Spectrophotometric analysis of lipid used to examine the phenology of the tick <i>Ixodes ricinus</i>

Ticks store lipid as an energy souce, which depletes progressively between blood meals. The amount of lipid and rate of lipid depletion can be used as a good indicator of the feeding history and assist in explaining the phenology of tick populations. However, existing gravimetric approaches to lipid measurement are relatively imprecise. To improve our ability to accurately measure lipid accumulation and metabolism in individual ticks, a microquantity colorimetric sulfophosphovanillan method of lipid estimation was standardised and used to explore the seasonal variations in the lipid content of I. ricinus nymphs.Lipid values for field-derived questing ticks, collected by blanket dragging, varied between 5-45 μg and clear patterns of lipid depletion were demonstrated under controlled laboratory conditions. For field populations collected monthly over two years, the results indicate that two different cohorts of nymphs enter the questing tick population in autumn and spring, with very few nymphs joining the population in summer.The data illustrate the seasonal change in lipid content of nymphal ticks, reflecting their feeding history and highlight the utility of the spectrophotometric technique for analysis of lipid in ticks in helping to improve our understanding of seasonal activity patterns

Contributions to the phylogeny of Ixodes (Pholeoixodes) canisuga, I. (Ph.) kaiseri, I. (Ph.) hexagonus and a simple pictorial key for the identification of their females

Background: In Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers. Results: Between 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2-100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5-100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5-100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes. Conclusions: A simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies