ICES CM 1999/Post: 9

Growth increments and biomineralization process in cephalopod statoliths Vera Bettencourt and Angel Guerra

Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo ti:lJTel: +34-986-231930, fax: +34-986-292762, e-mail: verab@iim.csic.es

Abstract A study on morphological, structural and biochemical composition of Sepia officinalis

and Loligo vulgaris statoliths and statocyst endolymph was undertaken with the aim of determine the major factors affecting the deposhion process of statolith formation and to clarify the cause for the poor definition of the growth increments in S. officinalis statoliths. The different biochemical composition of the statocyst endolymph found in both species accounted for a distinct statolith crystallisation process, which resulted in a different microstructure. This explains the better definition of growth increments in L. vulgaris comparing with S. officinalis ones. The protein content and Ca2+ and Mg2+ ions concentrations in the endolymph are more implicated in growth increments formation than Sr2+ ions concentration. Moreover the daily variations of the three factors mentioned, allowed us to formulate a working hypothesis to explain the daily deposition of growth increments: a dark ring (rich in organic matter) is deposited during daylight whereas a light ring (rich in CaC03) during darkness. These results are discussed on the light of the alternative hypothesis to explain the deposition mechanisms in statoliths.

Key words: biomineralization, cephalopods, growth increments, statoliths

Introduction Cephalopod statolith formation resulted from the deposition of a mineral structure in a

living creature. This mineralization process in a biological environment is called biomineralization. Gautron (1944) defined biomineralization as a sequence of events, where the secreted minerals by the cells crystallised and structured ensemble following a defined plan of construction. The obtained structures are composed by a mineral matrix and an organic matrix, generally make up by a mixture of proteins, phosphoproteins, glicoproteins, proteoglucids and polissacarids.

The presence of several substances on a mineral structure inhibits or favours the primary or the secondary nucleation and controls the crystalline structure formation. These regulation factors combined with the limits fixed by the space result in the elaboration of a methodical mineral structure of a determined configuration.

Although several works have been done on age and growth of cephalopods using growth increments in the statoliths (Morris & Aldrich, 1984; Natsukari et aI., 1988; Arkhipkin, 1993; Bettencourt et aI., 1996, Lipinski et aI., 1998, Jackson, 1998, among others) little is known'about the process involving the increment deposition in the statoliths. However, the clarification of this point is essential, since it could provide solutions to several doubts about the probably daily origin of the increments, allowing us to use the statoliths as an extreme useful tool for bioecological studies in the majority of cephalopod species.

Cephalopod statoliths are paired calcareous concretions essentially composed by calcium carbonate crystallised as aragonite with only a small percentage of organic material that ascertained to be protein (Radtke, 1983).

Presently, two alternative hypotheses to explain the deposition mechanisms in statoliths have been developed. Morris (1988, 1991a, 1991b) hypothesised that the low concentration of Mg2+ ions in the statocyst fluid allows the CaC03 precipitation in form of

aragonite, being the process controlled by the pH of the statocyst endolymph and the organic matrix. Lipinski (1993) proposed that the strontium will be the direct or indirect responsible for the definitions of growth layers and increments inthe statoliths.

Considering both hypotheses, a study on morphological, structural and biochemical composition of cuttlefish Sepia officinalis and the long-finned squid Loligo vulgaris statoliths and statocyst endolymph was undertaken. Our goals in this paper are to determine major factors affecting the deposition process on the statolith and to determine the causes for the poor definition of the growth increments in S. officinalis 'statoliths comparing with L. vulgaris ones.

Material and methods Specimen collection and terminology used Sepia officinalis (Linnaeus, 1758) and Loligo vulgaris (Lamarck, 1797) specimens

were collected from commercial fisheries in the Galician Rias (NW Iberian Peninsula) in summer 1996.

The terminology used in the present study is as follows: "focus" is defined as the starting point of crystallisation (Kristensen, 1980; Lipinski et aI., 1991). "Nucleus" is an area inside the first layer of proteinaceous template (Dawe etaI., 1985; Lipinski et aI., 1991). "Ring" or "line" is a general term which applies to all linear structures visible inside the statolith (Lipinski, 1993), the "light rings" are essentially composed of CaC03 and the "dark rings" are composed by organic matter. Finally, an "increment" or a "growth increment" is an area of the statolith composed by a light ring and a dark ring (Dawe et aI., 1985, Rodhouse et aI.,1994).

