Pattern of Oocyte Development and Batch Fecundity in the Mediterranean Sardine

Fisheries Research 67 (2004) 13–23

Pattern of oocyte development and batch fecundity in the Mediterranean sardine Konstantinos Ganias a,∗, Stylianos Somarakis b,c, Athanassios Machias c, Athanasios Theodorou a a Laboratory of Oceanography, University of Thessaly, Fytokou Street, GR 38446, N. Ionia, Magnisia, Greece b Department of Biology, University of Patras, 26500 Patra, Greece c Institute of Marine Biology of Crete, P.O. Box 2214, GR 71003, Iraklio, Crete, Greece Received 24 March 2003; received in revised form 6 August 2003; accepted 18 August 2003

Abstract In the present study, the pattern of oocyte development was investigated in the Mediterranean sardine (Sardina pilchardus sardina) in order to examine whether non-hydrated females could be included in batch fecundity measurements. Gonad histology and frequency distributions of oocyte diameters demonstrated that the Mediterranean sardine exhibits group-synchronous type of oocyte development. The spawning batch begins to separate in size from the adjacent population of smaller oocytes at the secondary yolk globule stage and a well-developed size-hiatus is established at the tertiary yolk globule stage. The spawning batch could be clearly identified prior to hydration and batch fecundity-on-fish weight relationships did not differ significantly between hydrated females and females at the tertiary and migratory nucleus stages. Thus, apart from hydrated females, batch fecundity in the Mediterranean sardine may also be measured by the use of females at the tertiary and migratory nucleus stages. Relative fecundity was shown to be independent of body weight and its estimates during the respective peak spawning months for the Aegean Sea and Ionian Sea stocks were 360 eggs/g (December 2000) and 339 eggs/g (February 2001). © 2003 Elsevier B.V. All rights reserved.

Keywords: Mediterranean sardine; Sardina pilchardus sardina; Oocyte development; Group-synchronous; Batch fecundity

1. Introduction pilchardus: Andreu and Pinto, 1957; Quintanilla and Pérez, 2000; Sardinops sagax: Claramunt and For multiple spawning ﬁsh species with indeter- Herrera, 1994; Macewicz et al., 1996; Sardinops minate annual fecundity the only useful fecundity melanostictus: Matsuura et al., 1991; Murayama measurement is the number of eggs produced in et al., 1994; Sardinops ocellata: Le Clus, 1979; a single spawning batch (batch fecundity) (Hunter Sardinella brasiliensis: Isaac-Nahum et al., 1988; et al., 1985). In all sardine and anchovy species and Engraulis mordax: Hunter et al., 1985; Engraulis subspecies studied so far (e.g. Sardina pilchardus capensis: Melo, 1994; Engraulis japonicus: Imai and Tanaka, 1994; Encrasicholina heteroloba: Wright, ∗ 1992; Encrasicholina punctifer: Maack and George, Corresponding author. Tel.: +32-421-093-053; fax: +32-421-093-054. 1999) females are multiple spawners exhibiting in- E-mail address: [email protected] (K. Ganias). determinate annual fecundity and continuous oocyte

