prevent_escape_chapter_5.2

5.2. ESCAPE THROUGH SPAWNING BY SEABREAM (Sparus aurata)

In Greece, the largest EU producer of seabream, both the number of fish farms and their production capacity increase spectacularly over the past decade, accompanied by a substantial decrease in the price of seabream. This industrial development has led to structural and functional changes in rearing processes. Farming durations increased from just 12 to 18 months before 1995 to durations of up to 40 months after 1999 (Dimitriou et al. 2007). The increased farming duration of gilthead seabream (Sparus aurata), a protandrous hermaphrodite, has resulted in the production of fish large enough to reach the stage of sex inversion and female sexual maturation, normally observed at an age of 2 – 3 years in the wild (Mylonas et al. 2011). Changes in rearing processes have resulted in the presence of large gilthead seabream individuals (larger than 500 g) in cages during the normal reproductive period of their wild counterparts (November – March: Mylonas et al. 2011). There is evidence that sex inversion and the production of both male and female gametes occur within cages under the present industrial rearing pattern (Dimitriou et al. 2007). However, in the Mediterranean region, information about spawning by fish kept in sea cages is sparse.

authors: Stelios Somarakis1, Christina Saapoglou1,2, Costas Tsigenopoulos1 & Michalis Pavlidis2

1 Hellenic Centre for Marine Research, Crete, Greece, 2 Department of Biology, University of Crete, Greece

Cite this article as: Somarakis S, Saapoglou C, Tsigenopoulos C, Pavlidis M (2013) Escape through spawning by seabream (Sparus aurata). In: PREVENT ESCAPE Project Compendium. Chapter 5.2. Commission of the European Communities, 7th Research Framework Program. www.preventescape.eu

INTRODUCTION

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ISBN: 978-82-14-05565-8

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The objectives of our work on gilthead seabream were to:

investigate whether large fish (i.e. fish beyond the size of sex reversal and onset of female maturation) produce eggs in sea cages, using the Greek seabream industry as a case study;

evaluate the quantity and quality of eggs released in gilthead seabream broodstock held in sea cages; and

assess whether eggs produced within sea cages disperse and survive in surrounding coastal waters.

Gilthead seabream have a complex reproductive biology that includes sex reversal, indeterminate annual fecundity and an extended spawning period. In general, sparid fishes are multiple spawners with asynchronous oocyte development (Mylonas et al. 2011).

To obtain and analyse seabream gonads and estimate daily and annual egg production, we sampled fish each month from five farms in Greece (Table 5.2.1). A total of 1262 large gilthead seabream were sampled from November 2009 to April 2010. Farms 1 – 3 were located in the Ionian Sea, whereas farms 4 – 5 were located in the Aegean Sea. Mean fish weight ranged from 0.820 to 1.75 kg and the percentage of females varied among farms from 38 to 81% (Table 5.2.1). We removed and weighed gonads and took subsamples for subsequent histological analysis and fecundity measurements.

The monthly development of the mean gonadosomatic index (gonad weight/fish weight or GSI) is illustrated in (Figure 5.2.1) for female fish. With the exception of farm 1, GSIs remained high up until February. Thereafter, gonadal regression occurred from February to April. In farm 1, the lowering of temperatures to below 13oC in January likely resulted in earlier gonadal regression. Changes in histological maturity stages of both males and females (e.g. males in Figure 5.2.2) matched closely with monthly variations in GSI.

Histological scoring of ovaries included the stage of development of the most advanced oocytes, the presence and histological characteristics of postovulatory follicles (POFs) and the incidence/prevalence of atresia (Hunter and Macewitz, 1985).

The most prominent characteristic of females with yolked oocytes (spawning capable fish) was a relatively high prevalence of atresia (absorption) of yolked oocytes. Alpha-stage atresia involves the resorbing of yolk and chorion (Figure 5.2.3a) which we quantified in all spawning

EVALUATION OF SEXUAL MATURATION AND EGG RELEASE IN SEABREAM REARED IN SEA CAGES

OBJECTIVES:

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capable females by measuring the percentage of oocytes affected. We measured batch fecundity (number of eggs produced in a spawning event) by counting the number of hydrated oocytes (Figure 5.2.3b) in pre-weighed subsamples from the ovary (Hunter et al. 1985).

After ovulation and egg release, post-ovulatory follicles (POFs) appear in the ovary (Figure 5.2.3c) which soon start to degenerate (Hunter and Macewitz 1985). POFs are an unambiguous marker of recent spawning and can readily be identified in the ovaries as long as their lumen is still visible (Hunter and Macewitz 1985). The POFs we observed in seabream ovaries were in a moderate degree of degeneration and always had a lumen. Given the temperature regimes in the farms sampled and information from POF degeneration experiments (Alday et al. 2008), we considered them to represent a single night’s (previous night’s) spawning.

