INTRODUCTIONTop

Sardine (Sardina pilchardus Walbaum, 1792) is one of the most important pelagic fish species due to its huge ecological (Cury et al. 2000Cury P., Bakun A., Crawford R.J.M., et al. 2000. Small pelagics in upwelling systems: patterns of interaction and structural changes in “wasp-waist” ecosystems. ICES J. Mar. Sci. 57: 603-618., Palomera et al. 2007Palomera I., Olivar M.P., Salat J., et al. 2007. Small pelagic fish in the NW Mediterranean Sea: An ecological review. Progr. Oceanog. 74: 377-396.) and economic role. It is distributed along the Atlantic continental shelves of Europe and Africa as well as in the Mediterranean, including the adjacent Black Sea. It is a fast-growing and short-living small migratory pelagic fish species. Sardine is a batch spawner with indeterminate fecundity (Blaxter and Hunter 1982Blaxter J., Hunter J.R. 1982. The biology of the clupeoid Fishes. Adv. Mar. Biol. 20: 1-123., Ganias et al. 2003Ganias K., Somarakis S., Koutsikopoulos C., et al. 2003. Ovarian atresia in the Mediterranean sardine, Sardina pilchardus. J. Mar. Biol. Assoc. UK 83: 1327-1332.), tending to spawn from September to March (Ganias et al. 2007Ganias K., Somarakis S., Koutsikopoulos C., et al. 2007. Factors affecting the spawning period of sardine in two highly oligotrophic Seas. Mar. Biol. 151: 1559-1569., Sinovčić et al. 2008Sinovčić G., Čikeš Keč V., Zorica B. 2008. Population structure, size at maturity and condition of sardine, Sardina pilchardus (Walb., 1792), in the nursery ground of the eastern Adriatic Sea (Krka River estuary, Croatia). Estuar. Coast. Shelf. Sci. 76: 739-744., Bouhali et al. 2015Bouhali F.Z., Lechekhab S., Ladaimia S., et al. 2015. Reproduction and maturation of the gonads of Sardina pilchardus from the Gulf of Annaba (North-Eastern Algeria). Cybium 39: 143-153.). This species is known as an omnivore that prefers zooplankton rather than phytoplankton prey items, and its diet changes seasonally in terms of its feeding activity and prey quantity (Costalago and Palomera 2014Costalago D., Palomera I. 2014. Feeding of European pilchard (Sardina pilchardus) in the northwestern Mediterranean: from late larvae to adults. Sci. Mar. 78: 41-54., Zorica et al. 2017Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.). In the Adriatic, the sardine is mainly targeted by purse seiners, which use artificial light to attract and aggregate the fish near the surface (Cingolani et al. 1996Cingolani N., Gianetti G., Arneri E. 1996. Anchovy fisheries in the Adriatic Sea. Sci. Mar. 60: 269-277., Kraljević et al. 2014Kraljević V., Čikeš Keč V., Zorica B. 2014. Analiza ulova ostvarenih plivaricom srdelarom u Jadranu. Croatian J. Fish. 72: 142-149.). Over the last few decades, the official sardine catch and biomass have fluctuated in the Adriatic area (FAO 2017FAO. 2017. Report of 20th session of the Scientific Advisory Committee on Fisheries. Fish. Aquac. Rep. 1245: 226.). Such fluctuations in sardine and small pelagic fish populations in general are common and most probably linked to numerous environmental factors to which incoming year-class individuals have to adapt (Fréon et al. 2005Fréon P., Cury P., Shannon L., et al. 2005. Sustainable exploitation of small pelagic fish stocks challenged by environmental and ecosystem changes: a review. Bull. Mar. Sci. 76: 385-462.).

The abovementioned fluctuations are scientifically intriguing and surely linked to the fish’s metabolism and its functioning, which is strongly correlated with fish life stages and the environment. Throughout its life span, the sardine (like all other living organisms) needs to accumulate sufficient energy reserves to ensure successful growth, reproduction and survival (Shulman and Love 1999Shulman G.E., Love R.M. 1999. The Biochemical Ecology of Marine Fishes. Adv. Mar. Biol. 36. Academic Press, San Diego, 350 pp.). The required energy reserves are not constant within the year or over the years, since the life cycle adapts along with the abiotic and biotic parameters of an ecosystem. The energy flow through one living organism is difficult to track and even more difficult to measure. In order to gain insight into this complex system, scientists have defined numerous ways of measuring metabolic rates. In the case of teleosts, the principal energy sources that can be used to measure the metabolic rate are lipids, proteins and carbohydrates (Shulman and Love 1999Shulman G.E., Love R.M. 1999. The Biochemical Ecology of Marine Fishes. Adv. Mar. Biol. 36. Academic Press, San Diego, 350 pp.).

