DOI: 10.5061/dryad.hhmgqnkrf
title: Data from: Inferring the feeding history of the castor bean tick, Ixodes ricinus from lipid and body measurements

description: 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.
description: Nymphs of <em>I. ricinus</em> 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.  <em>Tick
 body measurements</em> 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.
 <em>Measurement of tick lipid </em>
 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 <em>et al</em>., 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 <em>et al</em>.,
 2019<em>; </em>Alasmari and Wall<em>, </em>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. <em>Statistical
 analysis</em> 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 <em>post-hoc</em> 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).
description: # Dataset for Journal Article titled "Inferring the feeding history
 of the castor bean tick, Ixodes ricinus from lipid and body
 measurements"
 [https://doi.org/10.5061/dryad.hhmgqnkrf](https://doi.org/10.5061/dryad.hhmgqnkrf) ## Description of the data and file structure This study aimed to evaluate the use of a morphological index (using quantitative ratios derived from tick body measurements) to assess starvation in *I. ricinus*. The underlying rationale was to determine whether such measurements could provide a non-destructive approach to quantifying time since feeding, and to further explore this approach to determine whether morphological measurements change sufficiently predictably with starvation and, if so, whether these measurements can be applied to samples collected from the field to give an indication of the feeding history of *I. ricinus* ticks. ### **File 1: Data – Fig 2** A table showing tick morphological measurements used to calculate the hunger Index (ratio of the alloscutal index/scutal index) of nymphal *Ixodes ricinus* collected from the field at a single time point and starved for 9 weeks in a cooled incubator at 15 °C and 80% RH and measured at different time intervals (Fig 2). **Variables** **Alloscutum Length** - The midline distance from the posterior tip of the scutum to the posterior tip of the opisthosoma measured in millimeters **Scutal Index** - The product of the scutal length and width (SL*SW). Unit millimeter squared **Body Index** - The product of body length and width (BL*BW). Unit millimeter squared **Alloscutal Index** - The difference between the body index and the Scutal Index. Unit millimeter squared **Hunger Index** - The ratio of the Alloscutal Index/Scutal Index (AI/SI). Unit millimeter squared   ### **File 2: Data –Fig 3 and Fig 4** A table showing tick morphological and lipid measurements used to calculate median lipid values (µg) of individual *Ixodes ricinus* nymphs collected at a single time point and progressively starved for 9 weeks in a cooled incubator at 15 ⁰C and 80% RH, measured by spectrophotometric analysis at different time intervals (Fig 3). This data was also used for regression analysis to compare the Hunger Index of nymphal *Ixodes ricinus* at different lipid concentrations (Fig 4). **Variables** **Weight** – Dry body weight of nymphal ticks measured in milligrams. **SL** (scutum length)- The midline distance from the anterior edge to the posterior tip of the scutum in millimeters. **SW** (scutum width) - The lateral distance at the widest point of the scutum measured in millimeters. **BL** (body length) - The midline distance from the anterior edge of the scutum to the posterior tip of the opisthosoma measured in millimeters. **BW -** (body width) - The alloscutum width immediately posterior to the tip of the scutum measured in millimeters. **Alloscutum length** - The midline distance from the posterior tip of the scutum to the posterior tip of the opisthosoma measured in millimeters. **Lipid** – Lipid values of nymphal ticks measured in micrograms. ## Code/software All data can be viewed using Microsoft Excel