Comparison of the Effectiveness of Different Tags in the Sea Urchin Paracentrotus lividus (Lamarck, 1816)

Abstract

The marking of sea urchins was implemented with the main objective of being able to individually identify the urchins in the natural environment once released. In addition, it’s very useful to monitor individuals in studies of growth, movements, development, population dynamics, etc., that develop in the natural environment. Numerous different marking methodologies have been tested for sea urchins, either by physical marking (external and internal labels) or by using chemical marking methods consisting of the use of fluorochromes, which adhere to the calcified structures of the urchin. In this work, 5 different physical marks were used to mark 400 urchins of the Paracentrotus lividus species, which were kept for a month at the ECIMAT facilities in Toralla island. The efficacy of the methods used in each case was analyzed, comparing the survival rate and the tag retention rate of the tagged urchins obtained with each tagging methodology.

1. Introduction

The impoverishment of the health of coastal ecosystems in general increases due to overfishing, which has generated a rapid decrease in resources with repercussions on the economic sustainability of the global fishing sector, also leading to a decrease in biodiversity and a reduction in the food security [1].

The ecological importance of sea urchins is crucial, since they are the major regulators of the biomass of macroalgae on the seabed, which proliferate uncontrollably in habitats where sea urchin populations are depleted or completely disappeared [2, 3, 4, 5, 6], producing an imbalance in coastal ecosystems that affects the capture of other fishery products. The sea urchin is very sensitive to extraction due to the low population densities that it presents to the preference of individuals to inhabit shallow waters [7, 8, 9], and other factors such as human contamination or disease [10].

On the other hand, its economic importance has increased significantly in recent decades, due to the increase in demand for this product in the world market; conversely, the global catch of sea urchin from fisheries decreased from 117,000 tonnes in 1998 to 69,202 tonnes in 2014 [1]. This decrease was mainly due to the reduction in catches of the large world producers, such as Chile and the United States [11, 12], to counteract the overexploitation to which sea urchins were being subjected, although these measures were not effective for the recovery of natural populations.

Sea urchins’ gonads have been used as food since Roman times. Some species of sea urchins have been exploited commercially as food resources since the seventeenth century, being a highly valued resource in the market, where the highest quality gonads are a “gourmet” product that can reach a value of $ 300/kg. Asian countries are the major producers of sea urchins, with a total volume of 73,000 T per year [13], which in economic terms represents a total of between 200 and 300 million dollars [13].

In Europe, the most commercially important species is Paracentrotus lividus, Galicia is the main producing region of this species, with a total of 695 T of the sale in the fish markets during 2019 and an average sale price of € 8.28/kg, reaching peaks of up to € 24/kg of sea urchins [14].

As a consequence of this overfishing and the need to restore depleted natural populations, aquaculture production of the main commercial species has increased greatly in recent years [15, 16, 17], carrying out large-scale restocking tasks in different regions affected by the overexploitation of natural sea urchin banks around the world.

In order to monitor the urchins released to the natural environment and thus carry out subsequent studies on the effectiveness of the repopulation performed (survival, population dynamics, temporal evolution, etc.), it is necessary to identify the individuals released through the use of different types of individual brands. The tags used must be as less invasive as possible, so that they do not affect the growth or movement of individuals in the environment, and must present the maximum percentage of survival and retention rate possible for later recovery of the marked specimens.

There are various methods for marking sea urchins that have been studied and tested over the years in different species. Some of these studies are listed below, classified according to the type of marking used.

In general terms, the marking studies carried out with sea urchins were based on the use of five types of marks: 1) external labels or other markers of different shapes and colors inserted in the spines [18, 19, 20, 21, 22, 23]; 2) tags anchored by a perforation of the urchin’s test [18, 24, 25, 26, 27, 28, 29]; 3) passive integrated transponder (PIT) tags [30]; [13, 31] coded wire labels or CWT [31]; 5) marking using fluorochromes, such as tetracycline or calcein [32, 33, 34, 35, 36, 37, 38, 39]. All the methods used have advantages and disadvantages. The physical marking techniques of urchins, both external and internal, generally have low survival or retention rates of the mark, especially, when released to the natural environment, and may also affect the growth of marked sea urchins, and are not viable for the marking of small individuals [40].

