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Evaluation of the age of the red whelk Neptunea antiqua using statoliths, opercula and element ratios in the shell
Journal of Experimental Marine Biology and Ecology 325 (2005) 55 – 64 www.elsevier.com/locate/jembe
Evaluation of the age of the red whelk Neptunea antiqua using statoliths, opercula and element ratios in the shell C.A. Richardsona,*, C. Saurela, C.M. Barrosob, J. Thainc a
School of Ocean Sciences, University of Wales-Bangor, Menai Bridge, Anglesey LL59 5AB, UK b CESAM, Departamento de Biologia, Universidade de Aveiro, 3810 Aveiro, Portugal c CEFAS, Burnham laboratories, Burnham-on-Crouch, Essex, UK Received 8 December 2004; received in revised form 20 April 2005; accepted 25 April 2005
Abstract Growth rings present in whole and sectioned statoliths were used to determine the age of red whelks Neptunea antiqua, from the North Sea. Validation of the periodicity of the rings was established in four whelks by comparing the number of statolith rings with the number of seasonal Mg:Ca ratio cycles present in shell calcium carbonate samples drilled sequentially from along the growth axis. There was exact correspondence between the number of growth rings and the number of element ratio cycles in two of the shells and a 1-year difference in the estimated age between the two methods in the other two shells, evidence which is strongly indicative of an annual periodicity of deposition to the statolith rings. The estimated age of the whelks using the statolith rings varied between 4 years (shell length 102 mm) and 17 years (shell length 148 mm). The age of the whelks ascertained from the statoliths was compared with age estimates from the number of adventitious layers in sectioned opercula. The number of adventitious layers in whelks from 51 to 148 mm shell length ranged between 1 and 12 years. No significant difference was observed between the number of strongly defined statolith rings and number of opercula adventitious layers. D 2005 Elsevier B.V. All rights reserved. Keywords: Age determination; Opercula; Neptunea antiqua; Red whelks; Statoliths
1. Introduction For a comprehensive understanding of the population dynamics of gastropod mollusc populations it is important to be able to accurately determine the age of individuals within a population. Few studies on gas* Corresponding author. Tel.: +44 1248 382855; fax: +44 1248 716367. E-mail address: [email protected] (C.A. Richardson). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.04.024
tropod growth, however, have addressed the problem of determining the age of the species, and where this has been carried out the methods adopted have concentrated on the identification of modal size classes in size frequency distributions, and used surface striae on the operculum and external surface growth rings and checks (e.g. Buccinum undatum, Kideys, 1996). Estimating the age of gastropod shells, using conventional shell sectioning and growth line analysis techniques similar to those used in bivalve growth studies
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(Richardson, 2001), is complicated owing to the whorl-like shape of the shell. If sectioning is undertaken, then the process involves the piecing together of a complicated series of sections taken across each whorl of the shell. Some researchers have successfully used annual growth checks on the surface of gastropod shells, e.g. Monodonta lineata (Williamson and Kendall, 1981) and Fissurella crasa (Bretos, 1980) to estimate age. However the shell margin is easily damaged and repairs to the damaged shell occur frequently. For example, many Buccinidae are caught accidentally in trawls as by-catch and their shells can become damaged (Ramsay and Kaiser, 1998; Mensink et al., 2000) producing surface growth checks which can be difficult to distinguish from those of annual origin. A few studies have investigated the formation of semidiurnal tidal growth bands and annual growth lines at the margin of the shells of gastropods, including Littorina littorea and Nucella lapillus (Ekaratne and Crisp, 1982, 1984) and Nassarius reticulatus (Barroso et al., 2005a), respectively. Counting the number of surface striae on the operculum surface is a generally accepted method for determining the age of B. undatum and is based on a study by Santarelli and Gros (1985). They validated the periodicity of the striae (fine rings) present on the operculum surface by comparing the number of annual cycles of sea water temperature, obtained from the stable oxygen isotope record in the shell carbonate, with the number of striae on the opercula and found good correspondence between the number of striae (2–5) and the number of annual cycles (2–5) in three shells ranging in size from 24 to 63 mm. Based on this interpretation the operculum striae method has been used to estimate the age of B. undatum with only moderate success (Kideys, 1996) and without apparent difficulty in Neptunea antiqua (Powers and Keegan, 2001). Kideys (1996), for example, only successfully estimated the age of 16% of ~ 11,000 specimens of B. undatum using the technique. He observed a trend of increasing number of striae with increasing size, although overlaps in length at age occurred such that a 2-year-old whelk ranged in size between 26.4 and 86.8 mm. Like Kideys (1996) we found counting the number of surface striae on the operculum was a subjective method and did not produce consistent and reliable results. In view of the uncertainty in the use of operculum striae for age estimations we inves-
tigated alternative methods, including an analysis of the number of adventitious layers in operculum sections (see Fretter and Graham, 1994), for estimating the age of the red whelk N. antiqua. Growth increments have been identified in the statoliths of several molluscan groups including cephalopods (e.g. Clarke, 1978; Lipinski, 1993; Jackson, 1994) and larval gastropods (e.g. Bell, 1984; Granna-Raffucci and Appeldoorn, 1995; Zacherl et al., 2003). Richardson (2001) alluded to the possible value of using the growth rings present in post-larval, juvenile and adult gastropod statoliths to investigate ontogenetic changes in shell growth similar to those routinely afforded by cephalopod statoliths. The ratios of Mg:Ca and Sr:Ca incorporated into the aragonite and calcite molluscan shell varies in a temperature dependent way; the ratios are lower when sea water temperatures are minimal and are at a maximum when sea water temperatures are elevated (Klein et al., 1996; Putten et al., 2000; Richardson et al., 2003). In this paper we investigate and validate the periodicity of the growth rings in the statoliths of N. antiqua by comparing the number of rings with the number of annual cycles of Mg:Ca ratios in the carbonate of the shell. We investigate whether there is a relationship between the statolith rings and the number of surface striae and adventitious layers in the sectioned opercula and ascertain the most suitable method for determining the age of N. antiqua.