0165-7836/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2003.08.008 14 K. Ganias et al. / Fisheries Research 67 (2004) 13–23 size-frequency distribution, except in hydrated ovaries 2. Materials and methods where a clear size separation between the hydrated and non-hydrated oocytes is established. As a corol- Adult sardines were collected during two DEPM lary, the method of choice in assessing batch fe- (daily egg production method) surveys carried out in cundity in these species is the ‘hydrated oocyte the eastern Mediterranean during winter 2000–2001 method’ (Hunter et al., 1985). Nevertheless, due to at the respective peak spawning months for sar- the limited time course of hydration in small pelagic dine in the central Aegean Sea (December) and the fish and the aggregating behavior of spawning fe- central Ionian Sea (February) (Ganias et al., 2002; males (Hunter et al., 1985; Alheit, 1993), hydrated Anonymous, 2000). Such surveys provide fishery ovaries are often hard to obtain (e.g. Palomera and independent biomass estimates and involve the esti- Pertierra, 1993); thus, other more time-consuming mation of several biological variables of the popu- and often less accurate methods are applied (e.g. lation, like spawning frequency and batch fecundity the oocyte size-frequency method; Hunter et al., (Parker, 1985). Samples were collected on board 1985). both the commercial fishing fleet (purse seiners and In the present study, the pattern of oocyte develop- bottom trawlers) as well as the R/V “PHILIA” by ment was investigated in the Mediterranean sardine. means of an experimental pelagic trawl. A total of The latter differed from what has been described 19 commercial and 11 research samples were col- for other sardine and anchovy populations in dis- lected during the DEPM survey in the Aegean Sea playing a well-defined size-hiatus prior to oocyte (December 2000), and 21 commercial and 9 research hydration, which allowed for the precise micro- samples during the DEPM survey in the Ionian Sea scopic definition of the spawning batch since late (February 2001) (Fig. 1). Each sample consisted of a vitellogenic stages (i.e. tertiary yolk globule stage). random collection of 1.5–2 kg of sardines. Fish were Subsequently, batch fecundity of the Aegean Sea body-cavity slit and fixed in 10% neutral-buffered (December 2000) and Ionian Sea (February 2001) formalin, immediately after capture. sardine stocks was investigated by the use of females In the laboratory, 15–20 females were randomly with ovaries after the tertiary yolk globule stage of selected from each sample for histological analysis of development. the gonads. Whenever females with hydrated oocytes

Fig. 1. Map of the study area, indicating the location of adult sampling stations: (᭹) commercial fleet samples; (ᮀ) research pelagic trawl samples; (a) Aegean Sea (December 2000); (b) Ionian Sea (February 2001). K. Ganias et al. / Fisheries Research 67 (2004) 13–23 15 were macroscopically being detected in a sample, all than 500 oocytes per gonad. Oocytes are rarely per- such fish were processed in an effort to collect and fectly spherical in shape, so maximal and minimal analyze as many hydrated females as possible. Each oocyte diameters were averaged to decrease variance selected female was weighed (0.1 g) and its ovaries and avoid artificially increasing the overlap between removed, weighed (0.0001 g) and re-preserved in different groups of oocytes (West, 1990). formalin. Subsequently, a piece of tissue from the Batch fecundity was estimated using methods de- center of each ovary was subjected to histological scribed in Hunter et al. (1985). Briefly, individual analysis, considering that oocyte development in batch size was measured in three subsamples of sardine ovaries does not vary with position (Pérez 40–60 mg of tissue taken from the anterior, middle et al., 1992). Tissues were dehydrated, cleared in and posterior part of the gonad. Ovulated gonads xylol, and embedded in paraffin. Sections (4–6 ␮m) were not used in the fecundity analysis. Sardine go- were cut and stained with Mayer’s hematoxylin and nads are homogenous and there are no significant Eosin Y (Clark, 1981). Histological classification of differences in the number of hydrated oocytes per the ovaries was based on the developmental stage of unit weight between left or right ovary or among the most advanced oocytes (West, 1990). Stages of different parts of the same ovary (Pérez et al., oocyte development were adopted from Murayama 1992). et al. (1994) (Table 1 and Fig. 2). Ovary-free weight was corrected for the effect of In order to accurately assess the pattern of oocyte fixation in 10% buffered formalin using the equation: development, oocyte measurements were taken in fresh weight (g) = 0.945·(weight in preservative (g)) ovaries of the consecutive vitellogenic and final mat- (r2 = 0.99, n = 10, Ganias, 2003). Data on the num- uration stages. A random sample of five females per ber of eggs per batch (Fi) and the ovary-free weight stage of development and region/season were ana- (Wi) measured for the ith fish were used to fit a linear lyzed. Oocytes from small pieces of the ovaries were model: mechanically separated and those >0.2 mm were mea- Fi = a + bWi + i sured (±0.02 mm) with an ocular micrometer under a binocular microscope. The 0.2 mm was used as a Linear models explained data satisfactorily in terms of cutoff value in order to exclude the permanent stock residual properties and coefficients of determination of primary oocytes. Measurements included more (Zar, 1999). ANCOVA models were used to test for