Based on the above, the general aim of this study was to link energy reserves with the sardine’s reproductive cycle. Lipids (as energy reserves) were evaluated as they seem to play a major role in fish spawning. In fact, their levels have an effect on gonad development, egg quality, fecundity, fertilization, hatching rate and survival of larvae (Sargent et al. 1989Sargent J.R., Henderson R.J., Tocher D.R. 1989. The lipids. In: Halver J. (ed), Fish Nutrition, 2nd ed. Academic Press, London, pp. 153-217., 2002Sargent J.R., Tocher D.R., Bell J.G. 2002. The lipids, In: Halver J.E., Hardy R.W. (eds), Fish Nutrition, 3rd edition. Academic Press, San Diego, pp. 181-257., Rainuzzo et al. 1997Rainuzzo J.R., Reitan K.I., Olsen Y. 1997. The significance of lipids at early stages of marine fish: a review. Aquaculture 155: 103-115., Brosset et al. 2016aBrosset P., Lloret J., Muñoz M., et al. 2016a. Body reserves mediate trade-offs between life-history traits: new insights from small pelagic fish reproduction. R. Soc. Open Sci. 3: 160-202.). Within this study, the seasonality of lipid storage in the gonads, liver and muscle was studied over one year and analysed in the context of spawning. The findings from this investigation should provide a better understanding of sardine reproductive potential, which will be a step forward to sustainable management of this species.

MATERIALS AND METHODSTop

Biological sampling and processing

Sardine specimens (N=1085) were collected monthly from March 2017 to February 2018 (with the exception of January when the fishing ban for purse seine was in force) in the middle eastern Adriatic Sea by commercial purse seiner (mesh size of 14 mm) (Fig. 1). Immediately after capture, random samples of sardine specimens weighing approximately 1.5 kg were put on ice, landed and transported to the laboratory. Upon arrival in the laboratory, each sardine was measured (total length, TL) to the nearest 1 mm and weighed (total body weight, W) to the nearest 0.01 g. The sex of each specimen was determined macroscopically. The seasonality of sardine spawning was established by monthly changes in gonad weight (Wg±0.01 g) and the gonadosomatic index (GSI; monthly share of gonad weight (Wg) in total body weight (W). GSI=100 Wg/W) by sex and overall. The length-weight relationship was determined by sex on a monthly basis and overall, according to the logarithmic form of the standard equation log(W)=log(a)+b log(TL), where W is total fish weight in g; TL is the total length in cm; a is the proportionality constant; and b is the allometric coefficient.

Total lipid content analysis

A sub-sample was taken each month from the collected sardine sample for total lipid content analysis. Monthly sub-samples were made of ten sardine specimens of each sex, with the total length between 13.0 cm and 14.0 cm. Three pieces of tissue (1, whole liver; 2, piece of gonad; 3, piece of muscle fillet, with skin) were taken from each fish in the area between the anal opening and the caudal fin base for total lipid analysis. Each month, the tissue pieces of each organ were put together, weighed on an analytical balance (to the nearest 0.01 mg) and stored in plastic containers at –18°C.

The total lipid content was determined using a modified Van Handel extraction and analysis (Van Handel 1985Van Handel E. 1985. Rapid determination of total lipids in mosquitoes. J. Am. Mosq. Control. Assoc. 1: 302-304.). Tissue samples were homogenized using a Polytron PT 1600 E mixer (Kinematica AG, Luzern, Switzerland) and stored at −20°C until analysis. For the analysis, 50 mg of each tissue was mixed with 4 mL of chloroform:methanol mixture (1:1 (v:v)) using a Polytron mixer in a 50-mL polypropylene tube. The homogenate was centrifuged at 3000 rpm for 5 min, and the supernatant volume was recorded. A 250-µl aliquot was carefully transferred directly to the bottom of the 18×100 mm culture tube. The aliquot was placed at the bottom of the tube to minimize variability by preventing loss of lipids on the sides of the tube where they could not be properly digested.

The six-point standard curve was generated by setting up tubes containing 100 µL of the standards (0, 0.25, 0.50, 0.75 and 1.00 mg cod oil standard per millilitre of acetone). The samples and standards were set up in triplicate and placed in a heating block set at 100°C to allow the solvent to evaporate. After approximately 10 min, the solvent had evaporated and 100 µL of concentrated sulphuric acid was added to each tube by carefully depositing the acid at the bottom of the tube. The samples were vortexed, heated at 100°C for 10 min and after that allowed to cool to room temperature before adding 2.4 mL of vanillin reagent and vortexing. The reagent was prepared according to the standard method (Van Handel 1985Van Handel E. 1985. Rapid determination of total lipids in mosquitoes. J. Am. Mosq. Control. Assoc. 1: 302-304.): 600 mg of vanillin was dissolved in 100 mL of hot water after the solution had cooled down, and 400 mL of 85% phosphoric acid was added. The reagent was stored in a dark bottle at 4°C and used within two weeks. After incubation at room temperature for 5 min, the absorbance (A, nm; measured at 500 nm) was measured using a Perkin Elmer UV/VIS Lambda Bio 40 spectrophotometer (Perkin Elmer, Waltham, Massachusetts, USA). Solvent blanks were run throughout the procedure and used to “zero” the spectrophotometer.

In order to obtain the percent lipid content, the supernatant volume was multiplied by the concentration of lipid determined from the standard curve, and then divided by the sample weight (in mg) and multiplied by 100.

All solvents were HPLC grade. Sulphuric acid (98%), vanillin, o-phosphoric acid and cod liver oil were obtained from Sigma-Aldrich (St. Louis, MO, USA). The vanillin reagent was used within two weeks and stored in an amber bottle to reduce degradation.