2. Materials and methods Thirty-six red whelks N. antiqua, (size range 51 to 148 mm shell length) were collected by trawling from the North Sea from a site off the river Tyne, north-east England during February 2003 and individually frozen. Upon thawing shell length (SL) (Fig. 1A) was measured to the nearest 0.1 mm using vernier callipers and the flesh of each whelk removed by detaching the columellar muscle from the inner shell surface. The viscera and the dorsal portion of the foot, between the tentacles and the operculum, of each whelk were removed from the main body and examined. Each operculum was carefully prised from the foot, adhering flesh scrapped off and the operculum stored dry. Any epibiota were scraped and/or brushed from the outer shell surface.
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A
B
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C a
Shell Length (SL)
b
Fig. 1. (A) Drawing of the shell of the red whelk Neptunea antiqua to show the measurement of shell length. (B) The positions from which drilled shell samples were removed from the shell of Neptunea antiqua at ~ 0.5 cm intervals along the whorl of the shell. (C) Diagrammatic representation of the N. antiqua to show the striae; a–b direction of section.
Three methods were employed to estimate the age of the red whelks: (1) the number of striae on the surface of each operculum and the number of growth layers present in the sectioned operculum were counted, (2) rings present in whole and sectioned statoliths were counted and (3) the number of cycles of Mg:Ca ratios in samples of CaCO3 drilled from the shell surface along the shell spiral were determined. 2.1. Analysis of operculum rings Each operculum was placed in a 5 cm petri dish containing supersaturated sodium hydroxide solution for ~ 30 min to remove any residual flesh, rinsed with tap water and dried for 3 days between two glass slides held firmly together by placing a heavy weight (~ 1 kg) upon them. Once dry each operculum was examined externally for the presence of growth rings. In an attempt to enhance the appearance of the opercula they were illuminated using strong backlighting, a method that has been previously used to locate the position of the annual rings on the shells of Donax trunculus (Ramon et al., 1995) and surface pencil tracings of the opercula were made. Despite these approaches the clarity of the surface rings was equivocal and they could not be counted with any certainty. Instead each operculum was embedded in Metaset resin (Buehler U.K. Ltd.) and cut along its longest axis a–b
(Fig. 1C) using a rotating diamond saw. The cut surfaces were ground on progressively finer wet abrasive paper, polished with diamond paste on a polishing cloth, washed with detergent, rinsed with tap water and then left for 24 h to dry. Acetate peel replicas were prepared of the unetched and sectioned opercula, and the peels mounted between a glass slide and cover slip (see Richardson, 2001 and references therein). The slides were viewed under the light microscope and the number of adventitious layers (Fretter and Graham, 1994) counted in the peels of sectioned opercula. 2.2. Statolith growth ring analysis The main body (the viscera and the dorsal portion of the foot retained previously after removal of the shell) of each whelk was placed dorsal side up in a crystallising dish (125 or 250 ml depending on the size of the animal) containing supersaturated sodium hydroxide solution for N3 h. Upon digestion of the flesh the contents of the dish were examined microscopically and the pair of statoliths (each b0.2 mm in diameter) removed. Each statolith was rinsed twice in tap water to remove both sodium hydroxide and any remaining flesh, rinsed in 70% and then 85% alcohol and stored in an Eppendorf tube (500 Al) containing absolute alcohol (Saurel, 2002). Two methods were used to examine the statoliths.
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Method 1: On removal from alcohol the pair of statoliths was allowed to dry for a few seconds. One statolith was cleared in HistoclearR (Fisons laboratory Reagent: /0468/17) and then positioned in a drop (1–2 ml) of DPX mountant, (BDH microscopical reagent) on a slide. A short length (1 cm) of fishing line (10 kg breaking strain) was positioned in the DPX on both sides of the statolith to ensure a constant distance was maintained between the slide and cover slip (see Morales-Nin, 1992 for larval otoliths). The position of the statolith was orientated in the mountant using fine forceps and then covered with a coverslip and the slide placed for 12 h on a heating plate (30 8C) to harden the DPX. Method 2: A small drop of Metaset resin (~ 2–3 ml) was placed on a slide and allowed to dry for 24 h. The other statolith of the pair was immersed in a small drop (~ 2 ml) of liquid resin placed on top of the hardened resin drop and orientated using fine forceps and the resin allowed to harden for ~ 24 h. The statolith was then ground on progressively finer wet abrasive paper (400 and 1000 grit) under a dissecting microscope to monitor the progress of sectioning until the section passed through the midpoint of the statolith, i.e. the statolith was sectioned in half. The sectioned statolith was then polished with diamond paste on a polishing cloth, washed thoroughly with detergent and a drop of water placed on the section under a cover slip and observed directly using a compound microscope. Photomicrographs of the sectioned opercula and DPX whole mounted and resin-embedded sectioned statoliths were taken at 10 and 40 magnification using a digital ColorView II (3.3 MegaPixel colour using FireWirek) camera mounted on a microscope. Each statolith contained a series of growth rings (see Figs. 3–6) of differing intensity. Following photography of the sectioned statoliths, they were then etched in 0.1 M HCl for 15 s, rinsed in tap water, left to dry overnight and acetate peel replicas of the etched surfaces prepared as described previously for the opercula and photographed. The total number of strong and weak growth rings (maximum) and the number of strongly defined rings (minimum) were counted from whole mounted and sectioned statoliths. Whenever there was difficulty in resolving the rings, the peels of the statoliths were consulted, particularly where the rings at the statolith edge were narrowly spaced. Interpretation of the position of the rings was independently aided by ascertaining
variations in light transmission through the statolith along a traverse across the radius of the statolith. Each colour photomicrograph of the sectioned statolith was transformed to a grey scale and the pixel intensity along a horizontal line drawn through the radius of the statolith calculated using the software analySISR 3.2. The number of pixel intensity maxima was counted. 2.3. Seasonal variation in Mg:Ca ratios along the shell whorl The outer shell surfaces of four shells (SL 52.5, 70.2, 103.8 and 148.3 mm), covering the size range of whelks collected and whose ages had been estimated from the statoliths and opercula, were carefully cleaned by immersing them in a solution of 0.11 M HNO3 (69% Aristar) for 30 s. The shells were thoroughly washed in distilled water and then air dried (24 h). At intervals of 5 mm along the shell whorl (Fig. 1B) approximately 1.5 mg of shell was removed using a 0.6 mm diameter dental drill bit. The shell powder samples were placed separately in 12 ml centrifuge plastic tubes and dissolved with 10 ml of 0.11 M HNO3 (69% Aristar). The concentration of Ca and Mg in the aqueous samples was analysed using a JY138 Ultrace Inductively Coupled Plasma-Atomic Emission Spectrometer (ICPAES). The number of cycles of high and low element ratios present in the shell was counted and compared with the number of rings present in the statoliths.
