The ability to determine when ticks last fed and assign them to a specific feeding cohort is important in attempts to explain their population dynamics; the biochemical measurement of stored lipid, has been widely used for this purpose. However, when relating feeding history to behaviour or infection status, a non-destructive approach to its assessment would be of value and, to this end, previous studies have attempted to use morphometric indices. Within any instar, the sclerotised scutal components of the body will not vary with increasing starvation while the alloscutal components will, and the resulting ratio should provide a measure of time since feeding. Here, the aim was to determine whether such a morphological ratio (described here as the hunger index) changed predictably with starvation in Ixodes ricinus L. (Ixodida: Ixodidae). For this a cohort of 300 I. ricinus nymphs was collected from the field in February 2021 and starved in a humidified incubator at 15°C and 80% relative humidity (RH). Every 2 weeks, 50 nymphs selected at random were removed and killed by freezing; morphometric measurement was followed by the measurement of lipid using a standard spectrophotometric approach. Both hunger index and stored lipid changed significantly with increasing starvation and were positively correlated with each other. However, the change in morphometric ratio was relatively slight (11%) over 9 weeks and the variation was high. The data suggest therefore that morphological measurements could be used to provide, at best, only broad categorisation of the hunger status of individual I. ricinus ticks in the field.

Nymphs of I. ricinus were collected over a single day in February 2021.  The ticks were collected by blanket dragging from a single field site immediately west of the city of Bristol (UK); the collection site contains a mixture of woodland and grassland with large managed and wild deer populations.  Nymphs collected at this time of the year at this site have been shown previously to have relatively high lipid concentrations (Abdullah et al., 2018). Immediately after collection nymphs were transported to the laboratory. Three hundred ticks were transferred individually into labelled 0.5ml plastic Eppendorf tubes. Each tube was sealed with a piece of netting fastened with a rubber band. The tubes were placed in a humidified incubator (Versatile Environment Test Chamber, PHC Corporation, Japan) at 15 °C and 80% Relative humidity (RH). This environment was adequate to ensure good levels of survival of the ticks during the duration of study.

 Tick body measurements

Immediately following collection (Week 1) and subsequently every 2 weeks over a period of nine weeks, about 50 nymphs, were selected at random. These nymphs were first killed by freezing at -20°C.  Then a range of morphological measurements were made, to the nearest micrometer, using a binocular microscope (Leica M212, Wetzlar, Germany) with the aid of a calibrated graticule. These were: scutum length and width, total body length and width and the alloscutum length (Table 1, Fig 1). The objective was to measure a representative range of characters to allow the surface area of the alloscutal and scutal components of the body to be calculated, defined as the Scutal Index (SI) and the Alloscutal Index (AI) respectively. The ratio of the Alloscutal Index and Scutal Index was then calculated (AI/SI) – described here as the ‘Hunger Index’ (HI). Declining HI values indicate increasingly starved ticks.

Measurement of tick lipid

Following morphological measurement, within each batch, the lipid content of 30 of the measured ticks, selected at random, was quantified using a vanillin assay (Abdullah et al., 2019).  The spectrophotometric method used to measure lipid in recent studies is based on a microquantity colorimetric sulfophosphovanillan method (SPV), adapted from a method first proposed by Van Handel (1985) for use in mosquitoes. It has been shown to allow the lipid content for individual ticks to be estimated with accuracy (Abdullah et al., 2019; Alasmari and Wall, 2020). This technique is based on the principle that unsaturated lipids react with sulphuric acid to produce a carbonium ion; vanillin reacts with phosphoric acid to produce an aromatic phosphate; the carbonium ion then reacts with the activated carbonyl group of phospho-vanillin to produce a charged, coloured complex that is stabilised by resonance and absorbs light maximally at about 525nm.

Prior to lipid analysis, ticks were first weighed using an ultrasensitive microbalance (Sartorius-ME5, Göttingen, Germany), then placed individually in an oven (Heratherm Oven, Thermo Scientific, UK) and dried at 70 °C for 12 h and then re-weighed. Each individually dried and weighed tick was placed in a clean glass test tube and crushed with a glass rod in 0.5ml of a 1:1 chloroform-methanol mixture made up by adding equal parts of chloroform and methanol (Sigma-Aldrich, Gillingham, UK). Tick debris which had accumulated on the side of the tube were included by gently rocking the tube, after which 0.50ml of the mixture was transferred into a second clean glass tube. The glass tubes were then placed in a heating block (LSE Digital Dry Bath, Corning, USA) at 100 °C in a fume cupboard to evaporate the solvent. After evaporation, 0.1ml of 98% sulphuric acid (VWR International, Leicestershire, UK) was added and the tube was heated again for 10 min at the same temperature. The tube was then left to cool, after which 2.4 ml of vanillin reagent (Acros Organics, New Jersey, USA) was added to make a total volume of 2.5ml. After adding vanillin to the mixture, the reddish colour was allowed to develop over a period of 5 min. The absorbance of each tube was then read using a spectrophotometer (Biochrome, Biowave II, Cambridge, UK) at 525 nm against a blank which had been subjected to the same procedure as above, but without the tick material present. The lipid content of each individual tick was read from a calibration curve; the calibration curve of absorbance against known lipid concentration. The curve was obtained using analytical grade standard soybean oil (0.917g/ml) (Sigma-Aldrich, Gillingham, UK) diluted in chloroform-methanol (1:1). The mixture was subjected to the same procedure as described above.

Statistical analysis

Body measurement data were normally distributed, so to describe the relationship between the linear anatomical parameters of the tick with starvation, General Linear Models (GLM) were used. Bonferroni post-hoc tests were used to determine the differences between the pair-wise comparisons of the group means of the tick batches from different starvation time points. Linear regression was used to examine the relationship between hunger index and lipid content. Non-parametric Kruskal-Wallis tests were used to examine differences in lipid concentration at different starvation time points, due to the non-normal distribution of the lipid values. All means are reported ± SEM and all medians with their range or inter-quartile range.  All statistical analyses were carried out using SPSS (Version 28, IBM Corp, Armonk, N.Y, U.S.A).