Comparison of two external tagging methods used for the identification of individual adult Pacific oysters, Crassostrea gigas.
KEY WORDS: oyster, Crassostrea gigas, t-bar tags, glue-on laminated tags, longitudinal study
Tagging methods for marine and freshwater species of bivalve can be broadly classified into two categories: internal and external. The use of internal tags, such as passive integrated transponder (PIT) tags that are placed into the internal cavity of the bivalve between the pallial mantle and shell or within the pallial mantle, is much less common than the use of external tags (McMahon & Williams 1986, Kurth et al. 2007, Riley et al. 2010). Internal tags tend to be much more costly as an electronic reader is required to relocate the tags, they can be rejected by the organism, and insertion generally requires some form of relaxant or instrumentation, such as a micropipette, to hold open the valves while the tag is inserted into the internal cavity or tissues of the bivalve (Kurth et al. 2007).
External tags on the other hand are able to be visualized on the external surface of the bivalve and they do not require relaxation to apply. These tags tend to be more practical as the surface of the shell provides a hard and durable surface for tag application (Kurth et al. 2007). The methods described include shell engraving (Mattice & Wright 1986, McMahon & Williams 1986), paints and dyes (Buttner & Heidinger 1980), external PIT tags (Kurth et al. 2007, Tang et al. 2014, Hua et al. 2015), glue-on shellfish tags (Bayne 2002, Twist et al. 2016), self-adhesive tags (McMahon & Williams 1986, Riley et al. 2010), coded wire tags anchored within the external ligaments of the bivalve (Lim & Sakurai 1999), and nylon rivet tags anchored within the respiratory pores (Prince 1991, Lloyd-Jones et al. 2014). The main problem with external tagging methods are a decline in tag legibility over time and low tag recovery rates due to abrasion of tags on sediments or husbandry infrastructure, or biofouling of shells, particularly during long-term studies. T-bar anchor tags have been used in fish and crustacean studies, but apparently not in bivalve studies (Ogbum & Ruello 1999, Liu et al. 2013, Bodine & Fleming 2014, Meynecke et al. 2015). These tags may provide a solution for long-term studies on bivalves as they can be applied securely through a small hole created in the shell, and they then protrude from the shell rather than lying directly on the surface of the shell. In this location, the tags may be less subject to biofouling which may improve the long-term legibility of the identification number.
The aim of the current experiment was to compare two simple external identification methods in adult Crassostrea gigas (Thunberg, 1793): (1) a plastic-laminated glue-on number and (2) a t-bar anchor tag inserted into the upper shell, to determine the optimal methodology to use in longitudinal studies greater than 1 y in duration.
MATERIALS AND METHODS
Triploid Crassostrea gigas were recruited from several experimental oyster populations (Paul-Pont et al. 2013a, Paul-Pont et al. 2013b, Whittington et al. 2015, Hick et al., unpublished data). Briefly, these oysters originated from several batches of single-seed hatchery-reared spat (Shellfish Culture, Tasmania, Australia) and were initially maintained in two Ostreid herpesvirus-1-free estuaries: the Hawkesbury River [Broken Bay Oysters, Mooney Mooney, New South Wales (NSW)], prior to the detection of Ostreid herpesvirus-1 in that estuary in January 2013 (Paul-Pont et al. 2014), or the Shoalhaven River (Goodnight Oysters, Greenwell Point, NSW). When required, oysters were deployed in Woolooware Bay in the Georges River estuary, NSW, Australia. Woolooware Bay is located on the southern shore of Botany Bay, approximately 16 km south of the center of Sydney, NSW (Paul-Pont et al. 2013a, Paul-Pont et al. 2013b, Whittington et al. 2015).
Recruited oysters were maintained on plastic trays (see tagging and observation of oysters) fixed on wooden racks, at a standard intertidal growing height used by the local farmers. One hundred and fifty apparently healthy Crassostrea gigas [approximately 10- to 45.5- mo old; 60- to 160-mm shell length; 80-700 g closed, wet weight (shell + tissue)] were opportunistically, nonselectively sampled from this population in May 2014 for tagging and observation.
Tagging and Observation of Oysters
Each oyster was tagged with an individual identification number using two different methods. The first method was a plastic-laminated paper number glued to the top shell of the oyster using a two-part epoxy resin (Araldite 5 Minute Adhesive). Biofouling and over-catch present on the top shell was removed prior to application of the tag. The application area was dried with paper towel and each tag was glued onto the flattest part of the top shell, toward the midline of the oyster (Fig. 1). The second identification method was a standard, numbered t-bar anchor tag (TBA-1 Hallprint, Australia). A 3mm hole was drilled into the top shell of each oyster, approximately 10 mm in from the growing edge, using an electric rotary tool (Dremel 8200 10.8 V cordless rotary tool) and a t-bar tag was inserted into the hole using a standard tagging gun (Avery Dennison), taking care not to inject the tag into the soft tissues of the oyster. The hole was sealed with a layer of adhesive (Bostik Blu Tack) and a top coat of two-part epoxy resin (Fig. 1). These tags were monitored throughout the experiment to ascertain the best tag type to use in long-term studies.
Individually tagged oysters were maintained in two plastic trays (2 m x 1 m) fixed on wooden racks, at standard intertidal growing height, located at a farm site known as 'Site C' in Woolooware Bay (Site C: 34.033987[degrees] S, 151.1471544[degrees] E). Each tray was divided into eight segments (0.5 m X 0.5 m), and 6-10 oysters were opportunistically, nonselectively allocated to each segment at the beginning of the trial (depending on the size of the oysters). Tagged oysters were not intermixed with non-tagged oysters, which were maintained in separate plastic trays at the same site. Tagged oysters were examined on five occasions over a 15-mo observation period: May 2014, August 2014, November 2014, March 2015, and August 2015. Minimal husbandry interventions were provided to the oysters: over-catch and biofouling were removed from the trays and shells of the oysters on the observation dates specified above. Care was taken not to dislodge the labels during these procedures.