3. Results The sectioned opercula displayed a clear pattern of adventitious growth layers (Fig. 2). Generally the layers were clear to count (Fig. 2A and B) although the operculum margin was sometimes folded and trapped material enclosed between the growth layers making the location of individual layers more difficult (Fig. 2C). The estimated age of each whelk, based on the number of adventitious layers, ranged between 1 and 12 years for whelks ranging from 51.2 mm to 148.3 mm shell length. Examples of sectioned statoliths are shown in Figs. 3–6. Under low power magnification each one was observed to contain a series of circular growth rings with the increment between each ring progres-
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Fig. 2. Photomicrographs of sectioned opercula of Neptunea antiqua. (A) Six growth layers (one arrowed) from the mid-portion of the operculum of a 148.3 mm whelk (no. 1), (B) 12 growth layers (one arrowed) from the mid-portion of a 103.8 mm whelk (no. 13) and (C) the appearance of the growth layers in half of the operculum of whelk no. 1. The margin of the operculum is highly folded (arrow) and material is trapped within the folds.
sively narrowing towards the statolith circumference (e.g. Figs. 3A, 4A and 5A). At the statolith rim the rings were often difficult to observe due to refraction of transmitted light as it passed through the narrowly spaced increments and edge of the statolith. Acetate peel replicas, however, of etched sectioned statoliths revealed more detail and assisted in resolving the narrowly spaced rings (e.g. Fig. 5B and D). In some statoliths there was a pattern of weakly defined rings between the more distinct rings (e.g. Fig. 4C) which in a few statoliths (b5%) caused difficulties in resolving the position of the more prominent rings. The maximum (all strong and weak growth rings) and minimum numbers (all strongly defined rings) were recorded and the average number of rings calculated. The estimated age of the youngest whelk was ~ 5 years old (length 102.2 mm) whilst a whelk of 148.3 mm was estimated to be 20 years old (based on the maximum number of rings). Differences in light transmission through the statolith along a traverse across the radius, when converted to a grey scale, highlighted the positions of the darker growth rings, i.e. regions of low light transmission (Fig. 6). This method, when applied to all the statoliths and in conjunction with a visual inspection of the statolith section, assisted in evaluating and confirming the number of statolith growth rings. The maximum and minimum numbers of statolith rings and the operculum adventitious layers are compared in Fig. 7. No significant difference was found
between the minimum number of statolith rings and the operculum adventitious layers (normal operculum data, A 2 = 0.621 p = 0.097; non-normal statolith ring data, A 2 = 2.468, p b 0.001; Levene’s test of homogeneity of variance, t = 0.532 p = 0.589; Mann Whitney test; W = 1040.5 p = 0.408). A significant difference, however, existed between the maximum number of statolith rings and the number of operculum adventitious layers (normal operculum data, A 2 = 0.621 p = 0.097; non-normal statolith ring data, A 2 = 1.588, p b 0.001; Levene’s test of homogeneity of variance t = 0.532, p = 0.589; W = 649.5 p b 0.001). These analyses suggest that the adventitious lines and the strongly defined rings can be used to estimate the age of N. antiqua. However, there was exact correspondence of ages estimated using the two methods in only four whelks and in one large whelk there was disparity between the methods; four adventitious layers were counted in the operculum yet the statolith contained 15 rings. Validation of the seasonality of statolith ring deposition was undertaken in four whelks by constructing seasonal patterns of Mg:Ca ratios along the shell whorls and comparing the number of element ratio (ER) cycles with the number of rings. The ER cycles present in the shells compared favourably with the number of statolith rings. Whelk (no. 34) displayed 6 seasonal (Mg:Ca ratio) cycles along the shell whorl (Fig. 8A), exactly comparable with the number of
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of the aperture which varied seasonally (Fig. 8D) and cannot currently be explained.
4. Discussion Sectioning the operculum and counting the number of adventitious layers provided a method which could be used with confidence to age N. antiqua. Operculum sections revealed the presence of clear, countable adventitious growth lines, particularly in the early years of growth and when the number of operculum adventitious lines was compared with the number of strongly defined statolith rings and the number of Mg:Ca cycles in the shell there was good correspondence between the methods. However in one shell (no. 12) the adven-
Fig. 3. Photomicrographs of the statoliths of Neptunea antiqua (no. 1). (A) DPX-mounted whole statolith revealing a series of circular growth rings, (B) acetate peel replica of a section of a resinembedded and etched statolith, and (C) magnified portion of the DPX-mounted statolith to indicate the position of the annual growth rings (arrows). The age of the whelk was estimated to be 17 years.
statolith rings. Whilst shells (nos. 13 and 25) displayed 13 and 9 ratio cycles comparable to the 12 strong (average 9.5) rings and 11 strong rings (average 10), respectively, present in the statoliths (Fig. 8B and C). The largest and oldest shell (no. 1) displayed 17 ER cycles comparable with the average number of statolith rings (17.5) (Fig. 8D). Peaks in the element ratios varied between shells, attaining maximum concentrations in shell no. 34 of N 20 mmol mol 1 and towards the aperture in shell no. 1 of N35 mmol mol 1. Interannual variations were apparent in individual shells (Fig. 8) with little similarity between the amplitude of the ER cycles between shells; the exception being a pronounced low ER ratio during the 5th winter (shell no. 34) (Fig. 8A) and the 12th winter (shell no. 13) (Fig. 8C). The largest and oldest shell (no. 1) had unusually elevated Mg:Ca ratios at the shell margin
Fig. 4. Photomicrographs of the statoliths of Neptunea antiqua (no. 34). (A) DPX-mounted whole statolith, (B) magnified portion of the statolith to show the position of six growth rings (arrows) and (C) portion of the statolith to show the appearance of the weakly defined growth rings between the growth rings (arrows).