We collected females with hydrated oocytes and/or POFs, (i.e. actively spawning females) from December to February (Figure 5.2.4), depending on the farm, and used them to define the approximate duration of the spawning period (SP), i.e. the number of months with incidence of actively spawning females (Table 5.2.2). We used the average fraction of females with hydrated ovaries and ovaries with POFs to estimate S, i.e. the fraction of females spawning each day (Table 5.2.2). Farm 4 was excluded from the analysis due to small sample sizes. We then derived mean relative batch fecundities in the four farms (Table 5.2.2) as the marginal means from the general linear model:

log(RF) = Intercept + FARM + MONTH + ATRESIA, n = 37, p < 0.001, adj. r2: 0.846,

where RF: relative batch fecundity (eggs kg-1) and ATRESIA: prevalence of alpha stage atresia (% of oocytes affected).

Estimates of daily female-specific fecundity (number of eggs produced daily per kg of females in the cage, DFSF = S ! RF) and annual female-specific fecundity (number of eggs produced annually per kg of females, AFSF: DFSF ! SP; Table 5.2.2), decreased exponentially with sex ratio, R (Figure 5.2.5).

Figure 5.2.1. Monthly development of the gonadosomatic index (GSI) of female seabreams in the five farms.


Figure 5.2.2. Microphotographs of seabream functional testes. (A) Developing. (B) and (C) Spawning. (D) Regressing. (E) and (F) Regenerating. Note the ovarian tissue in (E). Green arrows: spermatogonia. Red arrows: spermatozoa. SC: spermatocytes. Sp: spermatids. Scale bar: 0.1 mm

in (A) and (B); 0.2 mm in C-F.

Figure 5.2.3. Key histological features of spawning capable female seabreams. (A) Ovary with yolked oocytes in alpha atretic stage (a). (B) Ovary with hydrated oocytes (H). (C). Post-ovulatory follicle. Scale bar: 0.2 mm in A-B; 0.1 mm in C.

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Figure 5.2.4. Upper panel: Fraction of females with yolked oocytes that had hydrated oocytes. Lower panel: Fraction of females with yolked oocytes that had postovulatory follicles.

farm R S RF DFSF SP AFSF

1 0.81 0.145 4000 579 30 17368

2 0.61 0.096 14256 1362 60 81740

3 0.38 0.186 16982 3158 90 284196

5 0.72 0.103 8110 833 60 49963

Number of fish sampled Sex ratio Average fish weight (kg)

farm 1 300 0.81 1.182

farm 2 300 0.61 1.435

farm 3 300 0.38 0.820

farm 4 120 0.63 1.410

farm 5 242 0.72 1.752

Table 5.2.2. Estimates of mean spawning fraction, S; mean relative batch fecundity, RF; sex ratio, R; daily female-specific fecundity (number of eggs produced daily per kg of female in the population, DFSF = S xRF, DFSF; duration of the spawning period in days approximated by the number of months with incidence of hydrated females and/or postovulatory follicles, SP, annual female-specific fecundity, (number of eggs produced annually per kg of female in the population, AFSF: DFSF x SP), AFSF.

Table 5.2.1. Summarized information on seabream sampling. Sex ratio: number of females / total number of fish. Farms 1-3 were located in the Ionian Sea; farms 4-5, in the Aegean Sea.


To evaluate the quantity and quality of eggs released in gilthead seabream held in sea cages, we designed, manufactured and installed an egg collector in a seabream cage.

We ran a study in a private fish farm (Karpasia) located in the island of Evia, Greece. It consisted of four phases: (a) design of an egg collector, (b) manufacturing of the collector, (c) installation on the net-pen sea cage, and (d) egg release monitoring. The egg collector was constructed in 2010 (Figure 5.2.6) and we performed a test experiment in January – February 2011.

To ensure the best in situ managerial conditions for spawning, we ran the experiment with an ideal sex ratio (1:1), at a low stocking density and a number of fish similar to that used in commercial seabream hatcheries. Sixty fish at a sex ratio of 1:1 (30 females and 30 males of a mean body weight of 990 and 577 g, respectively) were placed in a net pen cage (diameter 40 m, depth 10 m) in the middle of January 2011. Fish were left undisturbed for two weeks after which the egg collector was placed in the cage (Figure 5.2.6d and Figure 5.2.7a). We monitored eggs (Figure 5.2.7b and c) on a daily basis for two consecutive weeks (11 – 25 February 2011). There was a small daily release of eggs (1250 – 3250 eggs per day) apart from two consecutive days where a larger amount of eggs were collected (14/02/11: 30 375 eggs; 15/02/11: 13 250 eggs). We also monitored eggs for one-week in the middle of March, but no eggs were collected.

