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Age determination and growth of juveniles of the European hake, Merluccius merluccius (L., 1758), in the northern Tyrrhenian Sea (NW Mediterranean)
Fisheries Research 78 (2006) 211–217
Age determination and growth of juveniles of the European hake, Merluccius merluccius (L., 1758), in the northern Tyrrhenian Sea (NW Mediterranean) P. Belcari a , A. Ligas b,∗ , C. Viva b b
a Dipartimento di Scienze dell’Uomo e dell’Ambiente, Universit` a degli Studi di Pisa, via A. Volta 6, 56126 Pisa, Italy Centro Interuniversitario di Biologia Marina ed Ecologia Applicata “G. Bacci”, viale N. Sauro 4, 57128 Livorno, Italy
Received 23 June 2005; received in revised form 5 January 2006; accepted 9 January 2006
Abstract The aim of the present study was to provide an estimation of growth of juvenile European hake, Merluccius merluccius (L., 1758) (OSTEICHTHYES; MERLUCCIIDAE), by means of the analysis of otolith daily increments. Hake specimens were collected during trawl surveys carried out in the northern Tyrrhenian Sea (NW Mediterranean). The sagittae were removed from hakes ≤20 cm total length. Left otoliths were ground and polished to obtain thin frontal sections. Otolith microstructure was analysed under a compound green light-polarising microscope. A power curve with intercept was fitted to the length-age data to describe the growth of M. merluccius. According to the growth curve, a mean length of 18 cm was reached at the end of the first year of life. The validation of the otolith increment periodicity was performed by means of two indirect methods. © 2006 Elsevier B.V. All rights reserved. Keywords: Age determination; Otolith; Growth; Juveniles; Merluccius merluccius; NW Mediterranean
1. Introduction The European hake, Merluccius merluccius (Linnaeus, 1758), is a demersal finfish widely distributed in the Mediterranean and in the Atlantic, where it represents a basic component of the marine ecosystem and a resource of important economic value highly exploited by the mixed fisheries (Martin et al., 1999; Stergiou et al., 2003; Casey and Pereiro, 1995). The exploitation of this species is due to multigear fishery: most of the catches occur by means of trawl net, but also by other gears, such as long-line and gillnet (FAO, 2005). The bulk of trawl catches in the Mediterranean consists of immature hakes (<20 cm of total length), while artisanal gears mainly affect the adult fraction of population; this exploitation pattern shows a situation of over-exploitation of the resource, and may severely affect the yield per recruit and the long term sustainability of the stock and its production (Martin et ∗
Corresponding author. Tel.: +39 0586 260723; fax: +39 0586 809149. E-mail addresses: [email protected], [email protected] (A. Ligas).
0165-7836/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2006.01.006
al., 1999). In the northern Tyrrhenian Sea, the European hake is principally exploited by the trawl fleet, using a traditional bottom trawl net, locally called “volantina”; the wide vertical opening trawl net (“French” trawl net) and the gillnet are used by a large number of vessels as well. In this area, the recruitment of the hake takes place at depths of 100–200 m (Belcari et al., 2001), and the fishing ground exploited mostly by the trawl vessels produces a high amount of juvenile hake discards (Sartor et al., 2001). Data on age and growth of fishes are essential for the understanding of biological traits (e.g. lifespan, age at sexual maturity, etc.) and the study of population demographic structure and its dynamics (Panfili et al., 2002). In particular, knowledge of age and growth in the early life stages is fundamental to point out the effects of environmental changes on growth and survival, and can result in an improved understanding of the factors affecting recruitment (Stevenson and Campana, 1992). Estimating growth of juvenile M. merluccius is still a problem, owing to the difficult identification of the first
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annulus due to the presence of false rings (Morales-Nin et al., 1998). Moreover, recruitment to the fishing ground is almost continuous throughout the year due to multiple spawning, limiting the assessment of growth of juveniles based on length-frequency analysis (Morales-Nin and Aldebert, 1997). The aim of this study was to examine the growth rate of juvenile M. merluccius from the northern Tyrrhenian Sea by means of otolith microstructure analysis. This technique has proved to be a useful tool in resolving the issue of the first year of growth (Pannella, 1971) and it has been recently applied to the study of the European hake growth in the Mediterranean Sea and in the Atlantic Ocean (Morales-Nin and Aldebert, 1997; Arneri and Morales-Nin, 2000; Morales-Nin and Moranta, 2004; Kacher and Amara, 2005).
2. Materials and methods Hake specimens were collected by trawling in the northern Tyrrhenian Sea (Fig. 1). Four sampling trips were carried out in February, April, July and September 2001. Specimens were measured (fish total length to the 0.5 cm below) and sexed. The length-frequency distributions were broken down into normal components using the Batthacharya’s method performed by the FAO-ICLARM FiSAT package 1.1. Otoliths (sagittae) were removed from individuals ≤20.0 cm total length and stored dry in vials. A sub-sample of 616 left sagittae was taken for subsequent analyses. Otoliths were weighed to an accuracy of 10−4 g and length of the major axis of the otolith was measured (accuracy of 0.1 mm). The relation-
ship between otolith weight (WO ) and otolith length (LO ) was described through the power equation: WO = aLbO .