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Fig. 5. Photomicrographs of the statoliths of Neptunea antiqua (no. 25). (A) DPX-mounted whole statolith, (B) acetate peel replica of a section of a resin-embedded and etched statolith, (C) centre of the resin-embedded statolith with a putative natal ring (NR) indicated and 10 clear growth rings, and (D) acetate peel replica of a section of the resin-embedded and etched statolith indicating the first four growth rings (arrows).
titious layers noticeably underestimated the age of the whelk; four adventitious layers were counted in the operculum and 15 rings in the statolith. A possible explanation for the small number of lines in the sectioned operculum could be that at some stage during
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Fig. 6. Photomicrograph of a polished section of a statolith of Neptunea antiqua. Superimposed on the image is a trace of variations of light transmission through the statolith. Areas of low transmission are associated with the denser dark growth rings.
Fig. 7. Relationship between the number of operculum adventitious layers and the maximum (5) and minimum (E) numbers of statolith rings. The x = y line indicates where the number of operculum adventitious layers = the number of statolith growth rings.
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Mg:Ca ratio (mmol/mol)
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20 15 10 5 0
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Distance from apex to aperture (cm)
Fig. 8. Seasonal variations in Mg:Ca ratios in the shells of Neptunea antiqua (nos. 34, 13, 25 and 1) (A, B, C and D). Arrows indicate the position of the annual cessations in shell growth.
the life of the whelk the operculum had been lost and then re-grown. The position of the operculum on the foot when the body is withdrawn inside the shell aperture makes it vulnerable to loss during attack by predators and damage during feeding. Richardson (2001) proposed the possible use of growth rings in gastropod statoliths for investigating the age and growth rate of species that possessed them. Barroso et al. (2005b) provide some basic data on statolith structure and the relationship between statolith size and shell size in N. reticulatus. They found that the statoliths of N. reticulatus from the north-west coast of Portugal contained up to four well-marked growth rings, the first corresponding to a natal ring, formed during settlement, and the others corresponding to the first three winters of the whelk’s life. In this study we validated the periodicity of the statolith rings in four shells by comparing the number of rings with the number of element ratio cycles in the
shell. There was close correspondence between the statolith rings and the number of element ratio cycles strongly suggesting an annual periodicity to the rings. When the interpretation of the statolith rings was used to estimate the age of the whelks we ascertained that red whelks of ~ 150 mm in the North Sea can attain ages up to 17 years. Statoliths are internal structures and are not subject to external abrasion and damage like the shell and opercula. They are therefore potentially excellent recorders of annual changes in shell growth once a relationship between statolith diameter and shell length has been established. Correlation of statolith diameters with shell length, as demonstrated in N. reticulatus (Barroso et al., 2005b), would enable the length of N. antiqua to be estimated at the formation of each growth ring and thus allow the growth rate of this species to be determined. Comparisons of shell growth determined using the statolith rings could then be made between populations of N. antiqua. Statoliths
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are found in a number of gastropod taxa and the patterns of growth rings may contain information about past growth history. If the periodicity of the rings is understood then information can be extracted from statoliths in a similar way to that already being undertaken using squid statoliths. Squid statoliths contain daily growth rings that have been used to study daily growth rates from a variety of species including Alloteuthis africana and Alloteuthis subdula (Arkhipkin and Nekludova, 1993), Ilex illecebrosus (Hurley et al., 1985) and Sepioteuthis lessoniana (Jackson, 1990) (see also Richardson, 2001 and references therein). Further experimental investigations on gastropod statoliths are required to elucidate how factors such as feeding, reproduction and seasonal variations in seawater temperature affect deposition of the statolith rings. Once this has been accomplished it should then be possible to reconstruct the past history of growth from a range of gastropod species containing suitable statoliths. It has recently been demonstrated, for example, that the microstructure and chemical composition of the larval statoliths of the neogastropod Concholepas concholepas can be used in the reconstruction of their larval dispersal history (Zacherl et al., 2003). Such analyses should be possible using juvenile and adult gastropod species once the factors affecting deposition of the growth rings have been resolved.
Acknowledgements We are grateful to DEFRA for funding a small research project to investigate the age of the red whelk Neptunea antiqua. We are very grateful to G. Connolley from the Chemistry Department, University of Wales, Bangor for undertaking the Mg:Ca analyses using a JY138 Ultrace Inductively Coupled PlasmaAtomic Emission Spectrometer (ICP-AES). [RH]
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Ramon, M., Abello´, P., Richardson, C.A., 1995. Population structure and growth of Donax trunculus (Bivalvia: Donacidae) in the Western Mediterranean. Mar. Biol. 122, 665 – 671. Ramsay, K., Kaiser, M.J., 1998. Demersal fishing disturbance increases predation risk for whelks (Buccinum undatum L.). J. Sea Res. 39, 299 – 304. Richardson, C.A., 2001. Molluscs as archives of environmental change. Oceanogr. Mar. Biol.: An Annual Review 39, 103 – 164. Richardson, C.A., Peharda, M., Kennedy, H.A., Kennedy, P., Onofri, V., 2003. Age, growth rate and season of recruitment of Pinna nobilis (L) in the Croatian Adriatic determined from Mg:Ca and Sr:Ca shell profiles. J. Exp. Mar. Biol. Ecol. 299, 1 – 16. Santarelli, L., Gros, P., 1985. De´termination de l’aˆge et de la croissance de Buccinum undatum L. (Gastropoda, Prosobranchia)
a` l’aide des isotopes stables de la coquille et de l’ornementation operculaire. Ocean Acta 8, 221 – 229. Saurel, C., 2002. Age and growth rates of populations of dog whelks, Nucella lapillus determined from statoliths. MSc thesis, University of Wales, Bangor, 152 pp. Williamson, P., Kendall, M.A., 1981. Population structure and growth of the trochid Monodonta lineata determined from shell rings. J. Mar. Biol. Assoc. UK 61, 1011 – 1026. Zacherl, D.C., Manrı´quez, P.H., Paradis, G., Day, R.W., Castilla, J.C., Warner, R.R., Lea, D.W., Gaines, S.D., 2003. Trace elemental fingerprinting of gastropod statoliths to study larval dispersal trajectories. Mar. Ecol. Prog. Ser. 248, 297 – 303.
