Natural isotope signatures of host blood are replicated in moulted ticks

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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Ticks and Tick-borne Diseases 2 (2011) 225–227 Contents lists available at SciVerse ScienceDirect Ticks and Tick-borne Diseases journal homepage: www.elsevier.de/ttbdis Short communication Natural isotope signatures of host blood are replicated in moulted ticks Olaf Schmidt a,∗ , Hans Dautel b , Jason Newton c , Jeremy S. Gray d a UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland IS Insect Services GmbH, Haderslebener Straße 9, 12163 Berlin, Germany c NERC Life Sciences Mass Spectrometry Facility, Scottish Universities Environmental Research Centre, East Kilbride G75 0QF, United Kingdom d UCD School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland b a r t i c l e i n f o Article history: Received 10 December 2010 Received in revised form 7 July 2011 Accepted 8 September 2011 Keywords: Carbon isotopes Fasting Nitrogen isotopes Ticks Tick-borne diseases a b s t r a c t This proof-of-concept study demonstrates that stable isotope ratios of nitrogen and carbon (expressed as ␦13 C and ␦15 N) of host blood are faithfully reproduced in unfed nymphal Ixodes ricinus that developed from larvae fed on that host. Measured isotopic discrimination (i.e. the tick–blood spacing) was between −0.1 and 0.7‰ for ␦13 C and 3.8 and 3.9‰ for ␦15 N. Both ␦13 C and ␦15 N increased significantly with tick ageing. The isotopic analysis of unfed ticks has potential for determining the physiological age of unfed ticks, for identifying the season in which the previous stage had fed and for identifying the main hosts utilized by ticks. © 2011 Elsevier GmbH. All rights reserved. Introduction Materials and methods Ratios of naturally occurring stable isotopes, such as 15 N/14 N and 13 C/12 C, have been much used successfully in the study of food webs (Kelly, 2000; Crawford et al., 2008). Stable isotope ratio measurements make it possible to identify the components of food chains because isotopic ‘signatures’ are present in different foods ingested by animals and are passed on, with predictable modifications, in food chains (Hood-Nowotny and Knols, 2007; Martínez del Rio, 2007). The participation of haematophagous arthropods in food chains is comparable to that of a predator in that the isotopic signature of host blood, which reflects the food of the host, reoccurs in the tissues of the arthropod (Gómez-Díaz and Figuerola, 2010). Determination of natural isotope ratios in parasites, such as ticks, can therefore potentially provide information on the identity of hosts and, in specific cases, the season in which the parasite obtained its blood meal. Additionally, since whole-body N isotope ratios in fasting animals increase (Martínez del Rio, 2007), it should be possible to determine the period since the last blood meal of a tick. This proof-of-concept study investigated the relationships between the isotopic ratios present in the food of laboratory gerbils and a rabbit, in the blood of these hosts, and in nymphal Ixodes ricinus ticks that had fed on them as larvae. Larval I. ricinus (n > 100 per animal) were fed on laboratory rabbits (n = 1) or gerbils (n = 2). The rabbit was fed compound pellets and hay, the gerbils were fed a different type of compound pellets only. Larval I. ricinus were maintained at approximately 20 ◦ C and a high humidity (about 90% R.H.) until they had moulted to nymphs and frozen at −20 ◦ C upon sampling. To investigate the effects of ageing of these unfed ticks on the isotopic composition, they were sampled at intervals of 1, 7 and 10 weeks after they had moulted. All materials (host food, whole host blood, ticks) were freeze-dried and their C and N stable isotope composition (expressed as conventional delta values per mil, ı ‰) determined by Elemental Analysis–Isotope Ratio Mass Spectrometry (EA–IRMS). Dried host food and host blood were powdered using a mortar and pestle and analyzed in triplicate; ticks were analyzed whole. The effect of sample size was investigated by analyzing ticks singly or in pools of 3 ticks or 7 ticks (2 samples per host per date); the latter two being equivalent to minimum and standard sample masses for the IRMS method used. Data were analyzed by ANOVA (StatView, SAS Institute). ∗ Corresponding author. Tel.: +353 1 7167076; fax: +353 1 7161102. E-mail address: olaf.schmidt@ucd.ie (O. Schmidt). 