Scanning electronic microscopic (SEM) Ten statoliths of Sepia officinalis and ten of Loligo vulgaris were prepared for SEM

(PHILIPS SC-30). The statoliths were extracted from the cephalic cartilage (Lipinski, 1980) and stored in 96% ethanol. After removal, statoliths were cleaned in distilled water and then broken along the frontal plane (terminology after Clarke, 1978) by means of a scalpel. The statoliths were then etched in 1 % HCI for 2 - 3 minutes and again cleaned in distilled water. Posteriorly, statoliths were mounted on an aluminium stub and vacuum-coated with gold before viewing in the SEM. Magnifications usually did not exceed 1000x.

Energy dispersive x-ray analyses Twenty statoliths of S. officinalis and 10 statoliths of L. vulgaris extending to the

widest length range possible were used. Statoliths were prepared as for SEM. Energy dispersive X-ray analyses were performed on an EDAX analyser attached to the SEM. The analyses were taken in duplicate from two distinct points of each statolith. One point was located in the lateral dome at 50~m from the exterior margin and the other on a central point in the concave side of the wing.

Atomic absorption spectrophotometry Calcium, magnesium and strontium concentrations in the endolymph of both species

were determined using an atomic absorption spectrophotometer (VARIAN 250 Plus). Six samples of each species were taken. Three sample of40 individuals each were collected on 7 -10 h (morning specimens) in distinct days, and other three samples of 40 individuals each were taken on 19 - 21 h (afternoon specimens). After capture, individuals were immediately processed and 1O-20~1 of endolymph were extracted from the statocyst with the help of a microsyringe. The statoliths were also extracted and then frozen at -20°C until required for protein analysis.

Organic matter was removed from the endolymph with nitric acid. Lantan chloride was then added to avoid ionisation interference. The solutions thus obtained were taken to the

spectrophotometer to determined calcium and magnesium concentrations. Strontium was measured in a nitric/acetylene oxide flame under atomic emission stectrophotometry.

Organic matter determination Statolith were ashed in order to determine the percentage of organic matter present.

Water cleaned statoliths were dried at 60°C for 24h and then weighed individually. Dried statoliths were place in a muffle furnace at a temperature of 500°C for 3 h and again weighed. The organic content was expressed as the percentage of the weight lost due to ashing compared to the original dry weight.

Protein contents Protein content analyses were done in the endolymph and in the statoliths of both

species. The total protein content in the endolymph was determined by the Bradford & Smith method (Smith et aI., 1985). The protein content in the statolith was determined together, in the morning and in the afternoon specimens, since the protein in the statolith accumulated all over the growth. The total protein content was determined by quantification of the total amount of nitrogen using the Havilah et aI. (1977) method. The soluble protein content was determined by the Bradford & Smith (Smith et a1., 1985) method. However, firstly it was necessary to solublise and purify the sample, etching the statoliths in EGTA 1M for 32 hours. The solution was dialysed (MWCO: 3.500d) against ultra pure water for 24 hours and finally lyophilised (CHRIST Beta 2-16) at -80°C for 18 hours.

Biomineralization in vitro A comparative study of the crystallisation rate of dissolved calcium in the endolymph

was carried out in both species. The crystallisation rate was determined by measuring the pH (SENTRON 1001 pH) variation in time. The in vitro crystallisation method consists in to diffuse ammonia, coming from a deposit-solution of ammonium bicarbonate, through a solution of calcium chloride and endolymph, in a closed system. All the assays were realised in triplicate.

Results Specific morphological differences between the statoliths of both species were

observed. Thus, S. officinalis statoliths have the fourth main parts, lateral dome, dorsal dome, rostrum and wing, very well defined and separated by pronounced angles. However, these parts are not so well defined in L. vulgaris statoliths and they are not separated by pronounced angles, but for smooth outlines (fig. 1). The crystalline microstructure of the external surfaces of S. officinalis statoliths is very heterogeneous with crystals showing prominent angles and being deposited irregularly. Instead, the crystalline microstructure of the external surfaces of L. vulgaris statoliths is quite homogeneous with crystals regularly deposited and forming slight curvatures (fig.2). These crystal curvatures provide a surface relatively broad, continual and uniform that allows organic matter deposition, developing a more conspicuous and homogeneous dark ring than in crystal growth of S. officinalis statoliths where their pronounced angles prevent the formation of any broad and uniform surface. This seems to be the explanation for the better visualisation of increments in L. vulgaris statoliths than in S. officinalis ones (Fig. 3). The focus is very well visible in the statoliths of S. officinalis. Nevertheless, growth increments are very difficult to visualise, being only visible in the lateral dome (fig.4a). In this species, crystals are deposited concentrically with its large axes parallelly oriented to the statolith growth direction (fig 4b). The crystals in the rostrum of S. officinalis are jumbled; therefore a standard defined deposition does not exist (fig. 4c). Consequently, it is practically impossible to visualise growth increments in this part of the statolith.

