Only four out of 11 females (36.4%) produced eggs, with a mean number of 4036 ± 2967 with a REI of 60%. The mean weight of engorged detached females was 276 ± 105 mg. Hyalomma dromedarii fed in winter (January) reached an engorgement rate of 34.4% (11/32 females). In this study, we targeted the first point by feeding all life stages of H. In addition, the success rate reported for the in vitro feeding of ixodid ticks varies considerably and the addition of antibiotics to the blood meal to prevent microbial contamination may exert a negative effect on tick endosymbionts and tick fecundity 22, 23. There is for instance a lack of studies examining the artificial feeding of juvenile stages of one- and two-host tick species where ticks molt on the host to the subsequent life stage. However, extensive optimization of the artificial feeding method is still required. The development and optimization of methods to feed hard ticks artificially in the laboratory not only contributes to the 3Rs principle (to Reduce, Replace and Refine animal experiments) for humane animal research 20, but also provides researchers with a versatile tool to study various aspects of tick biology, pathogen transmission and drug discovery under controlled laboratory conditions 13, 21. In recent years, artificial tick feeding systems (ATFS) have been developed and adapted for different ixodid ticks such as Amblyomma hebraeum, Dermacentor reticulatus, Ixodes ricinus, Rhipicephalus appendiculatus as well as nymphs and adult Hyalomma species such as H. Studies on the biology of these ticks frequently require laboratory tick colonies that are reared and maintained on animals 29. The activity of this ticks species does not exhibit a seasonal variation and it can be found throughout the year on animals 11, 12. Hyalomma dromedarii can behave as a one-, two-, or three-host tick 8, 9, 10, whereby the immature stages can feed on camels, but also on rodents and birds. Larvae and nymphs of this species feed on rodents and the adults infest various hosts such as cattle, small ruminants and equids 8. In North Africa, it is active during the whole year with a peak in spring. Hyalomma excavatum can have a two-or three host life cycle tick depending on the availability of hosts. Hyalomma scupense collected in China were reported to behave as one- and two-host tick under laboratory conditions 7. scupense was found to have a one-host life cycle 6. scupense was found to behave as a two-host tick species with peak activity of the adults feeding on cattle in summer 5, whereas in other regions, a cold-adapted ecotype of H. temperature and relative humidity and by the availability of hosts 4. The duration of tick life cycle is strongly affected by environmental factors, e.g. This includes several species of the Hyalomma genus that are associated with the transmission of Crimean-Congo Hemorrhagic Fever virus in humans 1, 2 and Theileria annulata, the causal agent of tropical theileriosis in livestock 3. Ticks are hematophagous ectoparasites that may act as vectors for a variety of pathogens infecting humans and animals worldwide. The successful in vitro feeding of all stages of the three Hyalomma species makes this method a valuable tool for tick research, with potential applications in studies on the pathogens transmitted by these tick species such as Theileria annulata. Molting rates of the immature stages varied between 71.3% (216/303) and 100% (68/68) for the larvae and between 61.9% (13/21) and 96% (24/25) for the nymphs. The attachment and engorgement rates for the nymphs were 90.2% (129/143) and 47.6% (68/143) for H. For the larvae, the attachment and engorgement rates were 44.2% (313/708) and 42.8% (303/708) for H. scupense, respectively, with a reproductive efficiency index varying between 44.3 and 60.7%. The oviposition rates of the artificially fed females were 36.4%, 57.1% and 63.1% for H. dromedarii, 70% (21/30) and 34.4% (11/32) of the females attached and all attached females successfully fed to repletion. The attachment and engorgement rates of adults were 84% (21/25) and 76% (19/25) for H. This study represents the first report on the successful in vitro feeding of all life stages of two-host tick species, Hyalomma scupense and Hyalomma excavatum, and the three-host tick Hyalomma dromedarii. The life cycle of ticks can be completed under laboratory conditions on experimental animals, but the artificial feeding of ticks has attracted increased interest as an alternative method. Ticks are blood-sucking ectoparasites and can transmit various pathogens of medical and veterinary relevance.
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