Statistical analysis

In order to conduct a meaningful analysis, the physiology and the reproductive pattern of the investigated species were taken into account and three phases were defined: F, feeding; S, spawning; and R, resting. The dataset consisted of the gonadosomatic index (GSI) and measured fat content values obtained for each tissue (G, gonads; M, muscles; and L, liver). Given the results of the Draftsman Plot, whole data were log (x+1)-transformed. In order to determine the (dis)similarities in the dataset, principal component analysis was applied. A similarity matrix based on the Bray-Curtis index was then constructed and a one-way ANOSIM was applied to assess sex and phase differences in the dataset (Clarke and Warwick, 1994Clarke K.R., Warwick R.M. 1994. Changes in marine communities: an approach to statistical analysis and interpretation. Natural Environment Research Council. UK, 144 pp.). The PRIMER software routine (Plymouth Marine Laboratories, UK; Clarke 1993Clarke K.R. 1993. Non-parametric multivariate analyses of changes in community structure. Austral. J. Ecol. 18: 117-143., Clarke and Warwick 1994Clarke K.R., Warwick R.M. 1994. Changes in marine communities: an approach to statistical analysis and interpretation. Natural Environment Research Council. UK, 144 pp.) was used for all analyses and the level of significance was set to P<0.05.

RESULTSTop

Biological parameters

The total body length and mass of the observed sardine specimens (N=1085) varied from 11.5 to 16.9 cm (mean±SD: 13.9±0.03 cm) and from 11.30 to 54.03 g (mean±SD: 20.31±0.161 g), respectively. The monthly variations in mean total sardine length are shown in Figure 2.

The lowest mean monthly values of total body length were recorded in March (mean±SD: 13.2±0.77 cm), whereas the highest values were obtained in May (mean±SD: 14.8±0.60 cm). Sex was determined in 443 individuals (males N=179, mean TL±SD: 13.4±0.66 cm; females N=264, mean TL±SD: 13.9±0.85 cm). The overall sex ratio (m/f=0.404) deviated significantly from the hypothetical distribution of 1:1 (χ²=15.928, df=1, P<0.05), indicating that females were predominant over males. Seasonal oscillations in mean monthly gonadosomatic indices and gonad mass values, observed separately by sex or overall, showed the same trend (Fig. 3).

There was a slight increase in both values in October (GSI=0.568, Wg=0.11), after which both values increased sharply and stayed at high levels until February, when they reached their peaks (GSI=5.09, Wg=0.976 g) (Fig. 3). Afterwards, there was a sharp decline in the GSI and Wg.

The length–mass analysis demonstrated positive allometric growth (W=0.0029TL^3.3573, r²=0.8987), as the value of parameter b differed significantly from 3 [one-sample t-test (P<0.05)].

Fat content

The monthly fat content values in sardine gonads, muscles and liver measured separately for each sex are shown by Figure 4. Overall, the highest levels of fat content were recorded in the gonads, and specifically within male gonads from July (Fig. 4B; 17.6%). The oscillations in gonad fat content in males and females showed a similar trend, as they both reached high values during the warmer part of the year (Fig. 4; females, 4.11% in June and 3.89% in September; males: 9.72% in May and 12.61% in September), although gonad fat content was much higher in males than in females.

Slight oscillations in fat content values were observed in the muscles of both males and females. Furthermore, in both sexes the lowest level of fat in muscles was obtained at the end of the colder part of the year (Fig. 4; females, 1.35% in February; males, 1.02% in March), whereas the maximum values occurred at different times for each sex. In fact, the highest accumulation of fat in the muscles of females was recorded in November, and in males the highest accumulation was recorded in July (Fig. 4).

The fat content in the liver in the two sexes varied in a similar manner, and once again was higher in males than females. Both males and females showed higher storage of fat in the liver from March to August (females) or September (males), and maximum values for both sexes were recorded in April (Fig. 4).

Data were collected according to different life phases [spawning (S), feeding (F) and resting (R)]. Based on the high values of GSI and gonad mass (Fig. 2), spawning occurred from November to February, which was considered as the spawning phase. The resting phase occurred from March to June, coinciding with the lowest GSI and gonad mass values. Finally, the feeding phase occurred from July to October, as primary production is high during this part of the year (Kovač et al. 2018Kovač Ž., Platt T., Ninčević Gladan Ž., et al. 2018. A 55-Year Time Series Station for Primary Production in the Adriatic Sea: Data Correction, Extraction of Photosynthesis Parameters and Regime Shifts. Remote Sens. 10: 1460.) and sardines have to recover energy in order to continue reproducing.

By means of principal component analysis, 84.8% of the variability of four variables (fat content in gonads, liver and muscle; GSI) was explained by the first two components (Fig. 5). The most important components, PC1 and PC2, accounted for 65.0% and 19.8% of the total variation, respectively (Table 1). For PC1, the fat content within the gonads and gonadosomatic index gave the highest percentages, whereas for PC2, the highest percentages were obtained for the fat content within the gonads and liver (Table 1). In fact, the observed data were clustered into three groups that were more or less in line with the determined phases. The specimens in the spawning phase were thus grouped together with a high loading for PC1 and a low loading values for PC2 (highest GSI and lowest fat content in gonads). Although the resting and feeding phases were not as clearly separated as the spawning phase, they were differentiated on the basis of the fat content in the gonads and liver and the gonadosomatic index: lower values of all three factors defined the feeding phase, whereas a higher fat content in the liver and low fat content in the gonads defined the resting phase (Fig. 5).

