Efficacy of calcein and Coomassie Blue dyeing of shell growing-edges and micro growth-bands: Ageing juvenile of Pinctada mazatlanica (Pterioida: Pteriidae)
Eficiencia de la tinción de bandas de crecimiento con calceina y azul de coomassie, para estimar la edad de jóvenes de Pinctada mazatlanica (Pterioida: Pteriidae)
Patricia L. McCoy Loría1* & Leonardo Huato-Soberanis2*
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Abstract
Age validation is the first step to determine shellfish species age determination. This information is vital for different inferential models used in marine ecosystem ]]>
Pinctada mazatlanica. Thus the objectives of this study included: the evaluation of calcein to mark a shell growing-edge, and the efficacy of Coomassie Blue staining on posterior shell growth, to produce visible micro growth-bands that would enable age validation of juvenile mother-of- pearl oysters. Oysters were collected and cultivated at The Perlas del Cortez S. de R. L. MI. pearl-farming opera tion, in ]]>
=0.735mm) than at the higher one (=0.577mm) (not significant difference, p=0.198). Calcein marking of ]]>
Key words: age validation, calcein, coomassie blue dye, Gulf of California, micro growth-bands,
Pinctada mazatlanica, shellfish.
Resumen
La validación de la edad es el primer paso para determinar las edades de las especies de moluscos, esta información es de vital importancia para los diferentes modelos de inferencia utilizados en actividades de gestión de los ecosistemas marinos. Diversas técnicas de valida- ción se utilizan para marcar estructuras de carbonato de calcio, aunque la técnica de marcado de calceína en ostras nunca se había utilizado para la validación de la edad de P. mazatlanica. Los objetivos de este estudio fueron: evaluar la ]]>
=0.735mm) que con la de mayor concentración (=0.577mm), pero no significativamente (p=0.198). El marcado de conchas con calceína y tinción de matrices proteicas con azul de Coomassie posterior a su crecimiento, es recomendando como un método para la validación de la edad facilitando el conteo de micro bandas de crecimiento internas de la concha. Además, permitirá estimar edades con el fin de predecir fechas de colonización y ubicación de bancos naturales. ]]>
Palabras clave: calceina, Golfo de California, micro-bandas de crecimiento, ostras, madreperla, Pinctada mazatla nica, azul de coomassie, validación de edad.
Age determination of shellfish is ]]>
Techniques for ageing have been tested in several studies by using various chemicals as markers of different marine organisms containing calcium-carbonate (Nakahara, 1961; Hidu & Hanks, 1968; Jones, Thompson, & Ambrose, ]]>
Poly-anionic calcein (fluorescein complex) is a fluorescent compound that unites to CaCO3 in growth structures that are bio-mineralized (such as shells) of organisms. Once laid down in the CaCO3 growth-band, calcein ]]>
Natural growth marks can be observed on both the outer and inner parts of shells. How- ever, external marks are lost in older organisms because of shell erosion, thus leading to the use of internal marks. Internal mark use may require the preparation of thin sections using optical microscopy. Microscopic growth increases in some calcareous structures are ]]>
Pinctada mazatlanica, Class: Bivalvia, Order: Pterioida, Hanley ]]>
The objectives of the present study were to assess, in mother-of-pearl bivalve juvenile shells the: a) efficacy of fluorescent calcein incorporation to mark the growing-edge by injection and by submersion in two concentrations, b) survivorship of individuals and differences in shell growth submersed under two calcein concentrations, c) efficacy of Coomassie Blue dye to stain the ]]>
Materials and Methods
Study area: The research was carried out at The Perlas del Cortez S. de R. L. MI. pearl-farming operation, in Pichilingue (24°16’ N-110°19’ W), on the South-Eastern coast of La Paz Bay, in the state of Baja California Sur, Mexico. The median minimal sea surface temperature (SST) is 16.9°C from January to April, and the maximum SST is of 28°C from August to October. The shoreline climate is arid, with semidiurnal tides, annual precipitation of 250mm (IG-UNAM, 1992) and a median salinity of ]]>
Oyster source: We ]]>
We initially used 36 oysters for the growing-edge marking experiment with injected cal- cein in March ]]>
Calcein marking of shell growing-edge by injection: We injected 36 oysters with a ]]>
30H26N2O13, Sigma, Chem. Abs. No. 1461-15-0) dissolved in local marine water, into the mantle cavity thru the byssus opening until it overflow (Kaehler & McQuaid, 1999; Cáceres-Puig et al., 2011). Oysters were then placed immediately inside commercial triangular rearing cages attached to an underwater, suspended long-line inside La Paz Bay at an approximate 3m depth for 16d, with a SST at 21.7±1.2°C in March 2008.