(1)
Parameters a and b were estimated using ordinary leastsquare regression after transforming data into natural logarithms (Ricker, 1973). The Student’s t-test was applied to evaluate the isometry of the otolith growth. Left sagittae were ground on wet sandpapers, polished on abrasive clothes with alumina slurry and mounted external side up on glass slides, using a two component epoxy resin; a second grinding and polishing procedure was performed to obtain thin frontal sections. Otolith sections were analysed under a compound green light-polarising microscope with planapochromatic objectives, connected to an image analysis software (Optimas 6.2) through a video camera. The first discernible daily increment around the otolith core was considered to be deposited at hatching. Each otolith was read twice: from the core to the dorsal edge and vice versa. The mean of the two readings was considered the age of the specimen; a third reading was performed if the first two differed more than 10% (Arneri and Morales-Nin, 2000). The number of daily increments deposited within the nucleus was recorded to estimate the duration of the pelagic larval phase (Fischer, 1999; MoralesNin, 2000; Morales-Nin et al., 2005). Growth was described by computing a power curve with intercept: Lt = at b + c
(2)
where t is the age in days and Lt is the fish total length at the age t (Stevenson and Campana, 1992).
Fig. 1. Northern Tyrrhenian Sea.
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Otolith dorsal radius (from the core to the dorsal edge) was measured electronically. The linear regression between fish total length and otolith radius was estimated according to the formula: L = dR + c
(3)
where L is the fish total length and R is the otolith radius. Back-calculation of recent growth rate was carried out measuring the width of the last daily increments on the dorsal edge of otoliths (Suthers et al., 1989; MoralesNin and Moranta, 2004). For each survey, a sub-sample of 60 otoliths grouped according to the fish total length (<95 mm, 100–145 mm, 150–200 mm) was taken. The width of the 30 recent increments was measured electronically. RI (otolith radius 30 days before capture) was calculated subtracting the width of the 30 recent increments from the otolith radius. A body-proportional hypothesis (BPH) (Panfili et al., 2002) was used to back-calculate the fish length: LI = [(c + dRI )/(c + dRC )]LC
(4)
where LC is the fish total length (mm) at capture, RC the otolith radius (mm), LI and RI are the fish total length and the otolith radius 30 days before capture, c and d are the intercept and the slope of the fish total length on otolith radius linear regression, computed previously. The fish growth during the last 30 days before capture (L) was calculated subtracting LI from LC . For each survey, the linear regression of L on fish total length was computed; ANCOVA was used to test significant differences among the four groups. The hatch date distribution was back-calculated by subtracting the fish age from the date of capture and plotting monthly intervals. An age-length key was applied to the length-frequency distributions in order to obtain agefrequency distributions and calculate the mean age of the single cohorts caught on the different samplings. As an indirect validation method, the difference between the mean age of a cohort and the mean age of the same cohort collected in a subsequent sampling was compared with the number of days elapsed between the samplings (Arneri and MoralesNin, 2000; Panfili et al., 2002). As a further indirect validation of the periodicity of increment formation, the match between the spawning period of the species and the back-calculated distribution was performed (Campana, 2001; Panfili et al., 2002).
3. Results A total of 32,767 specimens of M. merluccius was caught during the trawl surveys in the northern Tyrrhenian Sea. Total length ranged from 4.0 to 84.5 cm. Length-frequency distributions of specimens ≤20 cm total length are shown in Fig. 2. A bimodal distribution was pointed out in all the samples (Table 1).
Fig. 2. Length-frequency distributions of juvenile Merluccius merluccius (≤20.0 cm total length) by cruise; n: number of specimens.
The otolith major axis ranged between 2.0 and 11.4 mm, while the weight ranged from 5 to 526 × 10−4 g. The morphometric relationship between otolith weight and otolith length resulted negative allometric (Table 2). The European hake sagitta presents a lengthened and flattened shape, developing more in length than in weight. A total of 579 otoliths from specimens ranging from 4.0 to 20.0 cm total length resulted interpretable on the basis of
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Table 1 Results of the length-frequency distribution analyses using the FAOICLARM FiSAT package 1.1 Month
Group
LT (cm)
S.D.
S.I.
February
1 2
n 4767 1377
10.12 13.52
0.96 1.09
– 3.317
April
1 2
175 1501
8.94 13.84
1.39 1.74
– 3.130
July
1 2
1501 1469
7.51 10.77
0.76 0.99
– 3.716
September
1 2
22333 2968
9.99 13.60
1.39 0.96
– 3.057
n, population; LT , mean total length; S.D., standard deviation; S.I., separation index.
Fig. 4. Core of the sagitta of M. merluccius. Magnification 400×; scale bar: 100 m.