The results of one-way ANOSIM showed that the four measured parameters (fat content in gonads, liver, muscle; GSI) did not significantly differ in relation to sex, whereas significant differences were observed according to the defined phases (spawning, resting and feeding) (Table 2).

DISCUSSIONTop

Biological parameters

The overall biological data obtained in this study for the sardines collected by commercial purse seiners in the eastern Adriatic Sea throughout a one-year period (March 2017-February 2018) were consistent with the review by Morello and Arneri (2009)Morello B.E., Arneri E. 2009. Anchovy and Sardine in the Adriatic Sea—An Ecological Review. Oceanogr. Mar. Biol. Ann. Rev. 47: 209-256. for this Adriatic sardine stock. The length distribution of the sardines investigated was within the ranges obtained for this type of fishing gear operating along the eastern side of the Adriatic, specifically the Croatian fishing grounds (Sinovčić et al. 2008Sinovčić G., Čikeš Keč V., Zorica B. 2008. Population structure, size at maturity and condition of sardine, Sardina pilchardus (Walb., 1792), in the nursery ground of the eastern Adriatic Sea (Krka River estuary, Croatia). Estuar. Coast. Shelf. Sci. 76: 739-744., Kraljević et al. 2014Kraljević V., Čikeš Keč V., Zorica B. 2014. Analiza ulova ostvarenih plivaricom srdelarom u Jadranu. Croatian J. Fish. 72: 142-149.), and their reproductive traits were consistent with those of previous studies (Brosset et al. 2015Brosset P., Fromentin J.M., Ménard F., et al. 2015. Measurement and analysis of small pelagic fish condition: a suitable method for rapid evaluation in the field. J. Exp. Mar. Biol. Ecol. 462: 90-97., Zorica et al. 2016Zorica B., Čikeš Keč V., Vidjak O., et al. 2016. Feeding habits and helminth parasites of sardine (S. pilchardus) and anchovy (E. encrasicolus) in the Adriatic Sea. Medit. Mar. Sci. 17: 216-229., 2017Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.). Females dominated the sardine population investigated in this study, in agreement with previous studies, with the exception of Mustać and Sinovčić (2010)Mustać B., Sinovčić G. 2010. Reproductive biology, length-weight relationship and condition of sardine Sardina pilchardus (Walb., 1792) in the eastern Adriatic Sea. Period. Biol. 112: 133-138., who reported that the sex ratio of the Adriatic sardine favoured males. This difference might be a matter of sampling strategies that covered limited areas of the Adriatic, as only two small areas of the middle Adriatic were sampled in the study of Mustać and Sinovčić (2010)Mustać B., Sinovčić G. 2010. Reproductive biology, length-weight relationship and condition of sardine Sardina pilchardus (Walb., 1792) in the eastern Adriatic Sea. Period. Biol. 112: 133-138.. The allometric coefficient (b=3.3573) obtained fits within the wide range (2.757<b<3.657) given for this population (Morello and Arneri 2009Morello B.E., Arneri E. 2009. Anchovy and Sardine in the Adriatic Sea—An Ecological Review. Oceanogr. Mar. Biol. Ann. Rev. 47: 209-256., Brosset et al. 2015Brosset P., Fromentin J.M., Ménard F., et al. 2015. Measurement and analysis of small pelagic fish condition: a suitable method for rapid evaluation in the field. J. Exp. Mar. Biol. Ecol. 462: 90-97.).

Fat content

The fluctuations in fat content in all the sardine tissues analysed in the present study (gonads, liver and muscles) were already reported for the same species by Garrido et al. (2007)Garrido S., Marçalo A., Zwolinski J., et al. 2007. Laboratory investigations on the effect of prey size and concentration on the feeding behaviour of Sardina pilchardus. Mar. Ecol. Prog. Ser. 330: 189-199., Nunes et al. (2015)Nunes B.S., Travasso R., Gonçalves F., et al. 2015. Biochemical and physiological modifications in tissues of Sardina pilchardus: spatial and temporal patterns as a baseline for biomonitoring studies. Front. Environ, Sci. 3. and Brosset et al. (2016b)Brosset P., Le Bourg B., Costalago D., et al. 2016b. Linking small pelagic dietary shifts with ecosystem changes in the Gulf of Lions. Mar. Ecol. Prog. Ser. 554: 157-171.. Due to the measured values of fat content, it seems that the sardine deposits its fat reserves mainly in the gonads, which presumably makes them the organs that are most affected by all physiological processes during the lifetime of an indeterminate batch spawner such as the sardine (Fig. 5).

