10
RESEARCH ARTICLE Age, growth and population structure of Modiolus barbatus from the Adriatic M. Peharda C. A. Richardson I. Mladineo S. S ˇ estanovic ´ Z. Popovic ´ J. Bolotin N. Vrgoc ˇ Received: 26 June 2006 / Accepted: 27 September 2006 / Published online: 20 October 2006 ȑ Springer-Verlag 2006 Abstract Age, growth and population structure of Modiolus barbatus from Mali Ston Bay, Croatia were determined using modal size (age) classes in length frequency distributions, annual pallial line scars on the inner shell surface, internal annual growth lines in shell sections of the middle nacreous layer and Calcein marked and transplanted mussels. The length frequency distributions indicated that M. barbatus attain a length of ~40 mm in 5–6 years indicating that a large propor- tion of the population in Mali Ston Bay is <5 years old. Some mussels of ~60 mm were predicted to be 14 years old using the Von Bertalanffy growth (VBG) equation. Up to the first 6 pallial line scars were visible in young (<6 years) mussels but in older shells the first scars became obscured by nacre deposition as the mussel increased in length and age. The age of the older shells (>6 years) was determined from the middle nacreous lines in shell section, which formed annually in winter between February and March; the wider dark incre- ments forming during summer (June to September). The oldest mussel, determined from the middle nacre- ous lines, was >12 years, with the majority of mussels aged between 3 and 6 years of age. The ages of mussels ascertained using the growth lines were not dissimilar to the ages predicted from the length frequency distribu- tions. Age at length curves produced using modal size class data were not different from the data obtained using the pallial scar rings and internal growth lines. Taken together these data suggest that M. barbatus attains a length of 40 and 50 mm within 5 and 8 years, respectively. Eighty one percent of individual M. barb- atus injected with a Calcein seawater solution (300 mg Calcein l –1 ), into their mantle cavity successfully deposited a fluorescent line, which was visible in suit- ably prepared shell sections under ultra violet light. Incorporation of Calcein into the mussel shells was seasonally variable with the lowest frequency of incor- poration in mussels marked in February and recovered in May. Seasonal shell growth was observed with sig- nificantly higher growth rates in mussels marked in May and removed in August (ANCOVA, F 3,149 = 23.11, P < 0.001). Mussels (~18 to 22 mm) marked in May and recovered in August displayed maximal growth rates of >2.5 mm month –1 compared with a mean mussel growth rate of 1.2 ± 0.6 mm month –1 . At other times of the year mussel shell growth ranged from immeasurable to 1.48 mm month –1 . Introduction The bearded horse mussel Modiolus barbatus (Linnaeus 1758) is a commercially important mussel and is Communicated by O. Kinne, Oldendorf/Luhe. M. Peharda (&) I. Mladineo S. S ˇ estanovic ´ Z. Popovic ´ N. Vrgoc ˇ Institute of Oceanography and Fisheries, S ˇ etalis ˇte Ivana Mes ˇtrovic ´a 63, 21000 Split, Croatia e-mail: [email protected] C. A. Richardson School of Ocean Science, University of Wales – Bangor, Menai Bridge, Anglesey LL59 5AB, UK e-mail: [email protected] J. Bolotin Institute for Marine and Coastal Research, University of Dubrovnik, Kneza Damjana Jude 12, 20000 Dubrovnik, Croatia 123 Mar Biol (2007) 151:629–638 DOI 10.1007/s00227-006-0501-3

Age, growth and population structure of Modiolus barbatus from the Adriatic

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Page 1: Age, growth and population structure of Modiolus barbatus from the Adriatic

RESEARCH ARTICLE

Age, growth and population structure of Modiolus barbatusfrom the Adriatic

M. Peharda Æ C. A. Richardson Æ I. Mladineo ÆS. Sestanovic Æ Z. Popovic Æ J. Bolotin Æ N. Vrgoc

Received: 26 June 2006 / Accepted: 27 September 2006 / Published online: 20 October 2006� Springer-Verlag 2006

Abstract Age, growth and population structure of

Modiolus barbatus from Mali Ston Bay, Croatia were

determined using modal size (age) classes in length

frequency distributions, annual pallial line scars on the

inner shell surface, internal annual growth lines in shell

sections of the middle nacreous layer and Calcein

marked and transplanted mussels. The length frequency

distributions indicated that M. barbatus attain a length

of ~40 mm in 5–6 years indicating that a large propor-

tion of the population in Mali Ston Bay is <5 years old.