Fig. 3. Frontal section of the sagitta of M. merluccius (8.0 cm total length). Magnification 25×; scale bar: 1 mm.
daily increment analysis (Fig. 3). Otolith counts ranged from 93 to 429 daily increments. The number of increments in the nucleus (Figs. 4 and 5) ranged between 39 and 71, with an average of 52 (±2). The parameters of the power equation with intercept computed from age (days)-length data to estimate the growth of M. merluccius are summarized in Table 3. According to the growth curve (Fig. 6), a mean length of 18.3 cm was reached at the end of the first year of life, with monthly growth rates ranging from 1.7 cm month−1 , in the first six months of life, to 1.3 cm month−1 , in the second half of the first year of life. The measurements of the dorsal radius of the otoliths used for the daily increment analysis ranged from 0.40 to 2.72 mm. The parameters of the linear regression between fish total length and otolith radius are shown in Table 4; the parameters c and d were used in the BPH back-calculation formula to estimate the fish length at 30 days before capture. The width of the 30 recent daily increments ranged from 0.09
Fig. 5. Prisms developed from the accessory primordia around the nucleus. Magnification 200×; scale bar: 100 m.
to 0.29 mm. Table 5 shows the mean width of the 30 distal increments for size class; the mean L, calculated from the difference between LC and LI , is shown as well. The results of linear regression analyses carried out from growth 1 month before capture (L) and fish total length data are summarised in Table 6. Growth showed a decreasing trend in comparison to total length. ANCOVA highlighted significant differences among the four groups (F3,235 = 2.85; P < 0.05). The birth-date analysis showed a continuous hatching throughout the year, with peaks in late summer (August–September ’00) and in winter ’01 (Fig. 7). Specimens collected in February and in April ’01 belonged mostly to the same cohort, hatched in late summer ’00, while hakes sampled in July and in September ’01 hatched mostly in
Table 2 Morphometric relationship between otolith weight (10−4 g) and otolith length (mm) n
Length range (cm)
r2
b
S.E. (b)
s1
s2
Males Females
171 175
11.0–20.0 11.0–20.0
0.965 0.959
0.278 0.086
2.455 2.549
0.469 0.523
**
**
**
**
Total
616
4.0–20.0
0.988
−0.239
2.708
0.296
**
**
a
n, number of specimens; r2 , determination coefficient; a, intercept; b, slope; S.E., standard error; s1 , significance level of the F-test (H0 : β = 0); s2 , significance level of the Student’s t-test (H0 : β = 3). ** P < 0.01.
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Table 3 Estimates of the parameters of the power curve with intercept fitted by age-at-length data n
a
S.E. (a)
b
S.E. (b)
c
S.E. (c)
r2
576
0.629
0.381
0.617
0.086
−5.426
2.382
0.937
n, number of otoliths; S.E., standard error;
r2 ,
determination coefficient.
Table 5 Mean width of the 30 distal increments on the dorsal margin of the otolith (µ width, mm) and mean fish length growth in the last 30 days before capture (µL, mm 30 days−1 )
Fig. 6. Growth curve of juvenile European hake in the northern Tyrrhenian Sea (: observed values). Table 4 Analysis of the regression fish total length–otolith radius n
r2
c
d
S.E. (d)
s
579
0.93
21.36
65.28
17.28
*
n, number of specimens; r2 , determination coefficient; c, intercept; d, slope; S.E., standard error; s, significance level of the F-test (H0 : δ = 0). * P < 0.05.
winter ’01, as shown in Fig. 8. These observations agree with the results of previous studies on spawning and recruitment of M. merluccius in the Tyrrhenian and Mediterranean (Biagi et al., 1995; Maynou et al., 2003; Abella et al., 2005). The cohort hatched in late summer, as shown by the birthdate analysis, was sampled both in February (mean length
Season
Size class
n
µ width
S.D.
µL
S.D.
Winter
L1 L2 L3
20 20 20
0.23 0.16 0.15
0.04 0.04 0.03
15.30 9.93 10.06
3.63 2.25 2.02
Spring
L1 L2 L3
20 20 20
0.22 0.16 0.15
0.04 0.02 0.01
14.70 10.43 10.59
3.24 1.24 1.01
Summer
L1 L2 L3
20 20 20
0.20 0.15 0.16
0.03 0.03 0.02
12.71 9.86 10.43
2.51 1.99 1.70
Autumn
L1 L2 L3
20 20 20
0.21 0.17 0.15
0.03 0.02 0.01
12.40 10.68 9.75
1.30 1.20 1.22
n, number of specimens; S.D., standard deviation; L1 , <95 mm; L2 , 100–145 mm; L3 , 150–200 mm.
Table 6 Analysis of the regression L – total length Winter Spring Summer Autumn
n
r2
a
b
S.E. (b)
s
60 60 60 60
0.52 0.41 0.10 0.44
20.65 17.47 13.17 13.81
−0.07 −0.05 −0.02 −0.02
0.07 0.05 0.05 0.30
** ** * **
n, number of specimens; r2 , determination coefficient; a, intercept; b, slope; S.E., standard error; s, significance level of the F-test (H0 : β = 0). * P < 0.05. ** P < 0.01.