Evaluation of the age of the red whelk Neptunea antiqua using statoliths, opercula and element ratios in the shell C.A. Richardsona,*, C. Saurela, C.M. Barrosob, J. Thainc a
School of Ocean Sciences, University of Wales-Bangor, Menai Bridge, Anglesey LL59 5AB, UK b CESAM, Departamento de Biologia, Universidade de Aveiro, 3810 Aveiro, Portugal c CEFAS, Burnham laboratories, Burnham-on-Crouch, Essex, UK Received 8 December 2004; received in revised form 20 April 2005; accepted 25 April 2005
Abstract Growth rings present in whole and sectioned statoliths were used to determine the age of red whelks Neptunea antiqua, from the North Sea. Validation of the periodicity of the rings was established in four whelks by comparing the number of statolith rings with the number of seasonal Mg:Ca ratio cycles present in shell calcium carbonate samples drilled sequentially from along the growth axis. There was exact correspondence between the number of growth rings and the number of element ratio cycles in two of the shells and a 1-year difference in the estimated age between the two methods in the other two shells, evidence which is strongly indicative of an annual periodicity of deposition to the statolith rings. The estimated age of the whelks using the statolith rings varied between 4 years (shell length 102 mm) and 17 years (shell length 148 mm). The age of the whelks ascertained from the statoliths was compared with age estimates from the number of adventitious layers in sectioned opercula. The number of adventitious layers in whelks from 51 to 148 mm shell length ranged between 1 and 12 years. No significant difference was observed between the number of strongly defined statolith rings and number of opercula adventitious layers. D 2005 Elsevier B.V. All rights reserved. Keywords: Age determination; Opercula; Neptunea antiqua; Red whelks; Statoliths
1. Introduction For a comprehensive understanding of the population dynamics of gastropod mollusc populations it is important to be able to accurately determine the age of individuals within a population. Few studies on gas* Corresponding author. Tel.: +44 1248 382855; fax: +44 1248 716367. E-mail address: [email protected] (C.A. Richardson). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.04.024
tropod growth, however, have addressed the problem of determining the age of the species, and where this has been carried out the methods adopted have concentrated on the identification of modal size classes in size frequency distributions, and used surface striae on the operculum and external surface growth rings and checks (e.g. Buccinum undatum, Kideys, 1996). Estimating the age of gastropod shells, using conventional shell sectioning and growth line analysis techniques similar to those used in bivalve growth studies
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(Richardson, 2001), is complicated owing to the whorl-like shape of the shell. If sectioning is undertaken, then the process involves the piecing together of a complicated series of sections taken across each whorl of the shell. Some researchers have successfully used annual growth checks on the surface of gastropod shells, e.g. Monodonta lineata (Williamson and Kendall, 1981) and Fissurella crasa (Bretos, 1980) to estimate age. However the shell margin is easily damaged and repairs to the damaged shell occur frequently. For example, many Buccinidae are caught accidentally in trawls as by-catch and their shells can become damaged (Ramsay and Kaiser, 1998; Mensink et al., 2000) producing surface growth checks which can be difficult to distinguish from those of annual origin. A few studies have investigated the formation of semidiurnal tidal growth bands and annual growth lines at the margin of the shells of gastropods, including Littorina littorea and Nucella lapillus (Ekaratne and Crisp, 1982, 1984) and Nassarius reticulatus (Barroso et al., 2005a), respectively. Counting the number of surface striae on the operculum surface is a generally accepted method for determining the age of B. undatum and is based on a study by Santarelli and Gros (1985). They validated the periodicity of the striae (fine rings) present on the operculum surface by comparing the number of annual cycles of sea water temperature, obtained from the stable oxygen isotope record in the shell carbonate, with the number of striae on the opercula and found good correspondence between the number of striae (2–5) and the number of annual cycles (2–5) in three shells ranging in size from 24 to 63 mm. Based on this interpretation the operculum striae method has been used to estimate the age of B. undatum with only moderate success (Kideys, 1996) and without apparent difficulty in Neptunea antiqua (Powers and Keegan, 2001). Kideys (1996), for example, only successfully estimated the age of 16% of ~ 11,000 specimens of B. undatum using the technique. He observed a trend of increasing number of striae with increasing size, although overlaps in length at age occurred such that a 2-year-old whelk ranged in size between 26.4 and 86.8 mm. Like Kideys (1996) we found counting the number of surface striae on the operculum was a subjective method and did not produce consistent and reliable results. In view of the uncertainty in the use of operculum striae for age estimations we inves-
tigated alternative methods, including an analysis of the number of adventitious layers in operculum sections (see Fretter and Graham, 1994), for estimating the age of the red whelk N. antiqua. Growth increments have been identified in the statoliths of several molluscan groups including cephalopods (e.g. Clarke, 1978; Lipinski, 1993; Jackson, 1994) and larval gastropods (e.g. Bell, 1984; Granna-Raffucci and Appeldoorn, 1995; Zacherl et al., 2003). Richardson (2001) alluded to the possible value of using the growth rings present in post-larval, juvenile and adult gastropod statoliths to investigate ontogenetic changes in shell growth similar to those routinely afforded by cephalopod statoliths. The ratios of Mg:Ca and Sr:Ca incorporated into the aragonite and calcite molluscan shell varies in a temperature dependent way; the ratios are lower when sea water temperatures are minimal and are at a maximum when sea water temperatures are elevated (Klein et al., 1996; Putten et al., 2000; Richardson et al., 2003). In this paper we investigate and validate the periodicity of the growth rings in the statoliths of N. antiqua by comparing the number of rings with the number of annual cycles of Mg:Ca ratios in the carbonate of the shell. We investigate whether there is a relationship between the statolith rings and the number of surface striae and adventitious layers in the sectioned opercula and ascertain the most suitable method for determining the age of N. antiqua.
