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Hard ticks are obligate hematophagous arthropods that occur throughout the world and parasitize humans and animals. They rank among the most important vectors due to their ability to transmit bacterial, as well as viral, protozoan and other pathogens. The blood meal provokes direct damage to the host (Uilenberg, 1992). For studies on ticks, including their biology, the pathogens they transmit or the development of acaricides and other control measures, a considerable number of laboratory animals are required to act as hosts (Kröber et Guerin, 2007a). A successful in vitro feeding system for hard ticks has the potential to reduce the number of required laboratory animals. Researchers have been trying to develop artificial feeding systems for different tick species for over 100 years. Despite the progress made, in vitro feeding is currently not routinely used in the laboratory rearing of ticks, as it has not been adapted to feed all life stages from important tick species and is associated with a reduced fecundity in adult ticks. All developed methods are also complex and labour intensive due to the long duration of the tick’s blood meal.
In Central Europe Ixodes ricinus and Dermacentor reticulatus represent the most important hard tick species (Karbowiak, 2014). Hence the aim of this study was to realize the in vitro feeding of these two species by improving an existing artificial feeding system using silicone membranes (Kröber et Guerin, 2007a). The feeding system was optimized for the feeding of all three developmental stages of I. ricinus by replacing the tick chambers made of acrylic glass by autoclavable glass chambers, using different attachment stimuli and reducing the membrane thickness for the feeding of juvenile stages. An average attachment rate of 74.8 % (95/127) and an engorgement rate of 40.2 % (51/127) with an average weight of 209.2 mg was obtained in the females. Of the replete females, 62.7 % (32/51) deposited egg batches with an average weight of 34.6 mg and 23.5 % (12/51) produced larvae. After optimization of the feeding system, 73.2 % (30/41) of the nymphs and 56.0 % (28/50) of the larvae fed to repletion on the membrane. There was a significant difference between the engorgement weights of nymphs that molted to males (2.7 mg) and those that molted to females (4.1 mg). The life cycle of I. ricinus was successfully completed in vitro by feeding on bovine blood through silicone membranes. The next step should be the observation of the development of the in vitro feeding success over several generations of I. ricinus.
The feeding technique was subsequently adapted for D. reticulatus and modified with the objective of feeding all life stages in vitro. Promising results were achieved with adult D. reticulatus: 179 (50.1 %) of the 357 females attached in the feeding system with glass tick chambers (K+G Glas) and 106 (29.7 %) engorged fully. Oviposition was observed in 91.5 % (97/106) of the engorged females and larvae hatched from 66.0 % (70/106) of the egg
batches. The average weights of the engorged females and the egg batches were 219.9 mg and 96.3 mg respectively. The in vitro feeding of D. reticulatus juveniles proved to be challenging. Only 0.07 % (20/27500) of the larvae and none of the nymphs (0/920) fed to repletion on silicone membranes. Efforts to optimize the composition and thickness of the membrane and to use different olfactory and mechanical attachment stimuli, blood from different animals and blood supplements were not successful. In another artificial feeding system which employed animal skins as membranes (FSB) (Bonnet et al., 2007) a slightly higher proportion of juveniles engorged: 1.49 % of larvae (57/3820) and 1.88 % of nymphs (15/800). A further optimization of the in vitro feeding of juvenile D. reticulatus is necessary.
Once the in vitro feeding of adult D. reticulatus had been established, further modifications were tested systematically in order to make the in vitro feeding more efficient and less laborious. This is a prerequisite for the adaptation of any in vitro feeding system for hard ticks by other laboratories. The use of previously frozen and of gamma-irradiated blood as well as of blood without the supplement of antibiotics for the tick feeding was investigated. In addition, different feeding systems including partially automated systems were tested. Although tick feeding and reproduction parameters did not differ significantly between ticks fed with blood stored at 4°C and blood stored at -20°C, a remarkably increased incidence of fungal growth was observed on tick chambers and membranes when previously frozen blood was used. The feeding of blood supplemented with fungicides or of freeze-dried blood could be possible alternatives. The utilization of gamma-irradiated blood for the in vitro feeding proved to be inappropriate since it resulted in an extensive microbial growth during the experiments. Although this did not prevent females from engorging, it resulted in a decreased fecundity. The use of blood without antibiotics also decreased the fecundity of the females, but to a lesser extent, while it increased the average engorgement weight. In a direct comparison between different feeding systems, females fed using the FSB noticeably showed lower attachment and engorgement rates, but also the highest mean engorgement weight of 291.1 mg. The in vitro tick feeding could be partially automated with two newly designed flow-through chambers made of polystyrene and glass (DFS PS and DFS Glas). Feeding of D. reticulatus adults using the DFS Glas system showed promising results, as it resulted in increased attachment, engorgement weights and egg hatch rates while the maintenance of the system was less laborious. After further optimizations and the feeding of other ixodid species, this partially automated feeding system could help replacing tick feedings on animal hosts by in vitro feeding.
The lengths of the hypostomes of all stages of I. ricinus and D. reticulatus were measured and a linear correlation between engorgement- and egg batch weight was demonstrated for D. reticulatus in both in vivo and in vitro feeding. It was furthermore observed that adult D. reticulatus mainly detached from the membrane during nighttime, in contrast to nymphal and adult I. ricinus which mainly detached during the day.