Table 1 S. pilchardus sardina: histological characteristics of oocytes at different developmental stages (adopted from Murayama et al., 1994) Stage Histological characteristics

Primary oocytes (Fig. 2(a)) At the beginning of this stage (chromatin nucleolar stage) oocytes consist of a limited cytoplasm densely stained with hematoxylin. A proportionally large and centrally located nucleus, occupies the greatest part of the oocyte. As the oocytes develop, the nucleo-cytoplasmic ratio decreases, and the nucleoli increase in number and arrange around the inner part of the germinal vesicle (perinucleolar stage) Yolk vesicle (Fig. 2(b)) Unstained ‘empty’ vacuoles (yolk vesicles) appear in the cytoplasm and then migrate to the periphery of the cytoplasm forming at first a single layer and then several peripheral rows (cortical alveoli). By the end of this stage the zona radiata appears Primary yolk globule (Fig. 2(c)) Yolk is incorporated to form small yolk globules within the peripheral ooplasm. Yolk globules extend 3/4 of the distance from the periphery to the perinuclear zone. Oil droplets begin to appear in the ooplasm Secondary yolk globule (Fig. 2(d)) Yolk globules increase in number and size, and consequently oocytes are filled with them. Oil droplets increase in number and fill the entire ooplasm Tertiary yolk globule (Fig. 2(e)) The oil droplets exist only in the periphery of the germinal vesicle Migratory nucleus (Fig. 2(f) and (g)) The germinal vesicle migrates toward the animal pole and the oil droplets begin to fuse to a single oil droplet Hydration (Fig. 2(h)) The oocyte dramatically increases in size, the nucleus membrane disintegrates, and yolk globules are fused into large yolk plates. The hydrated oocytes will be ovulated and then spawned 16 K. Ganias et al. / Fisheries Research 67 (2004) 13–23

Fig. 2. S. pilchardus sardina: consecutive stages of oocyte development—(a) primary oocytes; (b) yolk vesicle stage; (c) primary yolk globule stage; (d) secondary yolk globule stage; (e) tertiary yolk globule stage; (f) migratory nucleus stage (incipient); (g) migratory nucleus stage (fusion of oil droplets); (h) hydrated oocyte. n: nucleus, ca: cortical alveoli; zr: zona radiata; g: granulosa cells; od: oil droplets; yp: yolk plates. Scale bar = 0.1 mm. differences in the fecundity relationships. Slopes were was attributable to sampling times matching the peak tested first using a model that included an interaction of the spawning period in both the Aegean (Decem- term. If the interaction term was not significant (i.e. ber) and Ionian Seas (February) (Ganias et al., 2002; slopes were not significantly different among groups), Anonymous, 2000). Most yolked females were at the then the y-intercepts were tested using the ANCOVA secondary yolk globule stage (Table 2). model (Zar, 1999). The frequency distribution of oocyte sizes was continuous in all gonads up to the primary yolk globule stage (Fig. 3). However, in 20% of the ovaries 3. Results at the secondary yolk globule stage, the oocyte size-frequency distribution was interrupted by a dis- A total of 839 ovaries were examined histologi- tinct size-hiatus (Fig. 3). Histological examination cally. The classification of the ovaries with respect demonstrated that this hiatus is established between to the stage of the most advanced oocytes (Table 2) the advanced batch, which consists of oocytes syn- showed a low fraction of unyolked females which chronized at the secondary yolk stage, and the heterogeneous population of smaller oocytes, which Table 2 consists of primary oocytes and of oocytes at the yolk S. pilchardus sardina: percentage of females at different maturity vesicle stage (Fig. 4). At the subsequent developmen- stages tal stages, i.e. from the tertiary yolk globule stage Stage Aegean Sea Ionian Sea onwards, the spawning batch was fully separated in (December 2000) (February 2001) size in all females analyzed. Primary oocytes 0.48 0.00 In addition to the size-hiatus, the microscopic dis- Yolk vesicle 1.69 0.94 Primary yolk globule 22.71 12.47 tinction of the spawning batch at the tertiary yolk glob- Secondary yolk globule 48.07 66.82 ule and migratory nucleus stage was further facilitated Tertiary yolk globule 14.25 11.06 by the characteristic microscopic appearance of its Migratory nucleus 11.35 7.29 oocytes. The latter are highly opaque in relation to less Hydrated 1.45 1.41 developed oocytes (Fig. 5a) and contain a large group K. Ganias et al. / Fisheries Research 67 (2004) 13–23 17