It was clear that the majority of the fat/energy stores were accumulated in the feeding period (July–October), corroborating the food habits of sardines originating from the same area (Zorica et al. 2017Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.) and elsewhere (Costalago et al. 2012Costalago D., Navarro J., Álvarez-Calleja I., et al. 2012. Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species. Mar. Ecol. Prog. Ser. 460: 169-181., Le Bourg et al. 2015Le Bourg B., Bănaru D., Saraux C., et al. 2015. Trophic niche overlap of sprat and commercial small pelagic teleosts in the Gulf of Lions (NW Mediterranean Sea). J. Sea Res. 103: 138-146., Brosset et al. 2016bBrosset P., Le Bourg B., Costalago D., et al. 2016b. Linking small pelagic dietary shifts with ecosystem changes in the Gulf of Lions. Mar. Ecol. Prog. Ser. 554: 157-171.). In fact, Zorica et al. (2017)Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900. found that in the warmer period of the year (June–September), here defined as the feeding phase, sardines consumed higher energy prey such as fish eggs, cladocerans and decapod larvae, resulting in the highest stomach fullness index values. The pattern of acquiring energy reserves before the onset of spawning has previously been reported not only for the sardine (Mustać and Sinovčić 2009Mustać B., Sinovčić G. 2009. Comparison of mesenteric and tissue fat content in relation to sexual cycle of the sardine, Sardina pilchardus (Walb., 1792), in the eastern Middle Adriatic fishery grounds (Croatia). J. Appl. Ichthyol. 25: 595-599., Marin et al. 2010Marin M., Polak T., Gašperlin L., et al. 2010. Variations in the fatty acid composition and nutritional value of Adriatic sardine (Sardina pilchardus Walb.) through the fishing season. Acta Agric. Slov. 96: 95-101., Bandarra et al. 2018Bandarra N.M., Marçalo A., Cordeiro A.R., et al. 2018. Sardine (Sardina pilchardus) lipid composition: Does it change after one year in captivity? Food Chem. 244: 408-413.) and other small pelagics (Hunter and Leong 1981Hunter J.R., Leong R. 1981. The spawning energetics of female northern anchovy, Engraulis mordax. Fish. Bull. 79: 215–229., Cubillos et al. 2001Cubillos L.A., Arcos D.F., Bucarey D.A., et al. 2001. Seasonal growth of small pelagic fish off Talcahuano, Chile (37°S, 73°W): a consequence of their reproductive strategy to seasonal upwelling? Aquat. Living Resour. 14: 115–124., Pacetti et al. 2013Pacetti D., Balzano M., Colella S., et al. 2013. Effect of Spawning on Furan Fatty Acid Profile of Edible Muscle and Organ Tissues from Sardine (Sardina pilchardus) and Anchovy (Engraulis encrasicolus). J. Agric. Food. Chem. 61: 3969-3977.), but also for many other fish (El Kebir et al. 2003El Kebir M.V.O., Barnathan G., Siau Y., et al. 2003. Fatty Acid Distribution in Muscle, Liver, and Gonads of Rays (Dasyatis marmorata, Rhinobatos cemiculus, and Rhinoptera marginata) from the East Tropical Atlantic Ocean. J. Agric. Food Chem. 51: 1942-1947., Lloret et al. 2008Lloret J., Demestre M., Sanchez-Pardo J. 2008. Lipid (energy) reserves of European hake (Merluccius merluccius) in the north-western Mediterranean. Vie Milieu 58: 75-85., Grande et al. 2016Grande M., Murua H., Zudaire I., et al. 2016. Energy allocation strategy of skipjack tuna Katsuwonus pelamis during their reproductive cycle. J. Fish. Biol. 89: 2434-2448.).

The outcomes of the present study also showed that the fat content in gonads, liver and muscle decreased with the increase in the GSI. The decline in fat content was consistent with that reported by previous studies on small pelagic fish species (Pecquerie et al. 2009Pecquerie L., Petitgas P., Kooijman S.A.L.M. 2009. Modeling fish growth and reproduction in the context of the Dynamic Energy Budget theory to predict environmental impact on anchovy spawning duration. J. Sea Res. 62: 93-105., Nunes et al. 2011Nunes C., Silva A., Soares E., et al. 2011. The use of hepatic and somatic indices and histological information to characterize the reproductive dynamics of Atlantic sardine Sardina pilchardus from the Portuguese coast. Mar. Coast. Fish. 3: 127-144., 2015Nunes B.S., Travasso R., Gonçalves F., et al. 2015. Biochemical and physiological modifications in tissues of Sardina pilchardus: spatial and temporal patterns as a baseline for biomonitoring studies. Front. Environ, Sci. 3., Pacetti et al. 2013Pacetti D., Balzano M., Colella S., et al. 2013. Effect of Spawning on Furan Fatty Acid Profile of Edible Muscle and Organ Tissues from Sardine (Sardina pilchardus) and Anchovy (Engraulis encrasicolus). J. Agric. Food. Chem. 61: 3969-3977.). However, this decline did not continue for long, which was somewhat surprising considering the fact that the sardine has a relatively long spawning season during which it has to produce many batches of eggs (Ganias et al. 2004Ganias K., Somarakis S., Machias A., et al. 2004. Pattern of oocyte development and batch fecundity in the Mediterranean sardine. Fish. Res. 67: 13-23., Somarakis et al. 2006Somarakis S., Ganias K., Siapatis A., et al. 2006. Spawning habitat and daily egg production of sardine (Sardina pilchardus) in the eastern Mediterranean. Fish. Oceanog. 15: 281-292.). This is most likely due to the fact that the sardines continued to feed throughout the entire spawning season (Zorica et al. 2016Zorica B., Čikeš Keč V., Vidjak O., et al. 2016. Feeding habits and helminth parasites of sardine (S. pilchardus) and anchovy (E. encrasicolus) in the Adriatic Sea. Medit. Mar. Sci. 17: 216-229., 2017Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.) in order to obtain and maintain the required, although low, level of energy reserves in all tissues.