Calcein marking of shell growing-edge by submersion: In June 2010, 24 oysters were submerged in a 0.4g/L solution of calcein, using local marine water as a diluent (Fujikura et al., 2003). The remaining 26 oysters were submerged in a 0.7g/L solution of calcein (same diluent). All 50 ]]>
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All oysters were processed at the Laboratory of Fisheries Ecology at CIBNOR. Each of the 86 calcein-marked oysters were dorsal-ventral measured (height) before and after the cal- cein marking experiment, with an electronic digital vernier (Traceable Carbon Fiber Caliper 0.1mm, Ted Pella, Ultra-Cal Iv, USA).
Shell preparation: After extraction from rearing cages, each pair of shells was placed by individual on sticky plastic strips (3M Post-it Tape Flags 43x25mm, 3MTM, USA) that were labeled with a permanent marker for later individual identification. Each right shell valve was cleaned of internal tissues and prepared for examination; it was sliced and glued with cyanoacrylate ester (Instant Krazy Glue®, ]]>
®, USA).
All the shells were aligned to be ]]>
TM, USA); grain sizes used were 30, 12 and 3µm (Kennish, Lutz, Rhoads, 1980; Cerrato, 2000). Only one thin section was prepared for each individual oyster.
Because shell composition proportions affect micro growth-band coloration (Lutz & Rhoads, 1980), all sections were observed under an optical microscope (Olympus Bx41, Olympus America Inc., Melville, NY, USA) to count micro growth-bands, differentiating horizontal clear organic matrix lines from dark aragonite lines at 20x magnification (Taylor, 1973; Blank et al., 2003; Dauphin, 2003; Nudelman, Gotliv, ]]>
Additionally, fluorescent imaging was performed with the same microscope, but with a blue light filter, excited from 460 to 490nm. Calcein marks were detected at 20x magnification. Micro-photographs were captured of selected areas with a digitally- integrated camera (Cool Snap, Media Cybernetics, San Diego, CA, USA) with the Image-Pro Plus program, v.5 ]]>
Classification of calcein marks: ]]>
Coomassie Blue dye: Finally, all individual shell slices of calcein-marked oysters were submerged in a solution of Coomassie Blue R-250 (0.05% Coomassie Brilliant Blue R-250, 40% methanol and 7% acetic acid; the standard used for electrophoresis) for 15min. This dye binds to shell matrix proteins between successive calcium carbonate depositions. Excess dye was eliminated from shell slices with a cleanser solution (40% methanol and 10% ace- tic acid) for 5s.
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Counting micro growth-bands: Sections were microscopically observed, photographed and analyzed with the same equipment and software at the same magnifications as described above. Only one slice preparation per shell was observed. For each shell slice, a random field of view with clearly-stained micro growth-bands was chosen for counting. One count was made for each shell section on each of three successive days. Random, acceptable fields of view were chosen for each count. This gave three random counts for each ]]>
The dependence on type of calcein-stained mark produced by each of the two calcein concentrations used in submersion was estimated by contingency table analysis. Difference in mean 15-d growth increments between both calcein ]]>
A posteriori means comparisons were estimated with least significant difference, Tukey and Scheffe tests. Statistical tests were carried out with the program Statgraphics Centurion xvI (Statpoint Technologies 2007, ]]>
Results
Calcein-marked growing-edge by ]]>
None of the 36 calcein-injected oysters in 2008 presented any indication of an initially visible fluorescently-marked band. A dim green fluorescent light was emitted from the whole shell width. No mortality was observed.