Figure 5 shows the relationship between Sr/Ca ratios in the wing and in the lateral dome of S. officinalis and L. vulgaris. There was significantly more strontium in the wing

than in lateral dome of both species statoliths (t=3.55, p<0.05 in S. officinalis and t=2.64, p<0.05 in L. vulgaris). However, on the contrary, there was more calcium in the lateral dome than in the wing of both species, although these differences were not significant (p>0.05). Meanwhile, comparing the results between both species, there was more strontium in L. vulgaris statoliths and more calcium in S. officinalis statoliths although these differences were not significant (p>0.05). Furthermore, the Sr/Ca relationship was significantly higher in the wing than in the lateral dome of both species (t=3.66, p<O.OS in S. officinalis and t=2.71, p<O.OS in L. vulgaris). On the other hand, the Sr/Carelationship was higher in the wing (t=-2.09, p<0.05) and in the lateral dome (t=-1.81, p<O.OS) of all L. vulgaris than in the total of specimens of S. officinalis analysed. This relationship did not show any tendency with the length of the individuals, whoever the species considered.

Calcium, magnesium and strontium concentrations in the endolymph of S. officinalis and L. vulgaris are shown in Table 1. There were significantly higher values of calcium in the morning specimens than in the afternoon ones in both species (p<O.OOl). On the contrary, the magnesium ion showed opposite values (p<O.OOl). Comparing these ion concentrations between species and considering specimens captured at the same periods of the day it can be showed that both calcium and magnesium showed significant) y higher concentrations in S. officinalis than in L. vulgaris (p<O.OOl). Furthermore, strontium showed' different concentrations. Thus, it was more concentrated in the morning specimens of S. officinalis, but more concentrated in the afternoon individuals of L. vulgaris (p<O.OOl).

The percentage of organic matter in the statoliths of both species decreased potentially with the size of the individual (fig.6). However,' the percentage of organic matter was significantly higher in the statolith of L. vulgaris than in S. officinalis ones (t=2.27, p<0.05).

The total protein content in L. vulgaris endolymph was significantly higher than in S. offic ina lis, independently of the hour of the day (p<O.OOl) (Table 1). On the other hand, protein contents were significantly higher in the morning specimens than in the afternoon ones, in both species (p<O.OOl). The total protein content was higher in the statoliths of L. vulgaris than in S. officinalis ones, but not significant (p=0.S3). However, comparing the soluble protein in both species, it was observed that L. vulgaris statoliths contained significantly higher (p<O.OOl) concentrations of soluble protein that the S. officinalis ones (p<O.OOl).

The rate of crystallisation was measured by the pH variation in time (fig. 7). The increase of pH was very fast in the blank assay, reaching a maximum of 9.48 after 10 minutes. Posteriorly, pH decreased progressively. This revealed an induction time of nucleation, very short, followed by a rapid crystallisation rate. The pH variations experienced a different evolution in the S.officinalis endolymph assay. Firstly, the pH decreased a little and posteriorly it starts to increase gradually until reach a maximum of 7.89 after 120 minutes. The induction time was larger and the crystallisation rate slower than in blank assay. The pH variation in L. vulgaris endolymph assay was similar to that on the S. officinalis one. However, the increase of pH was slower and not so constant. Thus, ISO minutes were needed to have induction of nucleation. After that, the crystallisation process occurred very slowly as shown for the almost not pH decreasing.

Discussion The crystalline structure of S. officinalis statoliths has a very irregular form. This

irregular form coupled with the minor percentage of organic matter in S. officinalis statoliths compared with L. vulgaris could explain the' badly visualisation of growth increments in S. officinalis statoliths. Furthermore, the statoliths of both species become, more calcified as individuals grow since the organic matter declined with age. Consequently, ageing resulted in a lost of increments definition.

The results of this study showed a higher level of strontium in the wing than in the lateral dome of the statoliths of both species. Nevertheless, increments did not showed a better definition in the anterior or in the adjacent area of the wing than in the lateral dome. On the contrary, Durholtz et al (1997) found a better visualisation of the increments lin the surroundings areas of the wing corresponding with zones of high strontium concentration. This, supporting Lipinski' hypothesis. On the other hand, results of the present study also showed that growth increments are more visible in the lateral dome of S. officinalis statoliths and in the rostrum of L. vulgaris ones, than in the adjacent areas of the wing. These findings contradict Lipinski's hypothesis. Moreover, as growth increments are better defined in L. vulgaris statoliths than in S. officinalis, according with this hypothesis it would be expected that L. vulgaris statoliths should have more strontium than the S. officinalis ones. However, strontium differences are not significant neither in the wing nor in the lateral dome.