Aegean 0.0% 0.1-10.0% Primary 10.1 - 20.0% yolk stage 20.1-30.0% >30% Ionian

Aegean * Secondary yolk stage

Ionian *

* * Aegean * * * Tertiary * stage * * * Ionian * * * * * * * * * * Aegean * * * Migratory * * * nucleus stage * * * Ionian * * * * * * * * * * * * * * * * * * * * Aegean * * * * * * * * * * * * * * * * * * * * * * Hydration * * * * * * * * * * * * * * Ionian * * * * * * * * * * * * * * * * * * * * * 0.2- 0.25- 0.3- 0.35- 0.4- 0.45- 0.5- 0.55- 0.6- 0.65- 0.7- 0.75- 0.8- 0.85- 0.9- 0.95- 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 Oocyte diameter (mm)

Fig. 3. S. pilchardus sardina: evolution of oocyte size-frequency distribution in ovaries at consecutive stages of vitellogenesis and ﬁnal maturation. Asterisks indicate size-hiatus between the advanced batch and the population of smaller oocytes. 18 K. Ganias et al. / Fisheries Research 67 (2004) 13–23

Fig. 4. S. pilchardus sardina: microphotograph of a female gonad at the secondary yolk globule stage indicating the group-synchronous pattern of oocyte development, i.e., a “fairly synchronous population of larger oocytes (clutch), which is clearly separated from the more heterogeneous population of smaller oocytes from which the clutch is recruited” (Wallace and Selman, 1981).

Fig. 5. S. pilchardus sardina: microphotographs of whole oocytes from ovaries at the migratory nucleus stage: (a) simple transmitted light; (b) polarized light; od: oil droplets; scale bar = 1 mm. K. Ganias et al. / Fisheries Research 67 (2004) 13–23 19

14000 14000

12000 12000

10000 10000

8000 8000

6000 6000 batch size batch size 4000 4000

2000 2000

0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 (a)gonad free weight (g) (b) gonad free weight (g)