Fig. 7. Hatching date distribution of juvenile European hake in the northern Tyrrhenian Sea estimated by the birth-date analysis.
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Fig. 8. Hatching date distributions of the specimens collected in the four sampling trips carried out in the study area.
10.1 cm ± 0.9; mean age 184 days ± 8) and in April (mean length 13.8 cm ± 1.7; mean age 241 days ± 10). The Student’s t-test showed the agreement between the difference in mean ages in days and the difference in capture calendar date of 56 days (t = 0.83; P > 0.05).
4. Discussion and conclusions The validation of increment formation periodicity is a necessary prerequisite for the otolith study of any kind (Campana, 2001). The application of direct validation methods (rearing, capture marking, etc.) to determine the otolith increment periodicity in the European hake is still in progress (De Pontual et al., 2003; Morales-Nin et al., 2005). In the present study, the application of two indirect validation methods supported the daily periodicity of otolith increment formation. According to the growth model fitted to age-at-length data, European hake reaches a mean size of about 18 cm at the end of the first year of life, showing a mean growth rate of about 1.5 cm month−1 . The growth is higher in the first 6 months, decreasing in the second half of the first year of life. This was confirmed by the analysis of the recent growth: the specimens smaller than 10.0 cm, with an age less than 6 months according to the growth curve, show the fastest growth rate, as highlighted by the linear regression analysis of fish growth during the 30 days before capture on fish total length. Increment width is largest on the margin of the otoliths of the specimens smaller than 10 cm. These observations are supported by previous studies, which highlighted that the increments laid down along the prisms are progressively thinner, due to a change of metabolism with age, which could affect the otolith growth (Morales-Nin, 2000). A mean age of recruitment to the bottom of about 2 months, corresponding to a length of about 2.4 cm, was determined by the analysis of the nucleus daily increments. Similar observations were reported by Morales-Nin and Aldebert (1997), Arneri and Morales-Nin (2000), Belcari et al. (2002),
Morales-Nin and Moranta (2004) and Kacher and Amara (2005). The present results describe a fast growth rate of juvenile M. merluccius, as already reported in a preliminary study carried out in the same area (Ligas et al., 2003); previous papers based on the otolith microstructure analysis obtained estimations of the size at age 1 of about 16 cm in the Gulf of Lions (Morales-Nin and Aldebert, 1997) and in the Adriatic Sea (Arneri and Morales-Nin, 2000), and of 24 cm in the Atlantic (Kacher and Amara, 2005); Morales-Nin and Moranta (2004) observed monthly growth rates of juveniles ranging from 1.2 to 2.5 cm month−1 in the Catalan Sea. Morales-Nin et al. (2005) determined a growth rate of about 1.6 cm month−1 in a hatchery reared hake specimen. Previous estimations based on otolith annual ring analysis and on modal progression analysis reported average lengths at age 1 of 12 cm in the Ligurian Sea (Orsi-Relini et al., 1989) and of 17 cm in the Aegean Sea (Uc¸kun et al., 2000); Colloca et al. (2003) determined a length of about 15 cm in the central Tyrrhenian Sea. In the Gulf of Alicante, Garcia-Rodriguez and Esteban (2002), comparing otolith annual ring readings and length-frequency distributions, observed a fast growth rate, similar to that reported in the Atlantic (De Pontual et al., 2003; Pineiro and Sainza, 2003). Although faster growth rates should be more realistic (Morales-Nin et al., 1998), data on growth of M. merluccius are still debatable; differences between age estimations could be real and linked, for instance, to environmental conditions, or depend on inconsistencies in age determination methodologies (De Pontual et al., 2003). Difficulties in identifying the otolith first annual ring and restrictions in the applications of the modal progression analysis for the continuous spawning throughout the year of the species (Morales-Nin and Aldebert, 1997; Morales-Nin et al., 1998) make the analysis of otolith daily increments the most accurate estimations of growth in the first phases of life. However, estimation of age by means of otolith reading is made under the assumption of a coincidence between hatching and first increment, as information on the beginning of
P. Belcari et al. / Fisheries Research 78 (2006) 211–217
increment deposition is still in progress. Although the time elapsed between the two events is believed to be a few days at most, as observed by Morales-Nin et al. (2005), an underestimation of true age is possible (Morales-Nin, 2000). Further studies on the deposition of the first daily increment, on the improvement of direct validation methods, and on the physiological and environmental processes affecting the daily increment formation are requested to assess more accurately the growth dynamic of M. merluccius in the early phases of life and the related processes of recruitment and mortality.
Acknowledgements The authors like to thank Dr. Enrico Arneri from the CNRISMAR of Ancona, Italy, and Dr. Beatriz Morales-Nin from the CSIC/UIB-IMEDEA of Palma de Mallorca, Spain, for their most valuable technical and scientific support. The present research was partially supported by the European Commission, in the framework of the project “Estimation of trawl discards in the Western Mediterranean Sea. European hake (Merluccius merluccius) as case study (DG Fisheries study 00/09)”, and by the Italian Ministero delle Politiche Agricole e Forestali, in the framework of the project “Assessment of demersal resources (GRU.N.D.)”.