As soon as spawning ended, the sardines started their recovery in a resting phase that coincided with the start of spring and lasted until the start of summer. In the Adriatic, this period coincides with the peak of primary production (Ninčević Gladan et al. 2010Ninčević Gladan Ž., Marasović I., Grbec B., et al. 2010. Inter-decadal Variability in Phytoplankton Community in the Middle Adriatic (Kaštela Bay) in Relation to the North Atlantic Oscillation. Estuar. Coast. Shelf Sci, 33: 376–383., Kovač et al. 2018Kovač Ž., Platt T., Ninčević Gladan Ž., et al. 2018. A 55-Year Time Series Station for Primary Production in the Adriatic Sea: Data Correction, Extraction of Photosynthesis Parameters and Regime Shifts. Remote Sens. 10: 1460.), which leads to the increased feeding intensity of sardines (Zorica et al. 2016Zorica B., Čikeš Keč V., Vidjak O., et al. 2016. Feeding habits and helminth parasites of sardine (S. pilchardus) and anchovy (E. encrasicolus) in the Adriatic Sea. Medit. Mar. Sci. 17: 216-229., 2017Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.). The revitalization of fat stocks in all tissues was evident, although the most pronounced accumulation was noted in the liver, whose fat level was the lowest by the end of the spawning phase. It is known that the temporary energy storage within the liver is crucial for fish growth, reproduction and migration (Tocher 2003Tocher D.R. 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11: 107-184., Nunes et al. 2011Nunes C., Silva A., Soares E., et al. 2011. The use of hepatic and somatic indices and histological information to characterize the reproductive dynamics of Atlantic sardine Sardina pilchardus from the Portuguese coast. Mar. Coast. Fish. 3: 127-144.). Furthermore, the quality of energy stored in the liver most probably affects the development of future generations (health and proper development of eggs and larvae) (McBride et al. 2013McBride R.S., Somarakis S., Fitzhugh G.R., et al. 2013. Energy acquisition and allocation to egg production in relation to fish reproductive strategies. Fish and Fish. 16: 23-57.). Hence, the collection, storage and utilization of fat/energy stored in the liver might be one of the triggers that leads to the oscillation in spawning stock biomass of this and other small pelagic fish species.

In summary, the present results demonstrated that the fat reserves stored in gonads and liver were strongly affected by the spawning season. In general, gonads are widely used to determine the reproductive pattern, but, as shown within this study, the fat content in the liver might be of additional use. The fat content in the liver, other than being used as an indicator of spawning, may be an indicator of the recruitment success through the tracking of its temporal variations.

ACKNOWLEDGEMENTSTop

This work was part of the two projects: the Data Collection Framework (DCF), funded by the Ministry of Agriculture of the Republic of Croatia, and Exploration of ecologically sensitive areas with special emphasis on growth, development and protection of commercially important maritime organisms (ESAmar), funded by the Croatian Science Foundation (HRZZ). The referees’ comments and suggestions are greatly appreciated.

REFERENCESTop

Bandarra N.M., Marçalo A., Cordeiro A.R., et al. 2018. Sardine (Sardina pilchardus) lipid composition: Does it change after one year in captivity? Food Chem. 244: 408-413.
https://doi.org/10.1016/j.foodchem.2017.09.147

Blaxter J., Hunter J.R. 1982. The biology of the clupeoid Fishes. Adv. Mar. Biol. 20: 1-123.
https://doi.org/10.1016/S0065-2881(08)60140-6

Bouhali F.Z., Lechekhab S., Ladaimia S., et al. 2015. Reproduction and maturation of the gonads of Sardina pilchardus from the Gulf of Annaba (North-Eastern Algeria). Cybium 39: 143-153.

Brosset P., Fromentin J.M., Ménard F., et al. 2015. Measurement and analysis of small pelagic fish condition: a suitable method for rapid evaluation in the field. J. Exp. Mar. Biol. Ecol. 462: 90-97.
https://doi.org/10.1016/j.jembe.2014.10.016

Brosset P., Lloret J., Muñoz M., et al. 2016a. Body reserves mediate trade-offs between life-history traits: new insights from small pelagic fish reproduction. R. Soc. Open Sci. 3: 160-202.
https://doi.org/10.1098/rsos.160202

Brosset P., Le Bourg B., Costalago D., et al. 2016b. Linking small pelagic dietary shifts with ecosystem changes in the Gulf of Lions. Mar. Ecol. Prog. Ser. 554: 157-171.
https://doi.org/10.3354/meps11796

Cingolani N., Gianetti G., Arneri E. 1996. Anchovy fisheries in the Adriatic Sea. Sci. Mar. 60: 269-277.