2. Materials and methods Thirty-six red whelks N. antiqua, (size range 51 to 148 mm shell length) were collected by trawling from the North Sea from a site off the river Tyne, north-east England during February 2003 and individually frozen. Upon thawing shell length (SL) (Fig. 1A) was measured to the nearest 0.1 mm using vernier callipers and the flesh of each whelk removed by detaching the columellar muscle from the inner shell surface. The viscera and the dorsal portion of the foot, between the tentacles and the operculum, of each whelk were removed from the main body and examined. Each operculum was carefully prised from the foot, adhering flesh scrapped off and the operculum stored dry. Any epibiota were scraped and/or brushed from the outer shell surface.
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A
B
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C a
Shell Length (SL)
b
Fig. 1. (A) Drawing of the shell of the red whelk Neptunea antiqua to show the measurement of shell length. (B) The positions from which drilled shell samples were removed from the shell of Neptunea antiqua at ~ 0.5 cm intervals along the whorl of the shell. (C) Diagrammatic representation of the N. antiqua to show the striae; a–b direction of section.
Three methods were employed to estimate the age of the red whelks: (1) the number of striae on the surface of each operculum and the number of growth layers present in the sectioned operculum were counted, (2) rings present in whole and sectioned statoliths were counted and (3) the number of cycles of Mg:Ca ratios in samples of CaCO3 drilled from the shell surface along the shell spiral were determined. 2.1. Analysis of operculum rings Each operculum was placed in a 5 cm petri dish containing supersaturated sodium hydroxide solution for ~ 30 min to remove any residual flesh, rinsed with tap water and dried for 3 days between two glass slides held firmly together by placing a heavy weight (~ 1 kg) upon them. Once dry each operculum was examined externally for the presence of growth rings. In an attempt to enhance the appearance of the opercula they were illuminated using strong backlighting, a method that has been previously used to locate the position of the annual rings on the shells of Donax trunculus (Ramon et al., 1995) and surface pencil tracings of the opercula were made. Despite these approaches the clarity of the surface rings was equivocal and they could not be counted with any certainty. Instead each operculum was embedded in Metaset resin (Buehler U.K. Ltd.) and cut along its longest axis a–b
(Fig. 1C) using a rotating diamond saw. The cut surfaces were ground on progressively finer wet abrasive paper, polished with diamond paste on a polishing cloth, washed with detergent, rinsed with tap water and then left for 24 h to dry. Acetate peel replicas were prepared of the unetched and sectioned opercula, and the peels mounted between a glass slide and cover slip (see Richardson, 2001 and references therein). The slides were viewed under the light microscope and the number of adventitious layers (Fretter and Graham, 1994) counted in the peels of sectioned opercula. 2.2. Statolith growth ring analysis The main body (the viscera and the dorsal portion of the foot retained previously after removal of the shell) of each whelk was placed dorsal side up in a crystallising dish (125 or 250 ml depending on the size of the animal) containing supersaturated sodium hydroxide solution for N3 h. Upon digestion of the flesh the contents of the dish were examined microscopically and the pair of statoliths (each b0.2 mm in diameter) removed. Each statolith was rinsed twice in tap water to remove both sodium hydroxide and any remaining flesh, rinsed in 70% and then 85% alcohol and stored in an Eppendorf tube (500 Al) containing absolute alcohol (Saurel, 2002). Two methods were used to examine the statoliths.