Some mussels of ~60 mm were predicted to be 14 years

old using the Von Bertalanffy growth (VBG) equation.

Up to the first 6 pallial line scars were visible in young

(<6 years) mussels but in older shells the first scars

became obscured by nacre deposition as the mussel

increased in length and age. The age of the older shells

(>6 years) was determined from the middle nacreous

lines in shell section, which formed annually in winter

between February and March; the wider dark incre-

ments forming during summer (June to September).

The oldest mussel, determined from the middle nacre-

ous lines, was >12 years, with the majority of mussels

aged between 3 and 6 years of age. The ages of mussels

ascertained using the growth lines were not dissimilar to

the ages predicted from the length frequency distribu-

tions. Age at length curves produced using modal size

class data were not different from the data obtained

using the pallial scar rings and internal growth lines.

Taken together these data suggest that M. barbatus

attains a length of 40 and 50 mm within 5 and 8 years,

respectively. Eighty one percent of individual M. barb-

atus injected with a Calcein seawater solution (300 mg

Calcein l–1), into their mantle cavity successfully

deposited a fluorescent line, which was visible in suit-

ably prepared shell sections under ultra violet light.

Incorporation of Calcein into the mussel shells was

seasonally variable with the lowest frequency of incor-

poration in mussels marked in February and recovered

in May. Seasonal shell growth was observed with sig-

nificantly higher growth rates in mussels marked in May

and removed in August (ANCOVA, F3,149 = 23.11,

P < 0.001). Mussels (~18 to 22 mm) marked in May and

recovered in August displayed maximal growth rates of

>2.5 mm month–1 compared with a mean mussel

growth rate of 1.2 ± 0.6 mm month–1. At other times of

the year mussel shell growth ranged from immeasurable

to 1.48 mm month–1.

Introduction

The bearded horse mussel Modiolus barbatus (Linnaeus

1758) is a commercially important mussel and is

Communicated by O. Kinne, Oldendorf/Luhe.

M. Peharda (&) � I. Mladineo � S. Sestanovic �Z. Popovic � N. VrgocInstitute of Oceanography and Fisheries,Setaliste Ivana Mestrovica 63,21000 Split, Croatiae-mail: [email protected]

C. A. RichardsonSchool of Ocean Science, University of Wales – Bangor,Menai Bridge, Anglesey LL59 5AB, UKe-mail: [email protected]

J. BolotinInstitute for Marine and Coastal Research,University of Dubrovnik, Kneza Damjana Jude 12,20000 Dubrovnik, Croatia

123

Mar Biol (2007) 151:629–638

DOI 10.1007/s00227-006-0501-3

Page 2: Age, growth and population structure of Modiolus barbatus from the Adriatic

distributed along the Croatian coastline of the Adriatic

Sea (Benovic 1997; Zavodnik 1997). These mussels oc-

cur in the lower eulittoral–sublittoral fringe and extend

down to depths of 110 m where they attach by strong

byssus threads to rocky substrata. They occur from the

British Isles south to Mauritania, West Africa and are

found in the Mediterranean (Poppe and Goto 2000).

Despite their commercial importance and wide distri-

bution, there are no data on the age, growth and pop-

ulation structure of the species in Croatian coastal

waters. Such information is crucial for estimating sus-

tainable exploitation rates as well as for estimating the

potential of the species for aquaculture production.

Modiolid bivalves are common inhabitants of mangrove

systems in South East Asia where they attach to the

living and dead roots of mangrove plants (Morton

1977). In Panguil Bay, southern Phillippines, for

example the related species, Modiolus metcalfei is found

in sandy and muddy subtidal sediments away from the

fringes of the mangroves (Tumandara et al. 1997) where

they are harvested by local fisherman. Harvesting re-

sults in patchy distributions of the mussels, which reside

with the anterior axis of the shell buried up to two thirds

in sand and mud substrata.