Clarke K.R. 1993. Non-parametric multivariate analyses of changes in community structure. Austral. J. Ecol. 18: 117-143.
https://doi.org/10.1111/j.1442-9993.1993.tb00438.x

Clarke K.R., Warwick R.M. 1994. Changes in marine communities: an approach to statistical analysis and interpretation. Natural Environment Research Council. UK, 144 pp.

Costalago D., Palomera I. 2014. Feeding of European pilchard (Sardina pilchardus) in the northwestern Mediterranean: from late larvae to adults. Sci. Mar. 78: 41-54.
https://doi.org/10.3989/scimar.03898.06D

Costalago D., Navarro J., Álvarez-Calleja I., et al. 2012. Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species. Mar. Ecol. Prog. Ser. 460: 169-181.
https://doi.org/10.3354/meps09751

Cubillos L.A., Arcos D.F., Bucarey D.A., et al. 2001. Seasonal growth of small pelagic fish off Talcahuano, Chile (37°S, 73°W): a consequence of their reproductive strategy to seasonal upwelling? Aquat. Living Resour. 14: 115–124.
https://doi.org/10.1016/S0990-7440(01)01112-3

Cury P., Bakun A., Crawford R.J.M., et al. 2000. Small pelagics in upwelling systems: patterns of interaction and structural changes in “wasp-waist” ecosystems. ICES J. Mar. Sci. 57: 603-618.
https://doi.org/10.1006/jmsc.2000.0712

El Kebir M.V.O., Barnathan G., Siau Y., et al. 2003. Fatty Acid Distribution in Muscle, Liver, and Gonads of Rays (Dasyatis marmorata, Rhinobatos cemiculus, and Rhinoptera marginata) from the East Tropical Atlantic Ocean. J. Agric. Food Chem. 51: 1942-1947.
https://doi.org/10.1021/jf0204809

FAO. 2017. Report of 20th session of the Scientific Advisory Committee on Fisheries. Fish. Aquac. Rep. 1245: 226.

Fréon P., Cury P., Shannon L., et al. 2005. Sustainable exploitation of small pelagic fish stocks challenged by environmental and ecosystem changes: a review. Bull. Mar. Sci. 76: 385-462.

Ganias K., Somarakis S., Koutsikopoulos C., et al. 2003. Ovarian atresia in the Mediterranean sardine, Sardina pilchardus. J. Mar. Biol. Assoc. UK 83: 1327-1332.
https://doi.org/10.1017/S0025315403008774

Ganias K., Somarakis S., Machias A., et al. 2004. Pattern of oocyte development and batch fecundity in the Mediterranean sardine. Fish. Res. 67: 13-23.
https://doi.org/10.1016/j.fishres.2003.08.008

Ganias K., Somarakis S., Koutsikopoulos C., et al. 2007. Factors affecting the spawning period of sardine in two highly oligotrophic Seas. Mar. Biol. 151: 1559-1569.
https://doi.org/10.1007/s00227-006-0601-0

Garrido S., Marçalo A., Zwolinski J., et al. 2007. Laboratory investigations on the effect of prey size and concentration on the feeding behaviour of Sardina pilchardus. Mar. Ecol. Prog. Ser. 330: 189-199.
https://doi.org/10.3354/meps330189

Grande M., Murua H., Zudaire I., et al. 2016. Energy allocation strategy of skipjack tuna Katsuwonus pelamis during their reproductive cycle. J. Fish. Biol. 89: 2434-2448.
https://doi.org/10.1111/jfb.13125

Hunter J.R., Leong R. 1981. The spawning energetics of female northern anchovy, Engraulis mordax. Fish. Bull. 79: 215–229.

Kovač Ž., Platt T., Ninčević Gladan Ž., et al. 2018. A 55-Year Time Series Station for Primary Production in the Adriatic Sea: Data Correction, Extraction of Photosynthesis Parameters and Regime Shifts. Remote Sens. 10: 1460.
https://doi.org/10.3390/rs10091460

Kraljević V., Čikeš Keč V., Zorica B. 2014. Analiza ulova ostvarenih plivaricom srdelarom u Jadranu. Croatian J. Fish. 72: 142-149.
http://dx.doi.org/10.14798/72.4.764

Le Bourg B., Bănaru D., Saraux C., et al. 2015. Trophic niche overlap of sprat and commercial small pelagic teleosts in the Gulf of Lions (NW Mediterranean Sea). J. Sea Res. 103: 138-146.
https://doi.org/10.1016/j.seares.2015.06.011

Lloret J., Demestre M., Sanchez-Pardo J. 2008. Lipid (energy) reserves of European hake (Merluccius merluccius) in the north-western Mediterranean. Vie Milieu 58: 75-85.

Marin M., Polak T., Gašperlin L., et al. 2010. Variations in the fatty acid composition and nutritional value of Adriatic sardine (Sardina pilchardus Walb.) through the fishing season. Acta Agric. Slov. 96: 95-101.