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Age determination and growth of juveniles of the European hake, Merluccius merluccius (L., 1758), in the northern Tyrrhenian Sea (NW Mediterranean) P. Belcari a , A. Ligas b,∗ , C. Viva b b
a Dipartimento di Scienze dell’Uomo e dell’Ambiente, Universit` a degli Studi di Pisa, via A. Volta 6, 56126 Pisa, Italy Centro Interuniversitario di Biologia Marina ed Ecologia Applicata “G. Bacci”, viale N. Sauro 4, 57128 Livorno, Italy
Received 23 June 2005; received in revised form 5 January 2006; accepted 9 January 2006
Abstract The aim of the present study was to provide an estimation of growth of juvenile European hake, Merluccius merluccius (L., 1758) (OSTEICHTHYES; MERLUCCIIDAE), by means of the analysis of otolith daily increments. Hake specimens were collected during trawl surveys carried out in the northern Tyrrhenian Sea (NW Mediterranean). The sagittae were removed from hakes ≤20 cm total length. Left otoliths were ground and polished to obtain thin frontal sections. Otolith microstructure was analysed under a compound green light-polarising microscope. A power curve with intercept was fitted to the length-age data to describe the growth of M. merluccius. According to the growth curve, a mean length of 18 cm was reached at the end of the first year of life. The validation of the otolith increment periodicity was performed by means of two indirect methods. © 2006 Elsevier B.V. All rights reserved. Keywords: Age determination; Otolith; Growth; Juveniles; Merluccius merluccius; NW Mediterranean
1. Introduction The European hake, Merluccius merluccius (Linnaeus, 1758), is a demersal finfish widely distributed in the Mediterranean and in the Atlantic, where it represents a basic component of the marine ecosystem and a resource of important economic value highly exploited by the mixed fisheries (Martin et al., 1999; Stergiou et al., 2003; Casey and Pereiro, 1995). The exploitation of this species is due to multigear fishery: most of the catches occur by means of trawl net, but also by other gears, such as long-line and gillnet (FAO, 2005). The bulk of trawl catches in the Mediterranean consists of immature hakes (<20 cm of total length), while artisanal gears mainly affect the adult fraction of population; this exploitation pattern shows a situation of over-exploitation of the resource, and may severely affect the yield per recruit and the long term sustainability of the stock and its production (Martin et ∗
Corresponding author. Tel.: +39 0586 260723; fax: +39 0586 809149. E-mail addresses: [email protected], [email protected] (A. Ligas).
0165-7836/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2006.01.006
al., 1999). In the northern Tyrrhenian Sea, the European hake is principally exploited by the trawl fleet, using a traditional bottom trawl net, locally called “volantina”; the wide vertical opening trawl net (“French” trawl net) and the gillnet are used by a large number of vessels as well. In this area, the recruitment of the hake takes place at depths of 100–200 m (Belcari et al., 2001), and the fishing ground exploited mostly by the trawl vessels produces a high amount of juvenile hake discards (Sartor et al., 2001). Data on age and growth of fishes are essential for the understanding of biological traits (e.g. lifespan, age at sexual maturity, etc.) and the study of population demographic structure and its dynamics (Panfili et al., 2002). In particular, knowledge of age and growth in the early life stages is fundamental to point out the effects of environmental changes on growth and survival, and can result in an improved understanding of the factors affecting recruitment (Stevenson and Campana, 1992). Estimating growth of juvenile M. merluccius is still a problem, owing to the difficult identification of the first
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annulus due to the presence of false rings (Morales-Nin et al., 1998). Moreover, recruitment to the fishing ground is almost continuous throughout the year due to multiple spawning, limiting the assessment of growth of juveniles based on length-frequency analysis (Morales-Nin and Aldebert, 1997). The aim of this study was to examine the growth rate of juvenile M. merluccius from the northern Tyrrhenian Sea by means of otolith microstructure analysis. This technique has proved to be a useful tool in resolving the issue of the first year of growth (Pannella, 1971) and it has been recently applied to the study of the European hake growth in the Mediterranean Sea and in the Atlantic Ocean (Morales-Nin and Aldebert, 1997; Arneri and Morales-Nin, 2000; Morales-Nin and Moranta, 2004; Kacher and Amara, 2005).
2. Materials and methods Hake specimens were collected by trawling in the northern Tyrrhenian Sea (Fig. 1). Four sampling trips were carried out in February, April, July and September 2001. Specimens were measured (fish total length to the 0.5 cm below) and sexed. The length-frequency distributions were broken down into normal components using the Batthacharya’s method performed by the FAO-ICLARM FiSAT package 1.1. Otoliths (sagittae) were removed from individuals ≤20.0 cm total length and stored dry in vials. A sub-sample of 616 left sagittae was taken for subsequent analyses. Otoliths were weighed to an accuracy of 10−4 g and length of the major axis of the otolith was measured (accuracy of 0.1 mm). The relation-
ship between otolith weight (WO ) and otolith length (LO ) was described through the power equation: WO = aLbO .