The identification of the origin of strontium in the statoliths of cephalopods is controversial. As in fish otoliths, strontium in statoliths could be the result of a combination of exogenous factors (temperature, salinity, strontium dissolved in seawater and feeding) and of endogenous factors, as physiologic responses associated with growth, stress and reproductive cycles (Gallahar & Kingsford, 1996). Since all the individuals used in our analysis were collected at the same time and place it seems that environmental factors could be discarded as the potential source of strontium variation. Our results showed that Sr/Ca ratio have not relationship with growth in both species. Therefore the hypothesis that strontium is responsible for growth increments definition should be rejected.

Most of the authors that have studied the biomineralization process (Crenshaw, 1982; Weineer & Traub, 1984; Williams, 1984 and Simkiss & Wilbur, 1989; among others) indicated that the organic matrix could forms the substrate for first promote and after regulate the growth direction of the crystals and also could act as inhibitor of crystallisation. It has been demonstrated (Addadi & Weiner, 1985) that inhibition of crystal growth is done when proteins interact with some axes of the crystal, converting its form. Moreover, the existence of a glicoprotein in the soluble matrix of the otoliths that could inhibit the crystal nucleation, even after the crystallisation is started, was shown by Wright (1991). Our findings are in agreement with these facts. Thus, addition of endolymph to the crystallisation system increased the required time to begin the crystallisation process and decreased the precipitation rate. The delay in the increasing of pH and posteriorly slow decreased in the assay with L. vulgaris endolymph compared with the assay using S. officinalis endolymph pointed out a longer induction time and a posteriorly reduction in the crystallisation rate. This delay could be explained because the total protein content is higher in L. vulgaris endolymph than in S. officinalis ones, either in the morning or in the afternoon specimens. Furthermore, growth increments could be more defined because the aragonite crystal curved in presence of more proteins, allowing a uniform and continuous deposition of organic matter.

The proportion between calcium and proteins values in the endolymph of the morning and the afternoon individuals of both species was inverse. The afternoon specimens showed more calcium and less protein than the morning ones. This seems to support the existence of a rapid precipitation of the aragonite crystals during the night, which agrees with the increase of feeding activities of S. officinalis species during the darkness in Ria de Vigo (Castro & Guerra, 1989). Calcium concentration in the endolymph decreased towards dawn while protein contents increases. These proteins inhibit the CaC03 crystals precipitation and modify the form of the crystals allowing a uniform deposition of the organic matter during daylight. This modification accounted for the formation of growth increments constituted by alternate light rings (rich in CaC03) and dark rings (rich in organic matter). This results agree with the findings by Mugiya et at (1981) who indicated that the concentration of total calcium in the

endolymph of gold fish, Carassius auratus, shows a clear daily variation and calcium precipitation decrease or stop at sunshine and resume in 3 hours.

The role of Mg2+ and Sr2+ ions ih the deposition process of the statolith is also controversial. Thus, magnesium is considered by Morris (1988, 1991) a key factor in calcification, whereas statolith biomineralization would be controlled by strontium concentration in the endolymph, according to Lipinski (1993). With the results of the present study we can not reject any of the hypothesis, since the concentration of both ions in the endolymph were within the range of values that affects calcification on a crystallisation system. However, Sr2+ showed a different pattern of concentration in the endolymph of both species. Although Sr2+ ions are vitals for the development of cephalopods' statoliths (Hanlon, 1989), the Mg2+ ions seems to be closely related with growth increment definition, since its concentration change inversely in relation to calcium concentration, in the endolymph of both species,. In this way, the Mg2+ ions were more concentrated, and associated to low Ca2+ concentration at sunshine, allows organic matter deposition whereas calcification was delayed. However the inverse process will occurs at sunset when Ca2+ions were more concentrated, protein content was low and Mg2+ ions concentration was minor. Conjunction of all these factors gives the possibility of a rapid CaC03 precipitation in aragonite form.

Recently, Pauly ( 1998) related pH changes, generator of statolith growth increments in squid, with Lutz & Rhoads (1977) theory, who proposed that oxygen limitation during daytime activity (or of night-time activity in nocturnal species) increase oxygen requirements to a level that cannot be met by supply through the gills. The animal must revert to anaerobiosis, which lower tissue pH and with cause in bivalves, daily "etching" of the external shells.