Calcein-marked growing-edge by submersion: Both submersion treatments were successful with one visible ]]>
Fig. 1). Fluorescent bands were sometimes brighter and distinct at the greater calcein concentration (0.7g/L) (Fig. 2), but generally, results were found better at the lower calcein concentration (0.4g/L) with more “perfect” and ‘good” marks (
Fig. 1a, Fig. 1b) on the growing-edge (x2=16.0, df=3, p=0.0012) (Fig. 3). Additionally, calcein-stained growing edges were vis- ible with the naked eye as dark brown narrow bands, without the blue light that produced the fluorescent green band.
Survival rate after treatments was ]]>
Mean 15-day increment of shell height growth was slightly greater at the lower calcein concentration (
=0.735mm) than at the higher concentration (=0.577mm), but not significantly (F=1.71; df=1, 43; p=0.19).
Coomassie Blue staining: Observation of thin slices not stained by Coomassie Blue R-250 usually did not show distinct micro growth-bands. visible micro growth-band col- oration or distinction was not produced, consistently, with polishing, special filters to enhance any shell auto-fluorecences, or differences in shell composition proportion affects for this oyster species. Only staining by Coomassie Blue R-250 consistently produced distinctly ]]>
Fig. 2). It was effective for staining the organic protein matrix between micro growth-bands, allowing for easy recognition and count. However, prolonged exposure (more than 15min) with Coomassie degraded thin and younger thin slices, as well as calcein marks. Calcium carbonate layers were degraded over short time periods (hours).
Micro growth-band ]]>
Mean number of additional micro growth- bands, after calcein marking, was almost equal to number of days in rearing cages (15d). However, the mean number of additional micro growth-bands was slightly greater (=14.9, SE=0.20) for the 0.4g/L calcein concentration, when compared to the 0.7g/L calcein concentration ]]>
=14.5, SE=0.23), but not significantly (F=1.61; df=1, 102; p=0.21) for the 15-d growth period. Additionally, the mean number of micro growth-bands did not vary between the three successive daily counts, when combining both calcein concentrations (=14.56, 14.53, 14.89; F=0.52; df=2, 102; p=0.60).
Since the number of micro growth-bands produced by oysters was almost equal to the number of days in the rearing cages, we can safely assume that each micro growth-band was produced daily.
Discussion
Calcein used as a CaCO3-stainer was successful by immersion as a method for age validation producing a concise bright lime-green florescent band along the growing-edge with ]]>
Adamussium colbecki (Heilmayer et al., 2005; Lartaud et al., 2010), Comptopallium radula (Thébault, Chauvaud, Clavier, Fichez, & Morize, 2006), Mesodesma donacium (Riascos et al., 2007), Donax hanleyanus (Herrmann et al., 2009), Cerastoderma edule (Mahé, ]]>
Pinctada margaritifera (Linard et al., 2011), Loripes lacteus (van der Geest, van Gils, van der Meer, Olff, & Piersma, 2011) and other bivalves (Rowley & Mackinnon, 1995).
However, Fujikura et al. (2003) ]]>
Ruditapes philippinarum obtained unclear calcein boundaries and preferred strontium chloride (SrCl2) staining for age validation in this species. Herrmann et al. (2009) compared calcein and SrCl2 marking efficacy with D. hanleyanus and only marking with calcein was obtained. ]]>
Regarding the calcein concentration impact on marking, our individuals produced better marking (100%) at the lower (0.4g/L) than higher concentration (73% for 0.7g/L), as well as, a better quality mark at the lesser concentration (more perfect and good marks). The 27% of oysters at the greater concentration that showed no marking, actually showed green fluorescence over the whole shell thickness, as if the greater concentration affected the complete aragonite layer. This could be an indication of the presence of ]]>
Pteria sterna (Cáceres-Puig, Huato-Soberanis, Melo-Barrera, & Saucedo, 2011).