Growth in bivalves is usually described in terms of

an increase in the dimensions of the shell valves (see

Gosling 2003 for review). Several methods for deter-

mining the age and growth rate of European and

Mediterranean bivalves have been developed e.g.

length frequency analysis (Manca Zeichen et al. 2002),

surface growth rings (Peharda et al. 2002; Leontarkis

and Richardson 2005), growth lines in shell sections

(Richardson 2001) and stable oxygen isotopes (Ken-

nedy et al. 2001; Richardson et al. 2004). Each of the

methods has its advantages and disadvantages. Certain

techniques may be more appropriate for one species

than for another, but the best approach would be to

employ a range of methods which providing a robust

estimate of growth than using any one method alone

(Seed and Brown 1978).

Analysis of modal size classes in size–frequency

distributions enables estimates of the population

structure and age structure of bivalve populations to

be assessed under undisturbed natural conditions, but

does not, however, allow individual growth rates to be

determined. The accuracy of the analysis also depends

on how well the collected samples reflect the size

structure of the actual population. Where recruitment

is seasonal and the life span is short and there is little

variability in individual growth rates, individual year

classes can often be identified as distinct modes.

However, in long-lived species with extended recruit-

ment and variable individual growth rates it is usually

not possible to use size–frequency distributions to

measure growth rate due to the merging of year

classes (see Gosling 2003). The use of surface growth

rings for estimating the age and growth of bivalves is

often problematic because of the difficulties of dis-

tinguishing between seasonally produced annual

growth lines and those resulting from predation or

fishing induced disturbances (e.g. Gaspar et al. 1994;

Ramsay and Richardson 2000). The analysis of inter-

nal growth lines in shell sections has been developed

into a powerful tool for investigating the past growth

history of individual bivalve shells and for determining

their age (Richardson 2001). Whilst this method pro-

vides an accurate estimate of age and growth rate,

usually only limited numbers (20–30 shells) can nor-

mally be processed and analyzed routinely. Variations

in the stable oxygen isotopic composition (e.g. Jones

et al. 1983; Krantz et al. 1984; Kennedy et al. 2001)

and elemental ratios (e.g. Richardson et al. 2004) of

bivalve shell carbonate have been used successfully to

determine annual cycles of shell growth in one or two

individual shells. The limitations of these techniques

for determining bivalve population structure are the

number of shells whose age can be determined (e.g.

Richardson et al. 1999). In the assessment of individ-

ual bivalve shell growth rates the marking of individ-

ual bivalves (Richardson 2001; Gosling 2003) by

incorporating the fluorescent marker Calcein into the

mineralizing shell and transplanting them into their

natural environment has been shown to be a reliable

technique for estimating seasonal growth rates in

Perna perna (Kaehler and McQuaid 1999a).

In this paper we applied several methods. Analysis

of length frequency distribution was used for deter-

mining population growth rates. Calcein marking and

transplanting of M. barbatus was applied for analyzing

individual growth rates and seasonal differences in

growth rates, while the analysis of pallial line scars and

growth lines in the middle nacreous layer were applied

for individual age and growth determination.

Materials and methods

Samples of between 189 and 559 Modiolus barbatus

were collected monthly between January and

December 2004 by SCUBA divers from depths of

between 3 and 5 m from Mali Ston Bay in the Adri-

atic Sea (Fig. 1). Mussel length (anterior–posterior

axis) was measured to the nearest 0.1 mm using ver-

nier calipers and monthly length frequency distribu-

tions plotted. To estimate the growth of the

component mussel size classes these distributions

123

630 Mar Biol (2007) 151:629–638

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were separated into their component size cohorts and

analyzed using the method of Bhattacharya (1967)

contained in the FiSAT statistical package (FAO-

ICLARM). The method of sum of squared errors

(SSE) was used to determine the curvature parameter

(k) in the modified von Bertalanffy growth equation

Lt = L11� (ekðt�t0Þ). The growth performance index

phi-prime (/¢) was estimated in order to compare the

growth parameters obtained in the present work with

those reported by other authors (Sparre and Venema

1992).