McBride R.S., Somarakis S., Fitzhugh G.R., et al. 2013. Energy acquisition and allocation to egg production in relation to fish reproductive strategies. Fish Fish. 16: 23-57.
https://doi.org/10.1111/faf.12043

Morello B.E., Arneri E. 2009. Anchovy and Sardine in the Adriatic Sea—An Ecological Review. Oceanogr. Mar. Biol. Ann. Rev. 47: 209-256.

Mustać B., Sinovčić G. 2009. Comparison of mesenteric and tissue fat content in relation to sexual cycle of the sardine, Sardina pilchardus (Walb., 1792), in the eastern Middle Adriatic fishery grounds (Croatia). J. Appl. Ichthyol. 25: 595-599.
https://doi.org/10.1111/j.1439-0426.2009.01285.x

Mustać B., Sinovčić G. 2010. Reproductive biology, length-weight relationship and condition of sardine Sardina pilchardus (Walb., 1792) in the eastern Adriatic Sea. Period. Biol. 112: 133-138.

Ninčević Gladan Ž., Marasović I., Grbec B., et al. 2010. Inter-decadal Variability in Phytoplankton Community in the Middle Adriatic (Kaštela Bay) in Relation to the North Atlantic Oscillation. Estuar. Coast. Shelf Sci. 33: 376-383.
https://doi.org/10.1007/s12237-009-9223-3

Nunes C., Silva A., Soares E., et al. 2011. The use of hepatic and somatic indices and histological information to characterize the reproductive dynamics of Atlantic sardine Sardina pilchardus from the Portuguese coast. Mar. Coast. Fish. 3: 127-144.
https://doi.org/10.1080/19425120.2011.556911

Nunes B.S., Travasso R., Gonçalves F., et al. 2015. Biochemical and physiological modifications in tissues of Sardina pilchardus: spatial and temporal patterns as a baseline for biomonitoring studies. Front. Environ. Sci. 3: 7.
https://doi.org/10.3389/fenvs.2015.00007

Pacetti D., Balzano M., Colella S., et al. 2013. Effect of Spawning on Furan Fatty Acid Profile of Edible Muscle and Organ Tissues from Sardine (Sardina pilchardus) and Anchovy (Engraulis encrasicolus). J. Agric. Food. Chem. 61: 3969-3977.
https://doi.org/10.1021/jf400555u

Palomera I., Olivar M.P., Salat J., et al. 2007. Small pelagic fish in the NW Mediterranean Sea: An ecological review. Progr. Oceanog. 74: 377-396.
https://doi.org/10.1016/j.pocean.2007.04.012

Pecquerie L., Petitgas P., Kooijman S.A.L.M. 2009. Modeling fish growth and reproduction in the context of the Dynamic Energy Budget theory to predict environmental impact on anchovy spawning duration. J. Sea Res. 62: 93-105.
https://doi.org/10.1016/j.seares.2009.06.002

Rainuzzo J.R., Reitan K.I., Olsen Y. 1997. The significance of lipids at early stages of marine fish: a review. Aquaculture 155: 103-115.
https://doi.org/10.1016/S0044-8486(97)00121-X

Sargent J.R., Henderson R.J., Tocher D.R. 1989. The lipids. In: Halver J. (ed.), Fish Nutrition, 2nd ed. Academic Press, London, pp. 153-217.

Sargent J.R., Tocher D.R., Bell J.G. 2002. The lipids. In: Halver J.E., Hardy R.W. (eds), Fish Nutrition, 3rd edition. Academic Press, San Diego, pp. 181-257.

Shulman G.E., Love R.M. 1999. The Biochemical Ecology of Marine Fishes. Adv. Mar. Biol. 36. Academic Press, San Diego, 350 pp.

Sinovčić G., Čikeš Keč V., Zorica B. 2008. Population structure, size at maturity and condition of sardine, Sardina pilchardus (Walb., 1792), in the nursery ground of the eastern Adriatic Sea (Krka River estuary, Croatia). Estuar. Coast. Shelf. Sci. 76: 739-744.
https://doi.org/10.1016/j.ecss.2007.07.037

Somarakis S., Ganias K., Siapatis A., et al. 2006. Spawning habitat and daily egg production of sardine (Sardina pilchardus) in the eastern Mediterranean. Fish. Oceanog. 15: 281-292.
https://doi.org/10.1111/j.1365-2419.2005.00387.x

Tocher D.R. 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11: 107-184.
https://doi.org/10.1080/713610925

Van Handel E. 1985. Rapid determination of total lipids in mosquitoes. J. Am. Mosq. Control. Assoc. 1: 302-304.

Zorica B., Čikeš Keč V., Vidjak O., et al. 2016. Feeding habits and helminth parasites of sardine (S. pilchardus) and anchovy (E. encrasicolus) in the Adriatic Sea. Medit. Mar. Sci. 17: 216-229.
https://doi.org/10.12681/mms.1467

Zorica B., Čikeš Keč V., Vidjak O., et al. 2017. Seasonal pattern of population dynamic, spawning activities and diet composition of sardine (Sardina pilchardus W.) in the eastern Adriatic Sea. Turk. J. Zool. 41: 892-900.
https://doi.org/10.3906/zoo-1609-27