Following collection, we placed eggs in a 10 L bucket and transported them to land for quality evaluation. We then checked the quality and viability of floating eggs using a stereoscope. The measures of egg quality we used were: egg shape, number of oil droplets and symmetrical embryonic divisions. Egg viability was less than 5%.

Figure 5.2.5. Relationships between daily specific fecundity (DFSF: number of eggs produced daily per kg of female in the cage); annual specific fecundity (AFSF:

number of eggs produced annually per kg of female in the cage) and sex ratio.

MONITORING EGG RELEASES IN SEABREAM SEA CAGES

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Figure 5.2.6. (A) Phase 1. Construction of the upper part of the egg collector. Material used: non-toxic polyethylene; height of the construction: 1 m. (B) Completion of the construction. (C) Phase 2: construction of the egg collector–net complex. Size of the net 12 mm; depth: 4 m. (D) Installation of the egg collector on the sea cage.


Figure 5.2.7. (A) Experimental net-pen cage provided with the manufactured egg

collector. (B) Procedure for the collection of eggs – phase 1. (C) Procedure for the

collection of eggs – phase 2.

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To determine if eggs produced within sea cages disperse and survive in surrounding coastal waters, we carried out a fine-scale study of the distribution and abundance of sparid embryos around sea cages using vertical plankton tows. Sampling took place in the vicinity of a farm known to have large fish that could potentially spawn.

We carried out the site intensive ichthyoplankton survey for three consecutive days in 20 – 22 January 2011. Sampling took place onboard the 26 m Research vessel “PHILIA”. On each day, vertical hauls of a standard WP2 sampler (200 microns mesh-sized net) were done at 19 fixed stations to collect fish eggs and larvae (Figure 5.2.8). The seabream farm in the area contained a cage with large (>1.5 kg) seabream and another experimental cage with large fish that was used for the egg collector experiment (see above).

We sorted plankton samples onboard immediately after capture and all fish eggs potentially belonging to the family Sparidae (Divanach 1985) were staged and put individually in 96% alcohol for subsequent identification to the species level using molecular techniques.

We then subjected eggs from the plankton survey as well as 40 eggs collected with the experimental egg collector (see above) to molecular analysis. We extracted total genomic DNA and amplified a region of cytochrome oxidase subunit I (COI; ~670 bp) mitochondrial gene via PCR using universal primers FishF2 and FishR1 (Ward et al. 2005). PCR products were purified and we performed a single stranded sequencing with primer FishF2. We also used additional sequences for Sparus aurata and Diplodus vulgaris to align the sequences obtained in the present study and to assist molecular species identification.

We assigned staged sparid eggs to three age-classes: age-0 eggs (<1 day old), age-1 eggs (1-2 days old), and age-2 eggs (>2 days) based on a temperature–stage–age key devised from information in Divanach (1985), Polo et al. (1991) and Koumoundouros (1993). Finally, we estimated the weighted mean abundance of age-0, age-1 and age-2 eggs for each sampling day. We used station weighting factors that were proportional to their representative area. The difference in weighted mean abundance of age-1 eggs in sampling day 2 from age-0 eggs in sampling day 1 (the fraction age-1/age-0) was an estimate of daily eggs survival.

Based on the molecular identifications, we identified all sparid eggs to species level (Table 5.2.3). The great majority belonged to Sparus aurata and Diplodus vulgaris. The later was a wild species, so we considered it useful to compare the distribution, abundance and mortality of its eggs with those of S. aurata. Most gilthead seabream eggs were collected on day 1, whereas on day 2 and 3 their abundance was very low. Most D. vulgaris eggs were also collected on day 1. Furthermore, with the exception of two eggs that belonged to D. vulgaris, the remainder (95%) eggs collected with the egg collector (see above) were Sparus aurata, implying that spawning takes place inside the cages.

ASSESSMENT OF EGG DISPERSAL AND MORTALITY AROUND SEABREAM SEA CAGES


The fraction (age-1 eggs on day 2)–on–(age-0 eggs on day 1) (Table 5.2.4), expressed as a percentage, was 10% for S. aurata and 90% for D. vulgaris. Assuming that most eggs of both species had been spawned inside or very near to the cages (see below) and that no significant numbers of age-1 D. vulgaris eggs had been advected from outside the surveyed area, the survival of S. aurata eggs, most likely produced inside the cages, was very low compared to the wild population of D. vulgaris.

Plots of mean standardized egg abundance against distance from the cages (Figure 5.2.9) clearly showed that the abundance of age-0 eggs for both S. aurata and D. vulgaris was high in the close vicinity of the farm whereas age-1 eggs were more abundant away from the cages. These findings suggest that the fish farm was a site of increased egg production of both (farmed) gilthead seabream and other wild populations of the same family that were attracted close to the cages. The eggs were subsequently dispersed in the adjacent coastal habitat by the prevailing currents in the area.