Marking of individual M. barbatus with a fluorescent

Calcein mark was undertaken to investigate seasonal

changes in shell growth. In a preliminary study 10 live

M. barbatus (size range 30.2–35.1 mm) were marked by

injecting ~5 ml of a Calcein seawater solution (300 mg

Calcein l–1) into the mantle cavity using a small syringe

needle inserted between the shell valves in the area

around the pedal gape (see Kaehler and McQuaid

1999a). This location was chosen for entry into the

pallial cavity because the syringe needle could be in-

serted without opening the shell valves and it did not

damage the posterior shell margin. The mussels were

held in laboratory aquaria and supplied with seawater

for 1 month to allow further shell deposition to occur

beyond the expected Calcein mark. The flesh was re-

moved by immersing the mussel briefly in boiling water

and the shells air-dried and their length measured.

Each right shell valve was embedded in Epoxy resin

(Struers Ltd), sectioned from the umbo to the posterior

shell margin and the cut surfaces ground and polished

(see Richardson 2001). Polished shell surfaces were

observed under ultra violet radiation at a wavelength

of between 460 and 490 nm (U-MWIB Cube) using an

Olympus fluorescence microscope. Narrow band of

fluorescence were observed in all ten-mussel shells thus

demonstrating that M. barbatus incorporates Calcein

into its shell and the fluorescent band can be used as an

internal shell marker in shell growth studies of this

species (see Kaehler and McQuaid 1999a).

Between May 2004 and May 2005 a field experiment

was undertaken in Mali Ston Bay. Every 3 months ~50

to 60 M. barbatus (range 16.3–60.2 mm) were collected

by SCUBA diving and mussels injected in the field with

Calcein to mark the shell. After the injection mussels

were left out of the water for about 30 min to allow

incorporation of Calcein into the shell. Marked mussels

were placed in 2 mm mesh net cages and suspended

from a pier just above the bottom in a water depth of

5 m. After 3 months the mussels were removed and

replaced with another batch of similar size freshly

collected and Calcein marked mussels. The flesh of the

marked cage grown mussels was removed by boiling

and the shells sectioned. Mussel growth, measured

between the bright green fluorescent Calcein mark and

the growing posterior margin was measured using a

calibrated eyepiece graticule following the procedure

described by Kaehler and McQuaid (1999a). These

data were used for construction of a Gulland-Holt plot,

where growth rates were plotted on Y-axis and mean

shell length on X-axis (Gulland and Holt 1959). Von

Bertalanffy growth parameters were estimated from a

numerical value of the slope (k) and x-intercept (L¥)

(Sparre and Venema 1992). Differences in growth rates

Fig. 1 Location of Mali StonBay in the Adriatic

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Mar Biol (2007) 151:629–638 631

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between the quarterly periods were tested with a one-

way analysis of covariance (ANCOVA) with length as

covariate and study period as factor. Prior to the

analysis the growth increment data were log trans-

formed and tested for homogeneity of variance using

Levene’s test.

Further mussel age and growth data were obtained

from an analysis of the pallial line scars (see Peharda

et al. 2002) on the inner shell surface of the shells

marked with Calcein (N = 199) prior to their being

embedded in resin and from growth lines in the middle

nacreous layer of shell sections (Richardson 2001). To

assist in understanding the timing of growth line for-

mation in M. barbatus, small mussel shells (~20 mm)

from each monthly collection obtained for construction

of the length frequency distributions were embedded in

resin and sectioned. The growth lines were viewed di-

rectly from the sections, obviating the need to prepare

acetate peel replicas of the polished sections. The age

of each mussel was determined independently by two

readers and where discrepancies were found between

the ages these were either resolved following discus-

sion or an average estimated age calculated. In 8.5% of

shells, where the growth lines were either not clearly

visible or pronounced disturbance lines were present,

they were omitted from the analysis. Von Bertalanffy

growth curves were fitted using FiSAT to the size and

age data determined from the mussel shells. The dis-

tance between the clearly visible consecutive growth

lines was measured to the nearest 0.1 mm using a cal-

ibrated eyepiece graticule and these data were used in

the construction of a Gulland-Holt plot. In Mali Ston

Bay M. barbatus spawns between June and August,

with a peak in July (Mladineo et al. 2007), therefore 1st

August was chosen as the birth date.