Intact, legible tags were counted on the shells of both live and dead oysters at each observation, prior to the removal of dead oysters from the trial.
The two-sample binomial test (Genstat 15th Edition 2000-2015 VSN International Ltd., UK) was used to compare the retention rate of each tagging method.
Five oysters died as a consequence of the t-bar anchor tagging method. These mortalities were noted immediately after completion of the tagging procedure in May 2014. No oysters died on the day of application as a consequence of gluing plastic-laminated labels onto the top shell. During the study, another 51 oysters died, but as deaths were also observed in nontagged oysters from the same cohort, which were located in separate trays at the same site, death was unlikely to be caused by the tagging methods.
Observations of the tagging methods at each sampling time point indicated that t-bar anchor tags were better for use in long-term studies than plastic-laminated tags glued to the top shells of the oysters. All tags were applied in May 2014 and by August 2014, 3 mo after initial application, oysters with t-bar anchor tags had a higher retention rate of intact legible labels (142 of 143 oysters examined) compared with oysters with plastic-laminated labels glued to the top shell (93 of 143 oysters examined; P < 0.001; Table 1). By August 2015, 15 mo after the application of the tags, oysters with t-bar anchor tags still had a higher retention of intact, legible tags (86 of 94 oysters examined) compared with oysters with plastic-laminated labels (0 of 94 oysters examined; P < 0.001; Table 1).
Deaths due to the t-bar anchor tagging method occurred when the epoxy resin top coat drained into the internal shell cavity and contacted the soft tissues of the oysters. These deaths occurred only in small oysters (60-70 mm in length), as a result of technical deficiency while learning how best to apply the adhesive seal and epoxy resin top coat to these small oysters. No oysters died during or after application of the plastic-laminated labels. All of the adhesive seals placed over the t-bar tags in May 2014 had become dislodged by August 2015; however, the majority of t-bar tags had remained secure within the hole drilled into the top shell of the oyster. In many cases, the hole in the shell, in which the tag was placed, did not close up entirely, likely due to the movement of the t-bar tag in the water column. It is unknown what impact the 3-mm drill hole had on the survival of the tagged oysters, as this was not examined. It is possible that the holes acted as an entry point for marine parasites or pathogens; however, it is unknown whether such a small aperture would increase the risk of pathogen infection any further than that already caused by the normal filtering activities of the oysters (Ben-Horin et al. 2015).
Although t-bar anchor tags were more invasive than the plastic-laminated labels, the t-bar tags were more resistant to biofouling. The plastic-laminated labels either dislodged or were overgrown with biofouling rendering them illegible. T-bar tags also became biofouled; however, these tags were simpler to locate on the shell even when overgrown, and were more easily cleaned. T-bar tags can be easily read without the need for external hardware or equipment, in comparison with other methods such as external PIT or coded wire tags (Lim & Sakurai 1999, Kurth et al. 2007). The retention rate of t-bar anchor tags in this study was 91.5% after 15 mo in temperate marine field conditions (Table 1). In comparison, nylon rivet tags applied to the respiratory pores of Haliotis rubra had an annual tag shedding rate of 4%-35% (Prince 1991). Riley et al. (2010) demonstrated a 94% recovery rate for self-adhesive, vinyl-coated cloth tags applied to the top shell of Corbicula sp. after 440 days. Recovered tags were 100% legible; however, clams in that study were maintained in sand-bottom outdoor aquaculture raceways and not in open estuarine conditions. Thus, biofouling on the shells of the clams and on the adhesive tags was minimal (Riley et al. 2010).
The results of this study suggest that t-bar anchor tags are a better identification method for use in longitudinal studies (15 mo in length) involving adult Crassostrea gigas than plastic-laminated labels glued to the exterior of the shell.
This work was funded by the University of Sydney and in part by the Australian Government through the Fisheries Research and Development Corporation. Leonard Drake, Robert Hill and Keith Duggan of Endeavour Oysters, in the Georges River estuary, NSW are thanked for their assistance in the field.
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OLIVIA EVANS, PAUL HICK AND RICHARD J. WHITTINGTON *
Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, 425 Werombi Road, Camden, New South Wales, 2570, Australia
* Corresponding author. E-mail: email@example.com
TABLE 1. Comparison of the t-bar anchor tag and glue-on laminated label methods used to individually identify Crassostrea gigas adults over a period of 15 mo. Date of observation (number of oysters available for August November examination) May 2014 (150) 2014 (143) 2014 (143) Method Proportion of intact/legible tags T-bar anchor tags 1.000 0.993 0.958 Glue-on laminated tags 1.000 0.650 0.559 Results of two-sample binomial test P value Not applicable < 0.001 < 0.001 95% confidence interval * Not applicable 0.263-0.422 0.311-0.486 Date of observation (number of oysters available for March August examination) 2015 (139) 2015 (94) Method Proportion of intact/legible tags T-bar anchor tags 0.928 0.915 Glue-on laminated tags 0.094 0.000 Results of two-sample binomial test P value < 0.001 <0.001 95% confidence interval * 0.770-0.899 0.859-0.971 The proportion of intact-legible t-bar tags and glue-on tags observed on each date were compared using a two-sample binomial test. Each individual oyster was tagged with both a glue-on laminated label and a t-bar anchor tag. * 95% confidence interval for difference between proportions.
Please note: Some tables or figures were omitted from this article.
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|Author:||Evans, Olivia; Hick, Paul; Whittington, Richard J.|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2016|
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