichia\u003c/em\u003e ruminantium, an intracellular obligate bacterium that causes heartwater, and directly damages the skin, and causes losses in productivity. The tropical bont tick, which belongs to the \u003cem\u003eIxodidae\u003c/em\u003e family, has long mouth parts and feeding behaviors are characterized by prolonged blood meals during each life stage. The inability to control the tick and prevent the diseases it transmits is partly due to the necessity of rearing the tick on animals. Thus, the goal of this study was to develop an artificial membrane feeding system to complete the life cycle of \u003cem\u003eA. variegatum\u003c/em\u003e. All life stages of \u003cem\u003eA. variegatum\u003c/em\u003e were fed using fresh goat blood at 38\u0026deg;C, and blood replacement occurred every 12 hours. Key parameters, such as humidity, temperature, and membrane thickness, were optimized to mimic natural tick feeding conditions. The attachment of ticks to the artificial membranes was induced by synthetic pheromones and host hairs. The attachment and engorgement rates for immature tick stages exceeded 80%, demonstrating high feeding success using the artificial system. The reproductive capacity of \u003cem\u003eA. variegatum\u003c/em\u003e adult female ticks proved to be successful, with an oviposition rate of 35%. The larvae resulting from these eggs exhibited feeding patterns comparable to larvae derived from female ticks fed on goats. Collectively, these findings demonstrate the feasibility of using artificial feeding system to complete the breeding cycle of \u003cem\u003eA. variegatum\u003c/em\u003e without the use of live hosts for tick engorgement. Consequently, this innovative approach will facilitate further research to close the knowledge gap, including understanding the tick-pathogen interactions and feeding of other tick species or hematophagous arthropods of human and veterinary importance.\u003c/p\u003e","manuscriptTitle":"Successful completion of the life cycle of Amblyomma variegatum using tick artificial membrane feeding system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-12 02:05:11","doi":"10.21203/rs.3.rs-6867220/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-14T05:02:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-14T04:46:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T07:28:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-11T02:40:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aad14525-3785-4f9a-bcff-89c8e85fb4d1","owner":[],"postedDate":"June 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49915345,"name":"Biological sciences/Zoology/Entomology"},{"id":49915346,"name":"Biological sciences/Biological techniques/Biological models/Animal disease models"}],"tags":[],"updatedAt":"2025-11-24T16:00:43+00:00","versionOfRecord":{"articleIdentity":"rs-6867220","link":"https://doi.org/10.1038/s41598-025-23801-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-17 15:57:21","publishedOnDateReadable":"November 17th, 2025"},"versionCreatedAt":"2025-06-12 02:05:11","video":"","vorDoi":"10.1038/s41598-025-23801-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-23801-6","workflowStages":[]},"version":"v1","identity":"rs-6867220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6867220","identity":"rs-6867220","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}