DataDot^sup DNA^: an alternative marking system for tortoises of genus Testudo

Headnote

Abstract. It was analyzed the effectiveness of one method of individual and unique marking, alternative to the appli- cation of microchip, on 17 specimens of Testudo hermanni through DataDotDNA technology. This technology has prov- en to be an effective system of marking of Testudo spp., answering the need for unambiguous identification of indi- viduals. Further advantages are the easy application and reading, the long-term resistance as well as the difficulties of possible fraudulent tampering.

Keywords. Testudo hermanni, Testudo graeca, Testudo marginata, DataDotDNA, marking.

The species Testudo hermanni Gmelin, 1789, Testudo graeca Linnaeus, 1758 and Testudo marginata Schoepff, 1792 are enclosed in Annex A to Council Regulation (EC) No 338/97 and subsequent amendments and revi- sions. Council Regulation (EC) No 338/97 deals with the protection of species of wild fauna and flora by regulat- ing the trade therein, harmonising the implementation of the "Washington Convention on International Trade in Endangered Species of Wild Fauna and Flora" at Euro- pean level.

The legal obligation to mark all the living individuals of species included in Annex A has been introduced to control the pet trade, in accordance with the law 7 Febru- ary 1992 No 150 and the mentioned Council Regulation. The animals should be univocally identified by microchip ISO 11784:1996 (E) and 11785:1996 (E) compliant. Due to the commonly used microchip dimensions (mm 12 x 2.12), and in order to guarantee the animals welfare, the CITES Management Authority (in Italy the Ministry for Environment and Territory and Sea) provided, for indi- viduals under the age of five years, the possibility to use a photographic identification archive, instead.

The recent commercial availability of smaller micro- chip (mm 7 x 1.25) allowed the marking procedure of individuals within their first year of age, namely when their carapace reached the length of 5 cm. However, microchip inoculation is an invasive action that entails intrinsic risks, which are common to all the surgical pro- cedures, but more hazardous when performed on small and very young animals. Hence, in order to minimise all the risk factors, it has been tested the possible implemen- tation of the DataDotDNA technology for marking Testudo hermanni, which growth rate in the first years is lower than those of the other two species T. graeca and T. marginata.

Microdot identification technology has been applied for several years in Australia, United Kingdom, Italy and many other countries, to the prevention of car theft or to guarantee the authenticity of art and collector objects. More recently, it has been employed both to oppose the oyster theft (Honan, 2007; Sydney Morning Herald, 2007), and in entomological researches, to investigate the orchid pollination by wasps (Whitehead and Peakall, 2013). This technology is based on polymer discs (diam- eter one millimetre or less) assembled with a unique laser impressed alphanumeric code. Discs are secured on the involved surface using different kinds of adhesive.

The objectives of our research were: i) prove that DataDotDNA identification technology is an efficient alter- native, or complementary marking method for Testudo tortoises; ii) test its resistance to the removal by means of natural agents (rubbing or other), or by illicit manage- ment (removal and reuse of disks); iii) verify that all the procedures were in compliance with the animal welfare requirements, and in particular that they do not affect the individual growth rate.

Seventeen individuals of unspecified gender of Tes- tudo hermanni, between 1 and 3 years old, were divided into two homogeneous groups (A: experimental; B: con- trol), constituted by 9 and 8 individuals, respectively. During the trial period, the two groups were placed in large monitored enclosures, in semi-natural conditions, ambient temperature and same food supply.

An identification form was drawn up for each sub- ject. The main biometric measures (carapace length, width and height; plastron length; body mass) were recorded and filled in the form at the beginning of the trial. The shell of each subject of group A was cleaned and dried before placing the DataDotDNA technology id system. The identification system was composed by a 9 characters alphanumeric code, impressed in specular alternating rows on discs of 0.5 mm of diameter (Fig. 1). Discs were secured with three different adhesives based on: i) aqueous solution of dipropylene-glycol-mono- methylether (DGM); ii) methyl cyanoacrylate (CM); iii) n-butyl acetate (NB). The DGM based adhesive has UV fluorescence characteristics, which allow for easier detec- tion of discs glued on the shell. If necessary, the fluores- cent pigment (CIBA Uvitex NFW Optical Brightener) could be easily included in the composition of the other two kinds of glue, following the manufacturing instruc- tion for component ratios. For each subject, three poly- ester discs were separately suspended in each of the adhe- sive solutions and placed, with the aid of a small pad, on one vertebral and two coastal scutes. After the drying time, which ranged from 5 to 20 minutes (quicker for CM, longer for NB), the readability of the discs was test- ed with both a portable optical microscope (100×), and a portable digital microscope (27-108×) (Fig. 2). At the end of these operations, the animals were brought back to their enclosures.