The tags that performed the best so far in terms of survival and retention rate of the mark are the passive integrated transponder tags (PIT Tags), which consist of a cylinder fitted with a copper antenna and a microchip programmed with a number of identification (they contain millions of unique codes for the identification of the marked specimens), although they are not suitable for marking urchins with a test diameter less than 25 mm, and their effectiveness varies according to the species of sea urchin that is marked, reducing the survival rate of tagged urchins once released to the natural environment [41].

Chemical labeling with fluorescent substances works just as well as other physical labeling methods, also allowing the marking of large numbers of urchins of any size by immersing individuals in fluorochrome baths [42] or polyfluorochrome [42], although they present a significant disadvantage with respect to other marking techniques, being necessary the sacrifice of marked urchins to detect the mark, which makes it unfeasible to study the evolution of juveniles released for restocking purposes of overexploited areas. The objective of this work is to analyze the efficacy of 5 different physical marks for the identification of individuals of the species P. lividus.

2. Materials and methods

1. In June 2020, 400 juvenile urchins (P. lividus) were received from the coast of Cangas do Morrazo (Pontevedra, Galicia): 42° 16′40 ″ N 8° 47′23 ″ W, with an average of size 20 mm in diameter and an average weight of 5.4 g.

   The urchins were distributed in boxes of 50 liters of capacity, at a density of 20 urchins per box, with seawater filtered in an open circuit, continuous aeration, and feeding “ad libitum” with brown macroalgae of the genus Laminaria sp. The duration of the experiment was 1 month (started on June 23, 2020, ended on July 24, 2020). Five types of different tags were used for sea urchins:

   1. Colored stickers (Hallprint brand): FPN 8x4 (glue on shellfish tag), in 3 different colors (green, purple, and beige) with individual numbering.

   2. T-Bar labels (Hallprint brand): TBA (standard anchor T-Bar tag), in three different colors (green, purple, and beige) and individual numbering.

   3. Minitransponder (Trovan brand): 1′4 x 8 mm, high-performance ISO FDXA glass, with IM-200 1.4 Mini Tradi injector.

   4. Pieces of galvanized wire (3–4 mm long and 1 mm thick).

   5. PIT Tags (Hallprint brand): FDX Food-safe polymer (2.18 x 11.4 mm).

   All the treatments with three replicas per tag and a consistent control of unmarked urchins.

   The stickers were adhered to the urchin’s test with the help of Loctite glue (Figure 1), in an area of ​​the test where the spines of that area were previously sectioned, and the area was dried with absorbent paper. The galvanized wire sections were introduced into the coelomic cavity through the peristomial membrane of the urchin; Trovan Minitransponders and Hallprint PIT Tags were also introduced through the peristomial membrane of the urchin using a specific injector for each type of tag. The T-Bar labels were introduced in two ways into the urchins, half of the labels were introduced through the peristomial membrane and the other half through a hole drilled in the aboral half of the test with the help of a needle.

2. The 270 surviving urchins from the captive tagging experiment were housed in a tray belonging to the polygon of rafts of the San Xosé de Cangas do Morrazo Fishermen’s Association (Figure 2), on October 9, 2020, with an average size of 18 mm in diameter and an average weight of 2.98 g, in order to obtain the recapture rate of the marks in the natural environment.

Figure 1.

Different techniques for mechanical marking of sea urchins: a) Hallprint adhesive stickers; b) insertion of T-Bar tags through the peristomial membrane; c) insertion of T-Bar labels by drilling the aboral region of the test; d) insertion of galvanized wire through the peristomial membrane; e) injection of a mini transponder (Trovan brand) through the peristomial membrane; f) injection of PIT Tag (Hallprint brand) through the peristomial membrane.

Figure 2.

a) Urchins housed in the raft’s lantern; b) urchins taken from the lantern; c) Trovan Minitransponder reader; d) metal detector.