Fig. 6. Batch fecundity relationships for the eastern Mediterranean sardine: (a) Aegean Sea (December 2000); (b) Ionian Sea (February 2001). Black circles, solid line: tertiary yolk globule and migratory nucleus stage females (n2000 = 41, n2001 = 38); open circles, dotted line: hydrated ovaries (n2000 = 22, n2001 = 10). of closely-spaced oil droplets or a single large droplet, through zero (Table 3). The slope of the zero forced which become readily discernible when using polar- regression equals relative batch fecundity (Priede and ized light (Fig. 5b). Hence, oocytes of the potential Watson, 1993). Hence, estimated relative fecundity spawning batch could be easily identified by simple during the peak of the spawning period was 360 and observation, and batch fecundity measurements were 339 eggs/g for the Aegean Sea and Ionian Sea, respec- performed using both hydrated and non-hydrated fe- tively (Table 3). males, i.e., females at the tertiary or migratory nucleus stage. All batch size-fish weight relationships were best 4. Discussion described by linear models (Fig. 6). The slopes and the intercepts of the linear regressions did not differ sig- In multiple spawning fish with indeterminate annual nificantly neither between hydrated and non-hydrated fecundity, the size separation of the spawning batch females (slopes: F = 0.314, P = 0.577; intercepts: from the diverse population of the remaining oocytes F = 1.472, P = 0.288) nor between regions and usually occurs at hydration (Hunter et al., 1985). How- seasons (Aegean Sea and December and Ionian Sea ever, there are reports of certain multiple spawning and February) (slopes: F = 0.975, P = 0.326; in- species in which the spawning batch is separated in tercepts: F = 0.246, P = 0.621). Furthermore, for size and may be identified in earlier developmental every single regression line, the intercept was not sig- stages. For example, in the anglerfish (Lophius litu- nificant at the 0.05 level, hence the line was forced lon) the spawning batch is separated in size and may

Table 3 Batch fecundity (F) on ovary-free weight (W) relationships for the eastern Mediterranean sardine with respect to sampling region and gonadal stages useda Region Gonad stages br2 n Aegean Sea (December 2000) Hydrated 352.92 0.58 22 Tertiary and migratory nucleus 364.11 0.60 41 Hydrated and tertiary and migratory nucleus 359.91 0.59 63 Ionian Sea (February 2001) Hydrated 316.88 0.51 10 Tertiary and migratory nucleus 344.42 0.80 38 Hydrated and tertiary and migratory nucleus 339.03 0.74 48

a b: slopes of the zero forced linear models (F = bW); r2: coefficient of determination; n: sample size. 20 K. Ganias et al. / Fisheries Research 67 (2004) 13–23 be identified from the secondary yolk globule stage Based on the classification of Marza (1938), (Yoneda et al., 2001), and in Siganus canaliculatus Wallace and Selman (1981) recognized three types of since the tertiary yolk globule stage (Hoque et al., oocyte development, i.e. synchronous, asynchronous, 1999). In the case of the eastern Mediterranean sar- and group-synchronous, the latter being by far the dine, the spawning batch starts to fully separate in size most common situation among teleosts. According to from the adjacent population of smaller oocytes since Wallace and Selman (1981) in the group-synchronous the secondary yolk globule stage and the separation type: “at least two populations of oocytes can be is completed at the tertiary yolk globule stage. This distinguished at some time: a fairly synchronous pattern does not seem to vary spatially or seasonally, population of larger oocytes (clutch) and a more i.e. the pattern of clutch separation was similar in the heterogeneous population of smaller oocytes from Aegean Sea during December and the Ionian Sea dur- which the clutch is recruited”. Based on our obser- ing February. On the other hand, it differed from what vations, the Mediterranean sardine conforms to the has been described for other sardine and anchovy pop- group-synchronous pattern, as spawning batches de- ulations, in which the size separation and the clear velop and mature as synchronously developing oocyte distinction of the spawning batch is only achieved at populations, which are clearly distinguished from the oocyte hydration (see references in Section 1). heterogeneous stock of smaller oocytes from which Intra- or inter-specific differences in the frequency they recruit (Fig. 3). This type of development has distributions of oocyte sizes might be explained also been suggested for many multiple spawning fish in terms of differential rates of clutch recruitment. with indeterminate fecundity, such as the common Wallace and Selman (1981) report that increase in the snook, Centropomus undecimalis (Taylor et al., 1998), rate of clutch recruitment in female Apeltes quadracus the dusky grouper, Epinephelus marginatus (Marino injected with human chorionic gonadotropin changes et al., 2001), the white-spotted spinefoot, S. canalic- the frequency distribution of oocyte sizes, which ulatus (Hoque et al., 1999), the bleak, Alburnus al- becomes fairly continuous in ovaries prior to hydra- burnus (Rinchard and Kestemont, 1996), the white tion. In that respect, the exceptional, among batch bream, Blinka bjoernka (Rinchard and Kestemont, spawning clupeids, pattern of oocyte development in 1996), and the swordfish, Xiphias gladius (Taylor and the Mediterranean sardine, in which the spawning Murphy, 1992). batch is fully separated in size before hydration, can The existence of a well-defined size-hiatus in all be attributed to a low rate of clutch recruitment and females at the tertiary yolk globule and migratory nu- subsequently large inter-spawning interval. Indeed, cleus stages and the easy microscopic identification of female sardines in coastal central Greece were shown such oocytes (highly opaque appearance, presence of to spawn every 11–12 days (Ganias et al., 2003). This oil droplet(s) easily discernible with polarized light) value of spawning frequency is relatively low com- allows the potential use of females in these two stages pared to other sardine (range of spawning frequency of oocyte development for batch fecundity mea- values: 8–23%, Ganias et al., 2003) and anchovy surements using the simple method of enumerating stocks (range of spawning frequency values: 6.3–90%, advanced oocytes rather than applying the laborious Alheit, 1993). Size separation of the spawning batch ‘oocyte size-frequency method’ (Hunter et al., 1985). prior to hydration combined with large interspawning The inclusion of these females in the batch fecun- interval is also reported for other multiple spawning dity sample might prove very useful, because, due to species like L. litulon (which spawns once every one the limited duration of the hydration stage in small or more months; Yoneda et al., 2001) and S. canalic- pelagics, hydrated ovaries can often be hard to obtain, ulatus (which spawns every 26–30 days; Hoque et al., unless specifically designed sampling schemes can 1999). On the other hand, in species like nehu (En- be applied. At least for the Mediterranean sardine, crasicholina purpurea), which may spawn every other the simple identification of tertiary and migratory day (Clarke, 1987), the spawning batch is closely nucleus stages by microscopic examination of whole followed by subsequent oocyte batches and does not oocytes and their subsequent use in batch fecundity separate in size but at hydration (Maack and George, analysis offer a low-cost alternative to the ‘hydrated 1999). oocyte method’. K. Ganias et al. / Fisheries Research 67 (2004) 13–23 21