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Method 1: On removal from alcohol the pair of statoliths was allowed to dry for a few seconds. One statolith was cleared in HistoclearR (Fisons laboratory Reagent: /0468/17) and then positioned in a drop (1–2 ml) of DPX mountant, (BDH microscopical reagent) on a slide. A short length (1 cm) of fishing line (10 kg breaking strain) was positioned in the DPX on both sides of the statolith to ensure a constant distance was maintained between the slide and cover slip (see Morales-Nin, 1992 for larval otoliths). The position of the statolith was orientated in the mountant using fine forceps and then covered with a coverslip and the slide placed for 12 h on a heating plate (30 8C) to harden the DPX. Method 2: A small drop of Metaset resin (~ 2–3 ml) was placed on a slide and allowed to dry for 24 h. The other statolith of the pair was immersed in a small drop (~ 2 ml) of liquid resin placed on top of the hardened resin drop and orientated using fine forceps and the resin allowed to harden for ~ 24 h. The statolith was then ground on progressively finer wet abrasive paper (400 and 1000 grit) under a dissecting microscope to monitor the progress of sectioning until the section passed through the midpoint of the statolith, i.e. the statolith was sectioned in half. The sectioned statolith was then polished with diamond paste on a polishing cloth, washed thoroughly with detergent and a drop of water placed on the section under a cover slip and observed directly using a compound microscope. Photomicrographs of the sectioned opercula and DPX whole mounted and resin-embedded sectioned statoliths were taken at 10 and 40 magnification using a digital ColorView II (3.3 MegaPixel colour using FireWirek) camera mounted on a microscope. Each statolith contained a series of growth rings (see Figs. 3–6) of differing intensity. Following photography of the sectioned statoliths, they were then etched in 0.1 M HCl for 15 s, rinsed in tap water, left to dry overnight and acetate peel replicas of the etched surfaces prepared as described previously for the opercula and photographed. The total number of strong and weak growth rings (maximum) and the number of strongly defined rings (minimum) were counted from whole mounted and sectioned statoliths. Whenever there was difficulty in resolving the rings, the peels of the statoliths were consulted, particularly where the rings at the statolith edge were narrowly spaced. Interpretation of the position of the rings was independently aided by ascertaining
variations in light transmission through the statolith along a traverse across the radius of the statolith. Each colour photomicrograph of the sectioned statolith was transformed to a grey scale and the pixel intensity along a horizontal line drawn through the radius of the statolith calculated using the software analySISR 3.2. The number of pixel intensity maxima was counted. 2.3. Seasonal variation in Mg:Ca ratios along the shell whorl The outer shell surfaces of four shells (SL 52.5, 70.2, 103.8 and 148.3 mm), covering the size range of whelks collected and whose ages had been estimated from the statoliths and opercula, were carefully cleaned by immersing them in a solution of 0.11 M HNO3 (69% Aristar) for 30 s. The shells were thoroughly washed in distilled water and then air dried (24 h). At intervals of 5 mm along the shell whorl (Fig. 1B) approximately 1.5 mg of shell was removed using a 0.6 mm diameter dental drill bit. The shell powder samples were placed separately in 12 ml centrifuge plastic tubes and dissolved with 10 ml of 0.11 M HNO3 (69% Aristar). The concentration of Ca and Mg in the aqueous samples was analysed using a JY138 Ultrace Inductively Coupled Plasma-Atomic Emission Spectrometer (ICPAES). The number of cycles of high and low element ratios present in the shell was counted and compared with the number of rings present in the statoliths.
3. Results The sectioned opercula displayed a clear pattern of adventitious growth layers (Fig. 2). Generally the layers were clear to count (Fig. 2A and B) although the operculum margin was sometimes folded and trapped material enclosed between the growth layers making the location of individual layers more difficult (Fig. 2C). The estimated age of each whelk, based on the number of adventitious layers, ranged between 1 and 12 years for whelks ranging from 51.2 mm to 148.3 mm shell length. Examples of sectioned statoliths are shown in Figs. 3–6. Under low power magnification each one was observed to contain a series of circular growth rings with the increment between each ring progres-
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Fig. 2. Photomicrographs of sectioned opercula of Neptunea antiqua. (A) Six growth layers (one arrowed) from the mid-portion of the operculum of a 148.3 mm whelk (no. 1), (B) 12 growth layers (one arrowed) from the mid-portion of a 103.8 mm whelk (no. 13) and (C) the appearance of the growth layers in half of the operculum of whelk no. 1. The margin of the operculum is highly folded (arrow) and material is trapped within the folds.
sively narrowing towards the statolith circumference (e.g. Figs. 3A, 4A and 5A). At the statolith rim the rings were often difficult to observe due to refraction of transmitted light as it passed through the narrowly spaced increments and edge of the statolith. Acetate peel replicas, however, of etched sectioned statoliths revealed more detail and assisted in resolving the narrowly spaced rings (e.g. Fig. 5B and D). In some statoliths there was a pattern of weakly defined rings between the more distinct rings (e.g. Fig. 4C) which in a few statoliths (b5%) caused difficulties in resolving the position of the more prominent rings. The maximum (all strong and weak growth rings) and minimum numbers (all strongly defined rings) were recorded and the average number of rings calculated. The estimated age of the youngest whelk was ~ 5 years old (length 102.2 mm) whilst a whelk of 148.3 mm was estimated to be 20 years old (based on the maximum number of rings). Differences in light transmission through the statolith along a traverse across the radius, when converted to a grey scale, highlighted the positions of the darker growth rings, i.e. regions of low light transmission (Fig. 6). This method, when applied to all the statoliths and in conjunction with a visual inspection of the statolith section, assisted in evaluating and confirming the number of statolith growth rings. The maximum and minimum numbers of statolith rings and the operculum adventitious layers are compared in Fig. 7. No significant difference was found
between the minimum number of statolith rings and the operculum adventitious layers (normal operculum data, A 2 = 0.621 p = 0.097; non-normal statolith ring data, A 2 = 2.468, p b 0.001; Levene’s test of homogeneity of variance, t = 0.532 p = 0.589; Mann Whitney test; W = 1040.5 p = 0.408). A significant difference, however, existed between the maximum number of statolith rings and the number of operculum adventitious layers (normal operculum data, A 2 = 0.621 p = 0.097; non-normal statolith ring data, A 2 = 1.588, p b 0.001; Levene’s test of homogeneity of variance t = 0.532, p = 0.589; W = 649.5 p b 0.001). These analyses suggest that the adventitious lines and the strongly defined rings can be used to estimate the age of N. antiqua. However, there was exact correspondence of ages estimated using the two methods in only four whelks and in one large whelk there was disparity between the methods; four adventitious layers were counted in the operculum yet the statolith contained 15 rings. Validation of the seasonality of statolith ring deposition was undertaken in four whelks by constructing seasonal patterns of Mg:Ca ratios along the shell whorls and comparing the number of element ratio (ER) cycles with the number of rings. The ER cycles present in the shells compared favourably with the number of statolith rings. Whelk (no. 34) displayed 6 seasonal (Mg:Ca ratio) cycles along the shell whorl (Fig. 8A), exactly comparable with the number of
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of the aperture which varied seasonally (Fig. 8D) and cannot currently be explained.