Results

Length frequency analysis

The shell length of M. barbatus ranged between 1.7 and

65.5 mm. The smallest mussels (~5 mm) appeared in

the population between May and December, sugges-

tive of a prolonged settlement period (see Fig. 2). Only

0.8% of the M. barbatus were >55 mm in length. The

monthly length frequency distributions were polymo-

dal suggesting the presence of several age classes.

When the monthly distributions were separated into

their component size (age) classes and the length at age

data generated using the method of Bhattacharya fitted

to the VBG equation, an asymptotic length (L¥) of

66.11 mm and a k of 0.181 year–1 were obtained

(Table 1). From the VBG equation M. barbatus attain

a length of ~40 mm in 5–6 years indicating that a large

proportion of the population in Mali Ston Bay is <5

years old. However, some mussels were ~65 mm and

the VBG equation predicts that they are ~20 years old.

Analysis of Calcein marked Modiolus barbatus

shells

Two hundred and thirty two mussels were marked with

Calcein, of these 33 died during the 3 month growth

period (~14%) whilst 45 mussels (~19%) did not have a

Calcein mark. The highest mortality (18 mussels)

occurred in mussels marked and transplanted into the

cage in February and recovered in May 2005; for the

remaining periods mussel mortality ranged between 3

and 7 mussels. Mussels marked in February and

recovered in May were the least successful at incorpo-

rating a Calcein mark; 16 mussels failed to incorporate a

Calcein mark. The 154 successfully Calcein marked

M. barbatus demonstrated that growth was seasonal.

Equations obtained from Gulland-Holt plots of mussel

growth in each study period are shown in Table 2. The

estimated asymptotic lengths were variable ranging

between 49.3 mm (August–November) and 57.9 mm

(November–February), whilst k ranged between

0.063 year–1 (November–February) and 0.161 year–1

(May–August) (Table 1). There was no difference in

the length of the Calcein marked M. barbatus between

the four study periods (ANOVA, F3,150 = 1.86,

P = 0.138). Nevertheless statistically significant differ-

ences between the growth rates during the different

study periods (ANCOVA, F3,149 = 23.11, P < 0.001)

were observed. This was due to the high growth rates of

mussels marked in May and removed in August

(Fig. 3). No statistically significant differences in

mussel growth were observed in the three other

periods of the year. During each study period shell

growth was significantly and negatively related to shell

length (ANCOVA, F1,149 = 122.75, P < 0.001); growth

decreasing with increasing shell length. Two mussels,

initial length 17.9 and 21.5 mm, marked in May and

recovered in August, displayed rapid growth

(>2.5 mm month–1) compared with a mean mussel

growth rate of 1.2 ± 0.6 mm month–1. At other times

of the year growth ranged from immeasurable (a Calcein

mark on the shell margin) to 1.48 mm month–1.

Analysis of pallial line scars and middle nacreous

layer growth lines

Pallial line scars on the inner shell surface (Fig. 4) and

growth lines in the middle nacreous layer (Fig. 5) were

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632 Mar Biol (2007) 151:629–638

Page 5: Age, growth and population structure of Modiolus barbatus from the Adriatic

counted in 182 mussels (shell length 17.9–61.3 mm).

Each method had its advantages and disadvantages

although the first annual line was difficult to distinguish

using either method. The first six pallial line scars were

only visible in young (<6 years) mussels because in

older shells the first scars became obscured by nacre

deposition as the mussel increased in length and age.

Counting pallial scars in older mussels was not possible

Fig. 2 Monthly lengthfrequency distributions ofModiolus barbatus from MaliSton Bay, Adriatic to showthe appearance of smallmussels in the populationbetween May and October.The distributions are variablefrom one month to another

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Mar Biol (2007) 151:629–638 633

Page 6: Age, growth and population structure of Modiolus barbatus from the Adriatic

so instead their age was determined using growth lines

visible in the middle nacreous layer of shell sections.