The coordinates of the raft are as follows:

Latitude Lenght.

Vertex A 42° 16′ 31” N 08° 43′ 53” W.

Vertex B 42° 16′ 43” N 08° 43′ 30” W.

Vertex C 42° 16′ 42” N 08° 43′ 15” W.

Vertex D 42° 15′ 49” N 08° 43′ 22” W.

Vertex E 42° 15′ 35” N 08° 43′ 59” W.

The urchins were fed fortnightly with brown algae of the genus Laminaria sp.

The duration of the experiment was 5 months (started on October 9, 2020, ended on April 9, 2021).

3. Results

1. Below, you can see the survival and retention rates of the brand obtained with each type of tag employed.

   As can be seen in Figure 3, the survival obtained was high with all the tags used, except with the T-Bar labels inserted through a hole drilled in the aboral half of the test, since this perforation did not calcify and produced the death of more than 50% of the marked urchins.

   The retention rate obtained with each tag in captive conditions varied greatly, being insignificant in the case of the colored stickers and T-Bar labels introduced through the peristomial membrane of the urchin, which was totally expelled after a few days, and very high in the case of wire sections (93.33%) and Trovan Minitransponders (83.33%), which suggests the adequacy of these two types of marks for marking of individuals of P. lividus in captive conditions.

   In the case of the Hallprint stickers, it was observed that the urchins detached them with the help of the spines and pedicels, presenting the lowest retention rate of the mark together with the T-Bar labels and the Hallprint PIT Tags, therefore, these marks are not suitable for the identification of urchins released into the wild.

   Another drawback observed was that the glue produced abrasion injuries in the area of ​​the urchin’s test where it was applied (Figure 4), leaving important sequelae to the urchins marked with this technique, although it did not cause the death of the marked individuals.

   A Chi-square test (p-value < 0.05) was performed to statistically compare the efficacy of the tags used, resulting in significantly lower survival in urchins labeled aborally with T-Bar labels, while the retention rates of the wires and the Trovan Minitransponders were significantly higher than those of the rest of the tags employed.

2. Results obtained with the different labels after housing the marked urchins for 5 months in a culture structure (Figure 2a) suspended from a tray belonging to the polygon of rafts of the San Xosé Fishermen’s Association, in Cangas do Morrazo (Ría de Vigo).

   Recapture rate:

   • PIT Tags Trovan: 71%

   • PIT Tags Hallprint: 15%

   • Stainless steel wires: 0%

   • Stickers: 0%

   • T-Bar labels: 0%

Total Survival rate: 99,14%

Figure 3.

Percentage of survival and retention rate of the urchin’s tag with the different tags employed.

Figure 4.

Abrasive lesions on the urchin’s test caused by the glue used to fix the Hallprint stickers.

In the second part of the study performed with the urchins housed in a lantern suspended from the raft in the natural environment, very high survival rates were obtained, the total survival rate being 98.89% of the urchins housed in the raft.

The mark-recapture rates obtained were very low after 5 months, except in the case of the Trovan Minitransponders, with which a recapture rate of 71% was obtained, and a mark retention rate of 100% in recaptured urchins, which makes them the most appropriate type of tag for monitoring sea urchins of the Paracentrotus lividus species, both in captive conditions and in the natural environment and in short- and long-term studies.

4. Discussion

There are different marking techniques for sea urchins, developed over the last decades, both physical (external and internal) and chemical (different fluorochromes). The characteristics that an effective tag must meet for the identification of individuals released to the natural environment are the following: high survival rate of the urchin, high retention rate of the tag for at least a few months, ease of detection on the seabed for divers, ease of identification of tagged individuals, speed of tag application (in order to tag as many sea urchins as possible in a short period of time), and low cost.