Speciesday 1 day 2 day 3

N % eggs m2 N eggs m2 N % eggs m2

Sparus aurata 80 58.00 21.05 7 21.05 1.84 8 26.32 2.11

Diplodus vulgaris 86 58.00 22.63 38 73.68 10.00 24 31.58 6.32

Diplodus sargus 1 5.26 0.21 4 15.80 1.05 1 5.26 0.21

Boops boop 4 21.05 1.05

Table 5.2.3. Assignment of sparid eggs to species based on results of the molecular analyses. N = number of individuals collected in each day; % = per cent frequency

of occurrence; eggs m2= average abundance in the 19 stations.

Sampling day Age class Sparus aurata Diplodus vulgaris

1

age-0 3.22 6.92

age-1 1.86 2.99

age-2 1.94 NA

2

age-0 0.17 1.33

age-1 0.31 6.21

age-2 NA NA

3

age-0 0.99 0.39

age-1 NA 1.70

age-2 NA NA

Table 5.2.4. Weighted mean abundance (eggs m-2) of eggs per age class and sampling day. NA: no eggs were caught in this class. Note the difference

between age-1 eggs on day 2 and age-0 eggs on day 1.

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Figure 5.2.8. Map of the ichthyoplankton study area. Triangles indicate sampling stations. The location of the seabream farm is also indicated (red arrow).

Figure 5.2.9. Plots of mean standardized egg abundance (eggs m2) against distance from the farm (m). The abundance of eggs in each day was standardized by dividing the individual station abundances with the maximum abundance recorded during that day. Subsequently, for each station, we averaged the abundance of eggs for the three days of sampling.


The analysis of seabream gonads provides the first data showing that female seabream reach final maturation, ovulation and spawning in sea cages during the normal spawning period of the species. The fecundity estimates presented in Table 5.2.2 indicate that actual egg production is very low compared to commercial broodstock (Mylonas et al. 2011). Egg production seems to be down-regulated by atresia and decreases with sex ratio (i.e. with fish size in the cage).

The molecular identifications showed that the eggs caught inside the cage by the seabream egg collector were 95% Sparus aurata, which confirms the findings of the reproductive study. Egg collections show that eggs are produced inside the cages. Therefore, the production of S. aurata eggs caught in the plankton survey was most likely due to the farmed fish inside the cages rather than wild fish or escapees, although this possibility cannot be ruled out.

Day-to-day variability in egg production during the plankton survey was very high in Sparus aurata, which reflected our findings from the egg-collector experiment. This was also the case for Diplodus vulgaris which was attracted to and spawned in the close vicinity of the cages. Egg production was particularly high during the first day of the ichthyoplankton sampling and was much lower in the next two days, especially in S. aurata. As the owner of the fish farm informed us later, the fish in the cages were not fed at all for two days prior to the first day of plankton sampling, which may explain the high egg production recorded on day 1.

S. aurata and D. vulgaris eggs showed similar patterns of distribution and dispersal around the sea cages, with most eggs being recorded next to the cages and thereafter dispersing in the surrounding coastal waters. However, the estimate of daily egg survival calculated for S. aurata was very low (10%) compared to D. vulgaris (90%). Although there is a high degree of uncertainty in field estimates of egg mortality, the large difference in mortality estimates for S. aurata and D. vulgaris might imply that the eggs originating from farmed seabream can be of inferior quality. This is further supported by the low survival rates recorded in the egg collector experiment.

The results of this study demonstrate that seabream cultivated in sea cages beyond the size of sex reversal can reach female maturation, ovulation and release eggs during the normal spawning period of the species. However, egg production is very low, decreases considerably with sex ratio (i.e. with fish size in the cage) and varies considerably from day to day. In addition, the survival of fertilized eggs is likely to be low. These findings imply that the escape of eggs from seabream farms and subsequent ecological consequences might be low, depending on the size and intensity of farming within specific regions.

DISCUSSION

RECOMMENDATIONS

There are two cases in which the probability of producing more eggs or that larvae may recruit to the wild populations are increased:

sex ratio in cages is balanced (close to 1:1); and

farms are sited in areas where seabream can complete its life cycle, e.g. close to lagoons. Available information on gilthead seabream ecology is sparse, but indicates that it may be an estuary-dependent species.

The use of a curtain-like egg collector, such as used in the present study, could prevent some dispersion of eggs away from the cages.

A more precautionary mitigation measure would be to restrict the culture of large seabream (of sizes beyond that of sex reversal) in areas close to known nursery grounds of seabream, such as lagoons.

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