This method, however, was not without its difficulties

as the mussel shells had a highly variable shell color-

ation, which made it difficult to distinguish seasonal

growth lines from the dark or light patterns of color-

ation. Inspection of smaller mussels (<20 mm) col-

lected monthly showed that the narrow white middle

Table 1 Comparison of the estimated Von Bertalanffy growth parameters using different age determination methods

Method of analysis L¥ (mm) k year–1 t0 (year) /¢

Length frequency analysis 66.11 0.181 –0.071 6.67Calcein marking May–August 55.59 0.161 NA 6.21Calcein marking August–November 49.33 0.134 NA 5.79Calcein marking November–February 57.94 0.063 NA 5.35Calcein marking February–May 52.87 0.077 NA 5.37Pallial line scars and growth lines 59.78 0.210 –0.100 6.62Growth increment analysis (Gulland-Holt) 67.26 0.168 NA 6.63

L¥ asymptotic length, k growth constant, t0 initial condition parameter, /¢ growth performance index, NA not available

Table 2. Gulland-Holt equations estimated from measurements of shell growth in Calcein-marked Modiolus barbatus shells during 4periods between May 2004 and May 2005

Period of study N Gulland-Holt equation r2 P

May–August 41 Y = 8.967 – 0.161 X 0.450 <0.001August–November 39 Y = 6.605 – 0.134 X 0.394 <0.001November–February 47 Y = 3.673 – 0.063 X 0.456 <0.001February–May 27 Y = 4.148 – 0.081 X 0.420 <0.001

Y growth rate (mm/year), X mean total shell length (mm)

Fig. 3 Predicted Von Bertalanfy growth curves estimated fromthe modal classes in length-frequency (a) pallial line scars andgrowth lines in the middle nacreous layer (b) and analysis ofincrements between lines in the middle nacreous layer (c)

Fig. 4 Photomicrograph of pallial line scars (arrows) on theinner shell surface of Modiolus barbatus. Scale bar = 1 cm

Fig. 5 Photomicrographs of polished sections of the shell ofModiolus barbatus from Mali Ston Bay to indicate the appear-ance of the internal growth lines (arrows) in the middle nacreouslayer. Scale bar 200 lm

123

634 Mar Biol (2007) 151:629–638

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nacreous lines formed in the winter period between

February and March when sea water temperatures

were minimal (10�C) whilst the wider dark increments

represent summer shell growth.

The oldest mussel, determined using the middle

nacreous lines, was >12 years (L¥ = 60 mm), although

the majority of mussels were between 3 and 6 years of

age. The ages of mussels ascertained using the internal

growth lines were not dissimilar to the ages predicted

from the length frequency distributions; the VBG

curve predicted a mussel of ~60 mm to be ~14 years of

age (Fig. 6). When the shell scar and growth line age

data were fitted to the VBG equation an asymptotic

length of 59.78 mm and k of 0.21 year–1 were obtained

(Table 1); values not dissimilar from those estimated

when the length frequency distributions were sepa-

rated into their component modal size classes (see

Table 1). These data suggest that M. barbatus attains a

length of 40 and 50 mm within 5 and 8 years, respec-

tively. For comparison, the growth increment data used

in the construction of a Gulland-Holt plot produced an

elevated estimate of L¥ (67.26 mm), and a lower k

value of 0.168 year–1 (Gulland-Holt plot N = 317,

Y = 11.307 – 0.168 X, r2 = 0.313, P < 0.001) (see

Fig. 3) compared to the values determined using the

VBG equation (see Table 1). The Phi-prime index

(Table 1) overcomes the problem of correlation be-

tween k and L¥ and enables the comparison between

the different methods for determining the VBG con-

stants. The values of /¢ suggests no differences in the

growth parameters obtained using length frequency

distributions, pallial line scars and middle nacreous

growth lines, however the values obtained using the

Calcein marked mussels is smaller and is suggestive of

a difference.

Discussion

The appearance of juvenile Modiolus barbatus

(~5 mm) into the Mali Ston Bay population occurred

between May and December indicating a prolonged

settlement period. Settlement of a related species

M. metcalfi in Panguil Bay, southern Phillippines,

however, occurred during a discrete period between

May and July (Tumandara et al. 1997). Spat were

found attached to the byssal threads and shell surfaces

of the adult sized shells. In Mali Ston Bay, newly set-

tled M. barbatus spat were found amongst the clumps

of adults which were attached to each other.