Atresia would likely affect batch fecundity in ter- A comparison of the available batch fecundity-on- tiary yolk globule and migratory nucleus females. fish weight relationships for different Sardina and However, during peak sardine spawning, the intensity Sardinops stocks (Table 4) suggests lower fecundities of atresia is very low (<5% of yolked oocytes in af- for the eastern Mediterranean. The lower fecundity fected females (unpublished data), see also Zwolinski as well as lower spawning frequency (Ganias et al., et al., 2001) to significantly affect ‘potential batch 2003) in the eastern Mediterranean might be attributed fecundity’. In both batch fecundity-on-gonad free to the oligotrophy of this Sea especially in its east- weight relationships (Aegean Sea and Ionian Sea), ern basin (Stergiou et al., 1997). Such relationships the intercept was non-significantly different from between fecundity and the nutritional condition of zero, which indicated that relative fecundity (batch the individuals and the productivity of their habitat fecundity per unit body weight) did not change signif- are reported for several fish species. Hartmann and icantly with fish weight. Furthermore, the estimates Quoss (1993) report for the population of Coregonous of relative batch fecundity did not differ significantly lavaretus of the lake Constance the existence of a long between the Aegean Sea and the Ionian Sea in De- time-series of fecundity values, which correlate with cember and February, respectively. The latter can be the fluctuation of the degree of the eutrophication of attributed to the fact that both estimations took place the lake. In his review, Alheit (1989) reports that egg at months of maximum spawning activity for the reproduction in small pelagic spawners is very sensitive spective stocks (Ganias et al., 2002). In other sardine to climatic oscillations like El Niño, which affect the populations (e.g. Chilean sardine: Claramunt et al., productivity of the environment. The relationship of 1993; Plaza et al., 2002; Iberian sardine: Zwolinski fecundity with the nutritional/somatic condition of et al., 2001) batch fecundity varies seasonally. It is the individuals has also been supported by labora- therefore possible that batch fecundity relationships tory experiments; for several species food restriction for the eastern Mediterranean sardine also change resulted in a decline in fecundity (e.g. E. japonicus: with respect to season, being different outside the Kawaguchi et al., 1990; Tilapia zillii: Coward and peak of the spawning period than those estimated in Bromage, 1999; Gadus morhua: Lambert and Dutil, the present study. 1997).