Linard et al. (2011) also detected clear marks in all tested individuals (100%) at 0.150g/L calcein concentration, but observed less efficacy (65%) at 0.05g/L. However, Her- rmann (2009) got clear marks ]]>
Calcein was lightly toxic in our study (4% mortality) at 0.4g/L, and not toxic at all at 0.125g/L in the injection experiment. We did incur 15% mortality at 0.7g/L. ]]>
Fujikura et al. (2003) observed 33% (one out of three individuals) bivalve mortality after 24h exposure to calcein at 0.3g/L concentration, however, no mortality at 0.4 nor 0.7g/L. This greater mortality may be a result of the small sample size, of greater sensitivity of that species or the slightly longer exposure time to calcein. Linard et al. (2011) reported no mortality for P. margaritifera at 0.15g/L. Other ]]>
Nucella ostrina (Moran, 2000) and Mesodesma donacium (Riascos et al., 2007). Herrmann (2009) found exposure time (3h vs 6h) to calcein to produce slightly more mortality for D. hanleyanus than the actual calcein concentration used (from 4 to 6% mortality), and thus recommended calcein staining as a non-lethal method. Mahé et al. (2010) reported no mortality at 0.05 or 0.15g/L calcein ]]>
Cerastoderma edule). They observed clear marks at all concentrations and exposure times (submersion).
Regarding age-dependency and calcein marking, as a general observation, smaller oys- ters (<30mm shell height) in our study tended to have more perfect marks at the lower calcein concentration. In addition, staining efficiency was found ]]>
The calcein ]]>
D. hanleyanus, ]]>
C. concholepas and M. donacium.
Regarding calcein marking and micro growth-band periodicity, we observed calcein validation marks without aid of blue light from ]]>
CaCO3, because of our poor results during the injection experiment. This dark, brown calcein band has not been reported elsewhere.
Sub-littoral species are influenced by circadian rhythms, which regulate activities by external cues, like daylight exposure. Morton (1973, ]]>
Ligumia subrostrata (McCorkle, Shirley, & Dietz, 1979).
Shell deposition was ]]>
For other species of mollusks feeding and digestion is constant and simultaneous if food availability is also constant (Owen, 1974). Other bivalves present a double-phase feeding activity in a 24h period with intermediate digestion periods. In other studies these rates depend on ]]>
Growth was faster at ]]>
The major problem for ageing is the specific distinction of micro growth-bands on unstained thin sections (MacDonald & Thomas, 1980). The use of Coomassie Blue was crucial for growth-band counts for this species; it facilitated clear band boundaries and helped discriminate from blurred areas in the shell of the thin section. Checa, Rodríguez-Navarro, & ]]>
CaCO3 part of the band starts to degrade or dissolve because of the presence of acetic acid in the solution. Exposure time of thin sections with Coomassie Blue should be less than ]]>
CaCO3 layer presents an uneven relief on the surface of the thin section and aids in counting the micro growth-bands.
Additionally, some shells became ]]>
Concerning management implications, now that age of each micro growth-band is validated as pertaining to one 24-hr period, oyster spat collected at different dates, depths and ]]>
]]>
In conclusion, calcein is recommended as a non-lethal marking method for age validation for P. mazatlanica for the following reasons:1) is a simple method that enables the analysis of large samples; 2) it produced a bright fluorescent mark at low and high concentrations of calcein and a distinct brown ]]>
P. mazatlanica each internal micro growth-band is validated for a 24h growth formation. ]]>
Acknowledgments
This study was possible thanks to the Center for Biological Investigations of the North- west (Centro de Investigaciones Biológicas del Noroeste, CIBNOR) for providing laboratory facilities and equipment. We specially thank J. Cortes from ]]>
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1. Escuela de Ciencias Biológicas, Universidad Nacional (UNA), Campus Omar Dengo Avenida 1 Calle 9, Apartado Postal: 86-3000, Heredia, Costa Rica; pleemccoy@gmail.com 2. Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, México; lhuato@cibnor.mx
Received 02-xII-2013. Corrected 20-III-2014. Accepted 19-Iv-2014.
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=0.735mm) que con la de mayor concentración (
=0.735mm) than at the higher concentration (