Biofouling and a covering of periostracum obscured

any potentially visible external growth rings on the

shell surface of M. barbatus precluding their use as a

method for determining their age. Seed and Brown

(1978) reported difficulties in reading the surface

growth rings in the horse mussel Modiolus modiolus

and found that the thick black periostracum had to be

removed with dilute acid before the rings became vis-

ible and age determination was possible. Age estimates

of M. modiolus, based on surface rings demonstrated

high variability with ages varying from 6 to >20 years

of age for mussels of similar size (Seed and Brown

1978). In their study they encountered problems in

identifying different modal size (age) classes from size

to frequency distributions and noted that the distribu-

tions showed relatively little change throughout the

year with no clear regular modes. M. modiolus is a

species of known longevity (40–50 years) (Anwar et al.

1990) and the older year classes merge so it is therefore

perhaps not surprising that there is little evidence of

polymodal distributions within horse mussel length

frequency distributions. By contrast length frequency

distributions of M. barbatus, which we have shown to

be a younger mussel species (5–12 years), showed

evidence of polymodal size distributions in some

monthly population samples, which could be reliably

separated into their modal size classes by the method

of Bhattacharya, and evidence of a prolonged seasonal

recruitment between May and December.

Anwar et al. (1990) estimated the age of M. modi-

olus from internal growth lines in acetate peels of shell

sections what overcame the difficulties associated with

the use of surface rings. Shell sections revealed an

alternating pattern of light and dark annual growth

lines within the middle nacreous layer; lighter, more

translucent increments were deposited during the

summer (May–October) whilst the darker lines were

laid down during the winter (January–March). In older

slow-growing mussels, or those in which the outer shell

layer had been badly abraded, the regular alternating

Fig. 6 Von Bertalanfy growth curve fitted to the length and agedata determined from the pallial line scars on the inner shellsurface and growth lines in the middle nacreous layer

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Mar Biol (2007) 151:629–638 635

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pattern of summer and winter growth could still be

resolved even at the posterior margin of the oldest (48

years old) shells where the growth increments were

narrow and the lines compressed. Shell sections of

M. barbatus revealed a series of annually deposited

growth lines, although the first and second growth lines

were difficult to locate and the lines were narrow at the

margin in larger shells due to shell thickening and

compression of the most recently deposited lines. The

majority of the M. barbatus from Mali Ston bay were

<5 years old, although an occasional individual col-

lected was >12 years of age. The inner shell surface

pallial scars were clear for up to the first 6 years of life

but became progressively overlain with nacre deposi-

tion in older mussels. A combination of both methods,

pallial line scars and internal middle nacreous lines,

was therefore used to age the shells of M. barbatus.

The method used by Kaehler and McQuaid (1999a)

in which flourochrome Calcein is deposited into the

newly mineralizing shell to act as an in situ growth

marker in the brown mussel Perna perna was success-

fully used to mark M. barbatus. No mussel mortality,

resulting from Calcein injection, was observed by Ka-

ehler and McQuaid (1999a) who found that both

juveniles and adults incorporated Calcein into their

shells when it was administered at a concentration of

~80 mg l–1. By contrast we found ~14% mortality in

marked M. barbatus held in cages, with the highest

mortality occurring between February and May, and

~20% of mussels had not produced a fluorescent line

within the shell. The fastest growth rates in M. barba-

tus, (2.5 mm month–1), were about a quarter of the

rates observed by Kaehler and McQuaid (1999a) for

small P. perna (>10 mm month–1) and greater than

that achieved by larger mussels (<1 mm month–1). The

observed high mortality in M. barbatus between Feb-

ruary and May was not caused by Calcein injection but

was the result of successful attacks by the predatory

whelk Hexaplex trunculus. The cage containing the

mussels had been accidentally placed 0.5 m lower in

the water column during this spring period and H.

trunculus had been able to drill the mussels attached to

the mesh of the cage, from outside of the cage. The

dead shells had both drill holes and chipping marks

characteristic of H. trunculus (Peharda and Morton

2006). Caging marine organisms has its disadvantages

(Seed and Brown 1978) since there can be issues of

space and non-normal behavior and water flow through

the cage can be affected by the accumulation of epi-

bionts on the outside of the cage. Whilst we tried to

minimize these potential problems, the growth rates of

the caged mussels had a lower maximum value of k

than those determined using other methods in the

natural environment (Table 1). The clumping together

of caged mussels might have reduced their shell growth

through competition for food resources. Nevertheless

the data are a first contribution to understanding

seasonal growth rates in this little studied bivalve.