Although the aerobic and anaerobic metabolism in cephalopods has never been related with statolith increments formation, this complex metabolism shift could explain a rapid acid protein synthesis in high activity periods (Portner & Zielinski, 1998), thus accounting for the decrease of pH in the endolymph. Moreover, the Lutz & Rhoads theory could helps to understand the different definition of growth increments in cephalopods with distinct life style. Thus, the muscular pelagic squid like ommastrephids or short-finned squid, which are the cephalopods with highest metabolic rates (Well,s, 1994) and therefore the least hipoxia­tolerant, growth increments in the statoliths are very well defined. Whereas, in cuttlefish where the extensively use of the fins and the existence of a cuttlebone helping in neutral buoyancy save energy and make the aerobic - anaerobic metabolism shift more subtle, the increments in the statolith are badly defined. An intermediate case, will be the way of life of the long-finned squid (loliginids), accounting for the worse growth increments definition than in short-finned squid but better than in cuttlefish.

Acknowledgements The authors would like to express their gratitude to Dr. J.M Garcia Ruiz for his

hospitality and support during our research visit to the Instituto de Andalucia de Ciencias de la Tierra, Facultad de Ciencias, University of Granada, Spain in June 1997 for biomineralization studies. To C.A.C.T.I., University of Vigo, Spain for the analysis of elements. This work is a part of a PhD thesis by V. Bettencourt, funded by JNICT-Portugal (PraxisIBD/4561194) and supported by CICYT (MAR 95-1919-C05).

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"

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~,

Figure 1 - SEM pictures of a) anterior view and b) posterior view of Sepia officinalis statoliths (ML=105 mm) and c) anterior view and d) posterior view of Loligo vulgaris statolith (ML=152 mm).

•

Figure 2 - SEM pictures of external surface of a) Sepia officinalis and b) Loligo vulgaris statoliths.

Figure 3 - SEM picture showing the growth increments in the rostrum of a Loligo vulgaris statolith.

Figure 4 - SEM pictures of Sepia officinalis statoliths, a) lateral dome where it can be seen the focus and some in~rements can . be observed, b) lateral dome showing very sharp aragonite crystals concentrically deposited and c) rostrum where the irregular pattern of CaC03 deposition can be visualised.

Sepia officinais -'-wing

--- lateral dome 3

0 0 .-i

* 2 ,--., ce

U ...... '"' r:JJ '-'

I I I I I I I I I I I

5 21 50 84 103 146 171 199 204 210 ML(mm)

Loligo vulgaris -.-wing

0 ' --- lateral dome 0 .-i

* ,--., ce

U l:; r:JJ '-' .

I

98 122 143 168 171 220 232 253 324 327

ML(mm)

Figure 5 - Relationship between Sr/Ca ratio, in the wing and lateral dome of Sepia officinalis and Loligo vulgaris statoliths and the size ML (mm) of the individuals.

25

~ 20 ce E 15 u ·2 ~ 10 '"' o ~ 5

• L. vulgaris

• S. officinalis

•

y = 2867x-1.0905

l = 0.93 n=24

y = 1901.2x-1.1624

l = 0.83 n=25

• o +-------.-----~~-----,-------,------,

o 100 200 300 400 500 ML(mm)

Figure 6 - Relationship between the percentage of organic matter, in Sepia officinalis and Loligo vulgaris statoliths, and the size (ML, mm) of the individuals.

Table 1 - Ca2+, Mg2+, Sr2+ ').nd protein concentration. (means ± SD) in Sepia officinalis and Loligo vulgaris endolymph and total and soluble protein content in statoliths of both species.

Species

Sampling period

Concentration

Ca2+

Mg2+

Sr2+

Protein

Concentration

total protein

Protein soluble

10

8 pH

7

6·

Sepia officinallis Loligo vulgaris

endolymph

7 - 10 19 - 21 7 - 10 19 - 21

(j..Lg/ml) (j..Lg/ml) (j..Lg/ml) (j..Lg/ml)

502.55±36.1 835.82±24.7 372.57±9.9 602.53±3.5

89.03±2.55 71.9±2Al 42.8±3.] 1 8.35±OA3

4.27±0.64 lA6±0.14 1.57±0.19 3.75±0.3

1820±468.8 661.7±204.6 5566.0±552.2 2003 .1±553.0

(j..Lg/mg)

43.7±14.5

15.2±0.3

statolith

(!-Lg/mg)

53.3±10A

18.6±0.5

• blank

.. S. officinalis

• L. vulgaris

5 +-----------r----------,--~------_,--------__, o 50 100 150 200

Time (minutes)

Figure 7 - Rate of crystallisation measured by the pH variation in time in a closed system. a) blank assay, b) Loligo vulgaris endolymph assay, c) Sepia officinalis endolymph assay.