(1)
Parameters a and b were estimated using ordinary leastsquare regression after transforming data into natural logarithms (Ricker, 1973). The Student’s t-test was applied to evaluate the isometry of the otolith growth. Left sagittae were ground on wet sandpapers, polished on abrasive clothes with alumina slurry and mounted external side up on glass slides, using a two component epoxy resin; a second grinding and polishing procedure was performed to obtain thin frontal sections. Otolith sections were analysed under a compound green light-polarising microscope with planapochromatic objectives, connected to an image analysis software (Optimas 6.2) through a video camera. The first discernible daily increment around the otolith core was considered to be deposited at hatching. Each otolith was read twice: from the core to the dorsal edge and vice versa. The mean of the two readings was considered the age of the specimen; a third reading was performed if the first two differed more than 10% (Arneri and Morales-Nin, 2000). The number of daily increments deposited within the nucleus was recorded to estimate the duration of the pelagic larval phase (Fischer, 1999; MoralesNin, 2000; Morales-Nin et al., 2005). Growth was described by computing a power curve with intercept: Lt = at b + c
(2)
where t is the age in days and Lt is the fish total length at the age t (Stevenson and Campana, 1992).
Fig. 1. Northern Tyrrhenian Sea.
P. Belcari et al. / Fisheries Research 78 (2006) 211–217
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Otolith dorsal radius (from the core to the dorsal edge) was measured electronically. The linear regression between fish total length and otolith radius was estimated according to the formula: L = dR + c
(3)
where L is the fish total length and R is the otolith radius. Back-calculation of recent growth rate was carried out measuring the width of the last daily increments on the dorsal edge of otoliths (Suthers et al., 1989; MoralesNin and Moranta, 2004). For each survey, a sub-sample of 60 otoliths grouped according to the fish total length (<95 mm, 100–145 mm, 150–200 mm) was taken. The width of the 30 recent increments was measured electronically. RI (otolith radius 30 days before capture) was calculated subtracting the width of the 30 recent increments from the otolith radius. A body-proportional hypothesis (BPH) (Panfili et al., 2002) was used to back-calculate the fish length: LI = [(c + dRI )/(c + dRC )]LC
(4)
where LC is the fish total length (mm) at capture, RC the otolith radius (mm), LI and RI are the fish total length and the otolith radius 30 days before capture, c and d are the intercept and the slope of the fish total length on otolith radius linear regression, computed previously. The fish growth during the last 30 days before capture (L) was calculated subtracting LI from LC . For each survey, the linear regression of L on fish total length was computed; ANCOVA was used to test significant differences among the four groups. The hatch date distribution was back-calculated by subtracting the fish age from the date of capture and plotting monthly intervals. An age-length key was applied to the length-frequency distributions in order to obtain agefrequency distributions and calculate the mean age of the single cohorts caught on the different samplings. As an indirect validation method, the difference between the mean age of a cohort and the mean age of the same cohort collected in a subsequent sampling was compared with the number of days elapsed between the samplings (Arneri and MoralesNin, 2000; Panfili et al., 2002). As a further indirect validation of the periodicity of increment formation, the match between the spawning period of the species and the back-calculated distribution was performed (Campana, 2001; Panfili et al., 2002).
3. Results A total of 32,767 specimens of M. merluccius was caught during the trawl surveys in the northern Tyrrhenian Sea. Total length ranged from 4.0 to 84.5 cm. Length-frequency distributions of specimens ≤20 cm total length are shown in Fig. 2. A bimodal distribution was pointed out in all the samples (Table 1).
Fig. 2. Length-frequency distributions of juvenile Merluccius merluccius (≤20.0 cm total length) by cruise; n: number of specimens.
The otolith major axis ranged between 2.0 and 11.4 mm, while the weight ranged from 5 to 526 × 10−4 g. The morphometric relationship between otolith weight and otolith length resulted negative allometric (Table 2). The European hake sagitta presents a lengthened and flattened shape, developing more in length than in weight. A total of 579 otoliths from specimens ranging from 4.0 to 20.0 cm total length resulted interpretable on the basis of
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Table 1 Results of the length-frequency distribution analyses using the FAOICLARM FiSAT package 1.1 Month
Group
LT (cm)
S.D.
S.I.
February
1 2
n 4767 1377
10.12 13.52
0.96 1.09
– 3.317
April
1 2
175 1501
8.94 13.84
1.39 1.74
– 3.130
July
1 2
1501 1469
7.51 10.77
0.76 0.99
– 3.716
September
1 2
22333 2968
9.99 13.60
1.39 0.96
– 3.057
n, population; LT , mean total length; S.D., standard deviation; S.I., separation index.
Fig. 4. Core of the sagitta of M. merluccius. Magnification 400×; scale bar: 100 m.