Seed and Brown (1978) observed that file-marked

M. modiolus had somewhat slower growth rates par-

ticularly amongst older, larger individuals than natu-

rally growing mussels in Strangford Lough, Northern

Ireland. They suggested that the slower growth rate of

caged animals might be due to the conditions created

by the cages themselves and that the presence of mesh

plus the periodic fouling by algae probably impaired

feeding conditions within the cages by reducing water

circulation through them. In positioning the cage it

may not be possible to replicate the natural bed con-

ditions particularly if the cage is suspended in the water

column above the seabed, as was the case in this study.

It is known that water currents differ along the vertical

water column profile and that current speed affects

bivalve growth (Dame 1996). Bivalves living in current

dominated environments are the beneficiaries of

increased food availability and faecal waste removal

(Dame 1996).

Some M. barbatus shells collected during the study

were deformed. Cross-sections of the deformations

under SEM revealed the presence of endolithic cyano-

bacteria (Harper, personal communication). Kaehler

and McQuaid (1999b) found that endolith infestations

significantly reduced the strength and lowered the shell

growth of P. perna in South Africa. Differences in shell

growth rates of individual M. barbatus therefore may

have been partially attributable to endolith infestation.

The relationship between shell growth rates and inci-

dence, distribution and infestation of M. barbatus shells

by endolithic cyanobacteria would be an interesting

area for future study (see also Gray et al. 1999).

The VBG equation estimated a maximum shell

length of ~60 mm and a VBG growth constant (k) of

~0.21 year–1 using the length frequency distribution

data and the shell scar and growth line age data. These

data suggest that M. barbatus attains a length of

between 40 and 50 mm within 5 and 8 years, respec-

tively. The growth rate of M. metcalfei in Panguil Bay

in the Philippines, determined using length frequency

distribution analysis, yielded mean k and L¥ values of

2.04 year–1 and 62.5 mm, respectively (Tumandra et al.

1997); growth rates that were considerably higher than

those observed in M. barbatus from rocky substrates in

Croatian coastal waters. The Phi-prime (/¢) indices,

which ranged between 5.53 and 6.67 for M. barbatus in

this study were lower than those determined for

M. metcalfei (/¢ = 8.98). Tumandra et al. (1997), again

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636 Mar Biol (2007) 151:629–638

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indicating that the sediment dwelling Philippine spe-

cies is faster growing than the Croatian modiolid.

Owing to the rapid growth rate of M. metcalfei it is

therefore likely to be a successful species for maricul-

ture (Tumandra et al. 1997). The Croatian M. barbatus

takes about 12 years to attain a length of ~60 mm,

although the majority of mussels were smaller

(30–40 mm) and between 3 and 6 years of age. This

indicates that in Mali Ston Bay this mussel is a much

slower growing species than M. metcalfei in Philippine

coastal waters.

Bivalve shell growth can be site specific and it is

possible that M. barbatus grows faster in other parts

of the Adriatic. Previous study showed that the

Noah’s Ark shell (Arca noae), that often lives in

clumps with M. barbatus, has different growth rates at

three sampled locations in the eastern Adriatic Sea.

Individuals collected in the Mali Ston bay had the

intermediate growth rates (Peharda et al. 2002) and

they were similar to values recorded for M. barbatus

in the same area. Growth under culture conditions

can be faster than growth in the wild, and therefore,

it is necessary to conduct the experimental growth

study under aquaculture conditions in order to assess

the aquaculture potential of M. barbatus along the

Croatian coastline.

Acknowledgments This research was financed by the CroatianMinistry of Science and Technology. The authors are grateful toZeljko Bace, Marko Zaric, Nika Straglicic, Lovorka Kekez andMark Prime for technical assistance. Special thanks to BarbaraZorica for help with statistical analysis and Professor C.D.McQuaid for helpful suggestions for Calcein marking the musselshells. The experiments conducted comply with the current lawsof Republic of Croatia.

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