Table 4 Linear regression coefﬁcients for the relationship of ovary-free female weight on batch fecundity for several Sardina and Sardinops stocksa b Genus Species Subspecies Region Year Month abnFadj Sardina pilchardus pilchardus Portugal 1988c March −1097.60 437.17 126 7646 1999d January–February −89.30 437.10 55 8653 Spain 1988e April–May 1260.80 444.43 – 10149 1999d March 9046.00 243.00 21 13906 sardina Hellas 2000f December 0i 359.91 63 7198 2001f February 0i 339.03 48 6781 Italy 1994g November −655.00 455.84 91 8462 Sardinops sagax caerulea California 1986h – −21.02 495.76 44 9894 1987h – −21.09 531.83 56 10616 1994h April–May −10.59 439.53 51 8780 a Fadj: adjusted batch fecundity estimates for female weight equal to 20 g; a: intercept; b: slope; n: sample size. b Parrish et al. (1989). c Cunha et al. (1992). d Anonymous (2000). e Garc´ıa et al. (1992). f Present study. g Casavola et al. (1998). h Macewicz et al. (1996). i Zero forced regression. 22 K. Ganias et al. / Fisheries Research 67 (2004) 13–23

Acknowledgements Cunha, E.M., Figueiredo, I., Farinha, A., Santos, M., 1992. Estimation of sardine spawning biomass off Portugal by the We thank Dr. C. Papaconstantinou, E. Caragitsou daily egg production method. Bol. Inst. Esp. Oceanogr. 8 (1), 139–153. and A. Siapatis from the National Centre for Marine Ganias, K., 2003. Oceanographic and biological investigation of Research in Athens and Prof. K. Koutsikopoulos and ichthyoplanktonic production of sardine, Sardina pilchardus all the staff from the Laboratory of Zoology of the Uni- (Walbaum, 1792), in the seas of coastal central Greece. Ph.D. versity of Patras for their collaboration in the collec- Thesis. University of Thessaly, Volos, Greece (in Greek with tion of the samples. Dr. G. Koumoundouros is thanked English abstract). Ganias, K., Somarakis, S., Koutsikopoulos, C., Machias, A., for his assistance in microscopic photography and N. Theodorou, A., 2002. Seasonal variation of somatic and organ Papaconstantinou for her help in laboratory analysis. weight indices of sardine in the Aegean and Ionian Seas. In: The comments of two anonymous reviewers are much Proceedings of the Ninth ICZEGAR, vol. 37. appreciated. This research was funded by a EU Re- Ganias, K., Somarakis, S., Machias, A., Theodorou, A., 2003. search Project, DG XIV, Contract No. 98/0039. This Evaluation of spawning frequency in a Mediterranean sardine population. Mar. Biol. 142, 1169–1179. study represents part of a Ph.D. dissertation submit- Garc´ıa, A., Pérez, N., Lo, N.C.H., Lago de Lanzós, A., Solá, ted to the Department of Agriculture, Animal Produc- A., 1992. The egg production method applied to the spawning tion and Marine Environment, University of Thessaly, biomass estimation of sardine, Sardina pilchardus (Walb.) on Greece. the North Atlantic Spanish coast. Bol. Inst. Esp. Oceanogr. 8 (1), 123–138. Hartmann, J., Quoss, H., 1993. 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