1877-959X/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2011.09.006 Results and discussion The results established that increases in C and N stable-isotope ratios (␦13 C and ␦15 N) of host food, host blood, and unfed ticks up the food chain (Fig. 1) were analogous to those in other known food chains (Kelly, 2000; Hood-Nowotny and Knols, 2007; Crawford et al., 2008). Clearly, the isotopic composition of ticks reflected that Author's personal copy 226 O. Schmidt et al. / Ticks and Tick-borne Diseases 2 (2011) 225–227 Fig. 1. Stable-isotope composition of I. ricinus nymphs in relation to host blood and host feed (circles: rabbit; squares: gerbils, numbered 1 and 2). Rabbits were fed compound pellets and hay, gerbils were fed compound pellets only. of their host blood (Fig. 1). Measured isotopic discrimination (i.e. the tick–blood spacing) was invariable and close to expected values (Kelly, 2000; Hood-Nowotny and Knols, 2007): between −0.1 and 0.7‰ for ␦13 C and 3.8 and 3.9‰ for ␦15 N. The isotopic ratios of ticks fed on the 2 host species were significantly different (P < 0.001), even though ␦13 C and ␦15 N differences between host blood were less than 1‰ (Fig. 1). Previous attempts to identify the hosts that field-caught unfed ixodid ticks have fed on during the previous stage have focused on detection of host DNA in blood-meal remnants (Kirstein and Gray, 1996; Pichon et al., 2005; Morán-Cadenas et al., 2007; Allan et al., 2010). This methodology has met with some success, but is limited by its complexity, expense, and dwindling sensitivity as the tick ages. The measurement of naturally occurring stable isotope ratios may offer an alternative or complementary approach, especially since the isotopic signature is incorporated into tick tissues and will not weaken as blood meals are digested. To develop this method for field use, the blood–tick isotopic spacing should be investigated under controlled conditions for animals from major taxa of ecological significance in tick-borne diseases, such as birds, ruminants, and rodents. We know from the literature on the diets of large terrestrial predators (Crawford et al., 2008) that isotopic compositions are likely to be different between these host groups, which not only ingest different foods, but also assimilate them differently. Further laboratory and field studies are required first, to determine the variation in the isotopic composition between individual hosts and ticks and second, to establish if this variation is smaller than inter-species differences between hosts in natural habitats. If the isotopic signatures of potential host species are monitored over time in field studies, it may even be possible to focus on the season in which ticks fed in the previous stage, since their isotopic signatures will be a reflection of seasonal changes in host food (Ben-David et al., 1997; Crawford et al., 2008). Both ␦13 C and ␦15 N increased with tick ageing, with significant increases (P < 0.05) from week 1 to week 10 (Fig. 2A and B). If robustly calibrated, this remarkable finding could potentially be developed as a novel approach to estimate the age of unfed ticks and therefore to which seasonal activity cohort they belong. The effects of starvation on the isotopic composition of animals are, in general, insufficiently understood and require detailed physiological experimentation (Martínez del Rio, 2007). Our initial results suggest that ticks could serve as a suitable model group. Fig. 2. Stable-isotope composition of I. ricinus nymphs 1, 7, and 10 weeks after moulting (A) nitrogen and (B) carbon. Datapoints are means ± 1 SD (n = 3 measured samples with 3 or 7 ticks each). Author's personal copy O. Schmidt et al. / Ticks and Tick-borne Diseases 2 (2011) 225–227 A few surveys of isotopic compositions in other haematophagous parasites exist, for example fleas on seabirds, prairie dogs, and mice (Stapp and Salkeld, 2009; Gómez-Díaz and González-Solís, 2010), and mosquitoes that had fed either on chickens or humans (Rasgon, 2008). However, these have not been controlled studies, and none have attempted to apply the methodology to ticks for discrimination of different hosts or for parasite physiological age determination. In this proof-of-concept experiment, analytical precision for ␦13 C was comparable for standard-sized samples (7 ticks pooled, mean ± SD sample mass 0.65 ± 0.07 mg) and minimum-sized samples (3 ticks pooled, mean ± SD sample mass 0.28 ± 0.03 mg) (data not shown). Samples containing a single tick (mean ± SD sample mass 0.10 ± 0.01 mg) yielded significantly (P < 0.001) lower ␦15 N values (mean difference 0.75‰). However, the methodology is currently being developed such that single tick analyses will be attainable. This is important for determination of host identities since nymphs will only have fed once, as larvae. We have shown with unfed nymphal I. ricinus ticks that tick–blood isotope spacings are predictable. Therefore this methodology, pending suitable development for field use, has potential for identification of hosts utilized by a particular population of ticks. Additionally, we found that isotopic composition could potentially be used to determine the physiological age of unfed ticks, and this may be of use in further life cycle studies. Acknowledgements The NERC Life Sciences Mass Spectrometry Facility, East Kilbride, UK, provided access to isotope ratio mass spectrometry. Ticks were reared at IS Insect Services GmbH, under license from Landesamt für Gesundheit und Soziales, Berlin. 227 References Allan, B.F., Goessling, L.S., Storch, G.A., Thach, R.E., 2010. Blood meal analysis to identify reservoir hosts for Amblyomma americanum ticks. Emerg. Infect. Dis. 16, 433–440. Ben-David, M., Flynn, R.W., Schell, D.M., 1997. Annual and seasonal changes in diets of martens: evidence from stable isotope analysis. Oecologia 111, 280–291. Crawford, K., McDonald, R.A., Bearhop, S., 2008. 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