Fig. 3. Frontal section of the sagitta of M. merluccius (8.0 cm total length). Magnification 25×; scale bar: 1 mm.
daily increment analysis (Fig. 3). Otolith counts ranged from 93 to 429 daily increments. The number of increments in the nucleus (Figs. 4 and 5) ranged between 39 and 71, with an average of 52 (±2). The parameters of the power equation with intercept computed from age (days)-length data to estimate the growth of M. merluccius are summarized in Table 3. According to the growth curve (Fig. 6), a mean length of 18.3 cm was reached at the end of the first year of life, with monthly growth rates ranging from 1.7 cm month−1 , in the first six months of life, to 1.3 cm month−1 , in the second half of the first year of life. The measurements of the dorsal radius of the otoliths used for the daily increment analysis ranged from 0.40 to 2.72 mm. The parameters of the linear regression between fish total length and otolith radius are shown in Table 4; the parameters c and d were used in the BPH back-calculation formula to estimate the fish length at 30 days before capture. The width of the 30 recent daily increments ranged from 0.09
Fig. 5. Prisms developed from the accessory primordia around the nucleus. Magnification 200×; scale bar: 100 m.
to 0.29 mm. Table 5 shows the mean width of the 30 distal increments for size class; the mean L, calculated from the difference between LC and LI , is shown as well. The results of linear regression analyses carried out from growth 1 month before capture (L) and fish total length data are summarised in Table 6. Growth showed a decreasing trend in comparison to total length. ANCOVA highlighted significant differences among the four groups (F3,235 = 2.85; P < 0.05). The birth-date analysis showed a continuous hatching throughout the year, with peaks in late summer (August–September ’00) and in winter ’01 (Fig. 7). Specimens collected in February and in April ’01 belonged mostly to the same cohort, hatched in late summer ’00, while hakes sampled in July and in September ’01 hatched mostly in
Table 2 Morphometric relationship between otolith weight (10−4 g) and otolith length (mm) n
Length range (cm)
r2
b
S.E. (b)
s1
s2
Males Females
171 175
11.0–20.0 11.0–20.0
0.965 0.959
0.278 0.086
2.455 2.549
0.469 0.523
**
**
**
**
Total
616
4.0–20.0
0.988
−0.239
2.708
0.296
**
**
a
n, number of specimens; r2 , determination coefficient; a, intercept; b, slope; S.E., standard error; s1 , significance level of the F-test (H0 : β = 0); s2 , significance level of the Student’s t-test (H0 : β = 3). ** P < 0.01.
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Table 3 Estimates of the parameters of the power curve with intercept fitted by age-at-length data n
a
S.E. (a)
b
S.E. (b)
c
S.E. (c)
r2
576
0.629
0.381
0.617
0.086
−5.426
2.382
0.937
n, number of otoliths; S.E., standard error;
r2 ,
determination coefficient.
Table 5 Mean width of the 30 distal increments on the dorsal margin of the otolith (µ width, mm) and mean fish length growth in the last 30 days before capture (µL, mm 30 days−1 )
Fig. 6. Growth curve of juvenile European hake in the northern Tyrrhenian Sea (: observed values). Table 4 Analysis of the regression fish total length–otolith radius n
r2
c
d
S.E. (d)
s
579
0.93
21.36
65.28
17.28
*
n, number of specimens; r2 , determination coefficient; c, intercept; d, slope; S.E., standard error; s, significance level of the F-test (H0 : δ = 0). * P < 0.05.
winter ’01, as shown in Fig. 8. These observations agree with the results of previous studies on spawning and recruitment of M. merluccius in the Tyrrhenian and Mediterranean (Biagi et al., 1995; Maynou et al., 2003; Abella et al., 2005). The cohort hatched in late summer, as shown by the birthdate analysis, was sampled both in February (mean length
Season
Size class
n
µ width
S.D.
µL
S.D.
Winter
L1 L2 L3
20 20 20
0.23 0.16 0.15
0.04 0.04 0.03
15.30 9.93 10.06
3.63 2.25 2.02
Spring
L1 L2 L3
20 20 20
0.22 0.16 0.15
0.04 0.02 0.01
14.70 10.43 10.59
3.24 1.24 1.01
Summer
L1 L2 L3
20 20 20
0.20 0.15 0.16
0.03 0.03 0.02
12.71 9.86 10.43
2.51 1.99 1.70
Autumn
L1 L2 L3
20 20 20
0.21 0.17 0.15
0.03 0.02 0.01
12.40 10.68 9.75
1.30 1.20 1.22
n, number of specimens; S.D., standard deviation; L1 , <95 mm; L2 , 100–145 mm; L3 , 150–200 mm.
Table 6 Analysis of the regression L – total length Winter Spring Summer Autumn
n
r2
a
b
S.E. (b)
s
60 60 60 60
0.52 0.41 0.10 0.44
20.65 17.47 13.17 13.81
−0.07 −0.05 −0.02 −0.02
0.07 0.05 0.05 0.30
** ** * **
n, number of specimens; r2 , determination coefficient; a, intercept; b, slope; S.E., standard error; s, significance level of the F-test (H0 : β = 0). * P < 0.05. ** P < 0.01.