Biometric measurements were taken monthly dur- ing the trial period, according to the hibernation periods, for a total of 10 records for 13 individuals, and 11 records for the remaining 4. During these surveys, the conditions and readability of each disc were checked, as well as the presence of localised or generic growth aberrations of the shell, or on-going pathological conditions. Since the trial period started before for 4 tortoises - born between 2008 and 2009, and equally distributed in experimental and control groups - and later for the others (born in 2010 and 2011), an interval of 15 months, included one win- ter latency period, has been taken into consideration for each animal. The differences between the last and the first measurement, over such interval of time, were recorded for each biometric parameter (Table 1, 2).

ANOVA for repeated measures cannot be applied to analyse two groups of paired data (Huck and McLean, 1975). Feasible alternatives are: i) t-test for independent groups on the differences between last and first meas- urements (Hopkins, 1997), or ii) analysis of covariance (ANCOVA) with the experimental and control groups as independent variable, the last measurement as depend- ent variable and the first one as covariate. In these cases, the latter is considered the most powerful test (Huck and McLean, 1975). Covariate permits to remove the influ- ence of differences of the starting measurement within and between groups.

Neither abnormal development of carapace or plas- tron, nor local or systemic pathologies were found, in any subject, during the experimental period. The biometric data recorded were congruent with the age of any single tortoise, and the growth rates were in line with literature data (Cheylan, 1981).

All the alphanumeric code reading tests had positive result, for all three adhesives employed, but some discs were lost by three tortoises. Reading was reasonably easy with the portable optic microscope at 100×, while with the digital one at 108× it was pretty hard, due to difficul- ties to focus a bended surface, like the carapace. The only relevant reading problem was caused by discs applied on carapace black spots, because they were transparent with the code impressed in black.

ANCOVA gave not significant differences, at ? = 0.05, between the mean value of all biometric meas- ures (Table 3). Therefore, we can confidently conclude that DataDotDNA marking technology did not affect the growth of subjects included in group A.

DataDotDNA technology proved to be an efficient marking system for tortoises of Testudo genus, alternative, or complementary, to other currently adopted techniques (Stubbs et al., 1984; Guyot and Clobert, 1997), at least for tortoises raised in controlled conditions. This methodol- ogy meets the current regulation requirements in terms of unambiguous identification of individuals, durability on the marked animal, ease placement and reading and difficulty of possible illicit tampering of the mark.

Long-term durability tests of microdot discs on Tes- tudo individuals are currently in progress, as one of the objectives of our further research. However, the following tests on durability were carried out by an independent laboratory of Melbourne (Australia): accelerated aging treatments (high and low air temperature, high humid- ity) and removal tests (high pressure cold water/deter- gent cleaner test, high pressure hot water cleaner test). Those experiments gave permanence rates, in the range of 90-100% of the marking, for over 20 years (Johnson, 2010). The current (2013) price, excluding VAT, for a kit of 500 microdots (0.5 mm) is about 15 Euros.

The only remarkable limit of this method is the impossibility to read discs placed on black surfaces. How- ever, this problem could be solved by laser micro engrav- ing the code on different substrata, like ceramic. This new technique - 0.2 mm micro-tags, up to 15 alphanu- meric characters, better readability - has been recently developed by DataDot Technologies Ltd. (Australia), and should be tested in the field, as well.

Moreover, the fraudulent removal of discs is pos- sible, as is of micro-chips, but their reuse appear to be very hard. In fact, the removal process damages the discs, which get included into the adhesive layer used to fix them. Readability of discs was negatively affected by the thickness of the adhesive layer. The deterrent factors against illicit removal of marks are supported by: i) the dimensions of the polyester discs: 0.3 mm with 7 char- acters/row code, 0.5 mm with 11 characters/row code or 1.0 mm with 21 characters/row code. The smaller the disc, the more difficult will be their visual detection; ii) the quantity of discs, with the same code, that could be placed on each animal: the more the discs placed, the harder will be their complete removal. Hence, the ration- al could be using several small discs (0.3 mm), fixed on different parts of the shell. This should be a strong deter- rent against illicit marking removal.

ACKNOWLEDGEMENTS

The experimental animals belong to a group of Testudo her- manni that have been entrusted in judicial custody in the struc- tures of the State Forestry Corps. We thanks DataDot ITALIA S.r.l. for their kind willingness to share useful ideas during the design of the experiments, and for granting free-of-charge all the equip- ment, including adhesives and reading devices, used during the trials. We thanks two anonymous reviewers for their suggestions.

References

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