Mechanical marking techniques in sea urchins, either by placing external labels of different types or by intraperistomial insertion of internal labels, have the disadvantage of generally presenting low rates of tag retention [18, 43], which may also affect the survival of the marked population. Furthermore, they are not viable to mark small-sized urchins [40] and must be adapted to the morphology of the species to be marked, since there are important differences in the effectiveness of the marks depending on the size of the species, the length of thorns, etc. Tuya et al. [44] used fishing hooks attached to a cork buoy by means of a line to mark long-spined urchins of the Diadema antillarum species, obtaining a very high retention rate of the mark between 80 and 90% of marked urchins. Due to the low retention rate generally obtained with external labels, labels inserted in the test began to be used with variable success [25, 26]. Lees [45] tagged Strongylocentrotus purpuratus with stainless steel wire and obtained a 92% loss of tagged urchins after 9 months in the wild. Neill [21] marked sea urchins with anchor tags designed for marking fish and provided with a specific numbering, obtaining a very low survival rate from 11 days after the urchins were released to the natural environment.

The half-life of a cohort of Centrostephanus coronatus tagged with stainless steel wire introduced through the test was only 15 days [22]. Duggan & Miller (2001) marked individuals of the Strongylocentrouts droebachiensis species with both external and internal (anchor) tags, attached to the urchin’s test by means of a hole drilled in the test with the help of a needle, which caused a mortality of more than 50% of marked urchins within 1 month. Other authors who used anchor tags in the test [22, 25, 29, 46, 47] obtained a higher survival rate of urchins, although long-term survival terms in the natural environment remained low.

Passive Integrated Transponder Tags or PIT Tags are currently the most effective physical marking method in sea urchins, in relation to the uniqueness of the mark and its external readability, and do not affect the growth of urchins in the long term [30]. These labels have the advantage that they contain millions of unique codes, whereas, with chemical marking techniques such as the use of polyfluorochromes, only 4096 unique codes would be generated. Their main disadvantage is that they cannot be used to mark urchins smaller than 25 mm in diameter without causing the death of the individual.

The chemical marking method (through the use of fluorochromes) in sea urchins is also effective, also allowing the marking of a large number of urchins of any size by immersing individuals in fluorochrome baths [42]. This method is based on the incorporation of chemicals that bind to calcium, such as oxytetracycline, alizarin, calcein, etc., applied at the time of marking, which binds to the skeletal structures of various marine organisms [48]. Marking occurs through immersion, injection, or feeding.

This method has several advantages over other techniques used: 1) a large number of individuals can be marked at high speed in the natural environment; 2) minute growth increases can be detected; 3) very small individuals can be tagged by immersing them in baths containing the fluorochrome.

The main disadvantages of this technique are: 1) urchins must be slaughtered to detect the mark; 2) sample preparation is laborious and time-consuming; 3) the increase in test diameter cannot be directly measured, but is estimated from the growth increments of the individual skeletal structures; 4) in the case of skeletal resorption occurs in tagged urchins, negative growth would not be detectable.

The need to euthanize the tagged individual to detect the tag makes this tagging method unsuitable for the identification of large numbers of specimens released into the wild for repopulation purposes.

5. Minimum marking size

After verifying that the two tags that gave the best results in terms of survival and retention rate of the tag were the wires and the Trovan Minitransponders, consistent tests were carried out on marking juvenile urchins of different sizes, in a range of 10–30 mm in diameter, in order to determine the minimum size that juvenile urchins must have in order to be marked successfully without presenting mortality, obtaining a result of 13 mm in minimum diameter in the case of Trovan’s Minitransponders (8 mm in length), and only 11 mm in diameter for 4–5 mm long galvanized wire sections. These minimum marking sizes are lower than those found in the literature for physical labels, which are generally not less than 20 mm in diameter without excessive mortality, and is in the range of 10 to 15 mm in length of the urchins that de la Uz et al. [49] marked with coded wire tags (CWT), allowing the monitoring of juvenile P. lividus individuals from 5 to 6 months of age.

6. Conclusion

The results obtained in this work allow us to conclude that Trovan’s Minitransponders are an appropriate brand to monitor sea urchins of the P. lividus species in short and long-term studies, both in captivity and in the natural environment, presenting a high rate of retention and recapture in culture structures, and they are also suitable for marking juveniles of P. lividus from 13 mm of test diameter, with an approximate age of 5–6 months.