Fig. 7. Hatching date distribution of juvenile European hake in the northern Tyrrhenian Sea estimated by the birth-date analysis.
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Fig. 8. Hatching date distributions of the specimens collected in the four sampling trips carried out in the study area.
10.1 cm ± 0.9; mean age 184 days ± 8) and in April (mean length 13.8 cm ± 1.7; mean age 241 days ± 10). The Student’s t-test showed the agreement between the difference in mean ages in days and the difference in capture calendar date of 56 days (t = 0.83; P > 0.05).
4. Discussion and conclusions The validation of increment formation periodicity is a necessary prerequisite for the otolith study of any kind (Campana, 2001). The application of direct validation methods (rearing, capture marking, etc.) to determine the otolith increment periodicity in the European hake is still in progress (De Pontual et al., 2003; Morales-Nin et al., 2005). In the present study, the application of two indirect validation methods supported the daily periodicity of otolith increment formation. According to the growth model fitted to age-at-length data, European hake reaches a mean size of about 18 cm at the end of the first year of life, showing a mean growth rate of about 1.5 cm month−1 . The growth is higher in the first 6 months, decreasing in the second half of the first year of life. This was confirmed by the analysis of the recent growth: the specimens smaller than 10.0 cm, with an age less than 6 months according to the growth curve, show the fastest growth rate, as highlighted by the linear regression analysis of fish growth during the 30 days before capture on fish total length. Increment width is largest on the margin of the otoliths of the specimens smaller than 10 cm. These observations are supported by previous studies, which highlighted that the increments laid down along the prisms are progressively thinner, due to a change of metabolism with age, which could affect the otolith growth (Morales-Nin, 2000). A mean age of recruitment to the bottom of about 2 months, corresponding to a length of about 2.4 cm, was determined by the analysis of the nucleus daily increments. Similar observations were reported by Morales-Nin and Aldebert (1997), Arneri and Morales-Nin (2000), Belcari et al. (2002),
Morales-Nin and Moranta (2004) and Kacher and Amara (2005). The present results describe a fast growth rate of juvenile M. merluccius, as already reported in a preliminary study carried out in the same area (Ligas et al., 2003); previous papers based on the otolith microstructure analysis obtained estimations of the size at age 1 of about 16 cm in the Gulf of Lions (Morales-Nin and Aldebert, 1997) and in the Adriatic Sea (Arneri and Morales-Nin, 2000), and of 24 cm in the Atlantic (Kacher and Amara, 2005); Morales-Nin and Moranta (2004) observed monthly growth rates of juveniles ranging from 1.2 to 2.5 cm month−1 in the Catalan Sea. Morales-Nin et al. (2005) determined a growth rate of about 1.6 cm month−1 in a hatchery reared hake specimen. Previous estimations based on otolith annual ring analysis and on modal progression analysis reported average lengths at age 1 of 12 cm in the Ligurian Sea (Orsi-Relini et al., 1989) and of 17 cm in the Aegean Sea (Uc¸kun et al., 2000); Colloca et al. (2003) determined a length of about 15 cm in the central Tyrrhenian Sea. In the Gulf of Alicante, Garcia-Rodriguez and Esteban (2002), comparing otolith annual ring readings and length-frequency distributions, observed a fast growth rate, similar to that reported in the Atlantic (De Pontual et al., 2003; Pineiro and Sainza, 2003). Although faster growth rates should be more realistic (Morales-Nin et al., 1998), data on growth of M. merluccius are still debatable; differences between age estimations could be real and linked, for instance, to environmental conditions, or depend on inconsistencies in age determination methodologies (De Pontual et al., 2003). Difficulties in identifying the otolith first annual ring and restrictions in the applications of the modal progression analysis for the continuous spawning throughout the year of the species (Morales-Nin and Aldebert, 1997; Morales-Nin et al., 1998) make the analysis of otolith daily increments the most accurate estimations of growth in the first phases of life. However, estimation of age by means of otolith reading is made under the assumption of a coincidence between hatching and first increment, as information on the beginning of
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increment deposition is still in progress. Although the time elapsed between the two events is believed to be a few days at most, as observed by Morales-Nin et al. (2005), an underestimation of true age is possible (Morales-Nin, 2000). Further studies on the deposition of the first daily increment, on the improvement of direct validation methods, and on the physiological and environmental processes affecting the daily increment formation are requested to assess more accurately the growth dynamic of M. merluccius in the early phases of life and the related processes of recruitment and mortality.
Acknowledgements The authors like to thank Dr. Enrico Arneri from the CNRISMAR of Ancona, Italy, and Dr. Beatriz Morales-Nin from the CSIC/UIB-IMEDEA of Palma de Mallorca, Spain, for their most valuable technical and scientific support. The present research was partially supported by the European Commission, in the framework of the project “Estimation of trawl discards in the Western Mediterranean Sea. European hake (Merluccius merluccius) as case study (DG Fisheries study 00/09)”, and by the Italian Ministero delle Politiche Agricole e Forestali, in the framework of the project “Assessment of demersal resources (GRU.N.D.)”.
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