4. Discussion Sectioning the operculum and counting the number of adventitious layers provided a method which could be used with confidence to age N. antiqua. Operculum sections revealed the presence of clear, countable adventitious growth lines, particularly in the early years of growth and when the number of operculum adventitious lines was compared with the number of strongly defined statolith rings and the number of Mg:Ca cycles in the shell there was good correspondence between the methods. However in one shell (no. 12) the adven-
Fig. 3. Photomicrographs of the statoliths of Neptunea antiqua (no. 1). (A) DPX-mounted whole statolith revealing a series of circular growth rings, (B) acetate peel replica of a section of a resinembedded and etched statolith, and (C) magnified portion of the DPX-mounted statolith to indicate the position of the annual growth rings (arrows). The age of the whelk was estimated to be 17 years.
statolith rings. Whilst shells (nos. 13 and 25) displayed 13 and 9 ratio cycles comparable to the 12 strong (average 9.5) rings and 11 strong rings (average 10), respectively, present in the statoliths (Fig. 8B and C). The largest and oldest shell (no. 1) displayed 17 ER cycles comparable with the average number of statolith rings (17.5) (Fig. 8D). Peaks in the element ratios varied between shells, attaining maximum concentrations in shell no. 34 of N 20 mmol mol 1 and towards the aperture in shell no. 1 of N35 mmol mol 1. Interannual variations were apparent in individual shells (Fig. 8) with little similarity between the amplitude of the ER cycles between shells; the exception being a pronounced low ER ratio during the 5th winter (shell no. 34) (Fig. 8A) and the 12th winter (shell no. 13) (Fig. 8C). The largest and oldest shell (no. 1) had unusually elevated Mg:Ca ratios at the shell margin
Fig. 4. Photomicrographs of the statoliths of Neptunea antiqua (no. 34). (A) DPX-mounted whole statolith, (B) magnified portion of the statolith to show the position of six growth rings (arrows) and (C) portion of the statolith to show the appearance of the weakly defined growth rings between the growth rings (arrows).
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Fig. 5. Photomicrographs of the statoliths of Neptunea antiqua (no. 25). (A) DPX-mounted whole statolith, (B) acetate peel replica of a section of a resin-embedded and etched statolith, (C) centre of the resin-embedded statolith with a putative natal ring (NR) indicated and 10 clear growth rings, and (D) acetate peel replica of a section of the resin-embedded and etched statolith indicating the first four growth rings (arrows).
titious layers noticeably underestimated the age of the whelk; four adventitious layers were counted in the operculum and 15 rings in the statolith. A possible explanation for the small number of lines in the sectioned operculum could be that at some stage during
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Fig. 6. Photomicrograph of a polished section of a statolith of Neptunea antiqua. Superimposed on the image is a trace of variations of light transmission through the statolith. Areas of low transmission are associated with the denser dark growth rings.
Fig. 7. Relationship between the number of operculum adventitious layers and the maximum (5) and minimum (E) numbers of statolith rings. The x = y line indicates where the number of operculum adventitious layers = the number of statolith growth rings.
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Mg:Ca ratio (mmol/mol)
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20 15 10 5 0
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Distance from apex to aperture (cm)
Fig. 8. Seasonal variations in Mg:Ca ratios in the shells of Neptunea antiqua (nos. 34, 13, 25 and 1) (A, B, C and D). Arrows indicate the position of the annual cessations in shell growth.
the life of the whelk the operculum had been lost and then re-grown. The position of the operculum on the foot when the body is withdrawn inside the shell aperture makes it vulnerable to loss during attack by predators and damage during feeding. Richardson (2001) proposed the possible use of growth rings in gastropod statoliths for investigating the age and growth rate of species that possessed them. Barroso et al. (2005b) provide some basic data on statolith structure and the relationship between statolith size and shell size in N. reticulatus. They found that the statoliths of N. reticulatus from the north-west coast of Portugal contained up to four well-marked growth rings, the first corresponding to a natal ring, formed during settlement, and the others corresponding to the first three winters of the whelk’s life. In this study we validated the periodicity of the statolith rings in four shells by comparing the number of rings with the number of element ratio cycles in the
shell. There was close correspondence between the statolith rings and the number of element ratio cycles strongly suggesting an annual periodicity to the rings. When the interpretation of the statolith rings was used to estimate the age of the whelks we ascertained that red whelks of ~ 150 mm in the North Sea can attain ages up to 17 years. Statoliths are internal structures and are not subject to external abrasion and damage like the shell and opercula. They are therefore potentially excellent recorders of annual changes in shell growth once a relationship between statolith diameter and shell length has been established. Correlation of statolith diameters with shell length, as demonstrated in N. reticulatus (Barroso et al., 2005b), would enable the length of N. antiqua to be estimated at the formation of each growth ring and thus allow the growth rate of this species to be determined. Comparisons of shell growth determined using the statolith rings could then be made between populations of N. antiqua. Statoliths
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are found in a number of gastropod taxa and the patterns of growth rings may contain information about past growth history. If the periodicity of the rings is understood then information can be extracted from statoliths in a similar way to that already being undertaken using squid statoliths. Squid statoliths contain daily growth rings that have been used to study daily growth rates from a variety of species including Alloteuthis africana and Alloteuthis subdula (Arkhipkin and Nekludova, 1993), Ilex illecebrosus (Hurley et al., 1985) and Sepioteuthis lessoniana (Jackson, 1990) (see also Richardson, 2001 and references therein). Further experimental investigations on gastropod statoliths are required to elucidate how factors such as feeding, reproduction and seasonal variations in seawater temperature affect deposition of the statolith rings. Once this has been accomplished it should then be possible to reconstruct the past history of growth from a range of gastropod species containing suitable statoliths. It has recently been demonstrated, for example, that the microstructure and chemical composition of the larval statoliths of the neogastropod Concholepas concholepas can be used in the reconstruction of their larval dispersal history (Zacherl et al., 2003). Such analyses should be possible using juvenile and adult gastropod species once the factors affecting deposition of the growth rings have been resolved.
Acknowledgements We are grateful to DEFRA for funding a small research project to investigate the age of the red whelk Neptunea antiqua. We are very grateful to G. Connolley from the Chemistry Department, University of Wales, Bangor for undertaking the Mg:Ca analyses using a JY138 Ultrace Inductively Coupled PlasmaAtomic Emission Spectrometer (ICP-AES). [RH]
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