Scientific Cooperation to Support
Responsible Fisheries in the Adriatic Sea
General Fisheries Commission for the Mediterranean (GFCM)
Scientific Advisory Committee (SAC)
Subcommittee on Stock Assessment (SCSA) - Rome, 26-30 September 2005
Length at first maturity of the Adriatic anchovy (Engraulis
encrasicolus, L.)
R. Rampa 1, E. Arneri 2, A. Belardinelli 2, E. Caputo 1, N. Cingolani 2, S. Colella 2, F. Donato
2
, G. Giannetti 2, A. Santojanni 2
1
2
Università di Ancona (Italy)
Istituto di Scienze Marine - Sezione Pesca Marittima, Ancona (Italy)
SUMMARY
An histological analysis of gonads was performed during the spawning season of the most
commercially important clupeoid of Adriatic sea, Engraulis encrasicolus. Samples were collected
during the spring - summer 2003 (May – September) and the summer 2005 (June and July) in
northern and middle Adriatic Sea. Gonadic maturity for males and females was described by an
histological five maturity stages scale according to Billard (1986) and West (1990).
When two-year data were used the length at first maturity (L50) of females was found at about 8.1
cm; moreover the histological aspect of ovaries containing several oocyte batches in different
maturity stage, indicated an intermittent release of ripe eggs in the course of a prolonged spawning
season (April – August).
Although it wasn’t possible to fit a logistic curve for males, data suggest that length at first maturity
is about 7.5 cm. Over this length testes were always mature.
Our results confirm that the length at first maturity of anchovy may be different among different
areas of Mediterranean basin. This could be due to the diverse climatic and trophic parameters that
characterize the various areas of Mediterranean sea.
Key words: anchovy, Adriatic Sea, size at first maturity, histological analysis.
1
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Introduction
Anchovy, (Engraulis encrasicolus L.) is the most valuable clupeoid in the Mediterranean area and
in particular represents one of the most important resource for the fishing industry in the Adriatic
basin (Cingolani et al., 1996) . So several studies have dealt with aspects of reproduction of this
species particularly focusing on annual cycles of gonad development, fecundity, eggs size and
quantity in every spawning act. As a rule in little pelagic fishes, anchovy is a batch spawner with
protracted spawning season and a very high number of released eggs. The knowledge on these
aspects could be a valid ally to better guarantee a rational fishing effort without damaging the
continuity of the exploited stock.
Our work focused on the reproductive features of the anchovy populations sampled in northern and
middle Adriatic during a two year period; the gametogenesis was histologically investigated and
particular attention was paid in determining the length of first maturity of this species in the
considered area as a mean to better operate a control on the fixed capture-size thus assuring the
juveniles to reach the sexual maturity. Several authors underlined as anchovy attain sexual maturity
within the first year of life (Furnestin, 1945; Cort et al., 1976; Lucio & Uriarte, 1990); this means,
according to data obtained in various areas of west Mediterranean sea (Alboran sea) and east
Atlantic (Bay of Cadiz), the reaching of a length at for both males and females between 11 and 13
cm (Lucio & Uriarte 1990; Giraldez & Abad, 1995; Motos 1996; Millan, 1999) whereas Sinovcic
(1999) reported in east Adriatic a first maturity length at about 9 cm. These results differ from data
reported by Lisovenko & Andrianov, (1996) that showed for the subspecies Engraulis encrasicolus
ponticus endemic of the Black sea a first spawning length comprised between 6.5 and 7 cm.
This variableness might be attributed to the influence of various abiotic (temperature, salinity,
hydrodinamism) and biotic (nutrients and productivity) factors on different anchovy populations
(Sparre et al., 1989). Several studies on other clupeoids (Hunter & Goldberg, 1980; Abad &
Giraldez, 1992; Giraldez & Abad, 1995) demonstrated that these fishes carry out a number of
reproductive tactics aiming to guarantee, as much as possible, the surviving of eggs and larvae; in
particular Alheit (1989) verified that thanks their ecological plasticity, anchovy populations were
able to adapt their reproductive strategies (e.g. fecundity, frequency of spawning, first maturity) to
the diverse environmental conditions.
Materials and methods
A total of 189 anchovies (93 females and 96 males, tables 1 - 2) were collected in the coastal waters
of northern (P.to Garibaldi, Goro, Rimini) and middle (P.to Recanati) Adriatic sea during the
spring-summer 2003 (May - September) and the summer 2005 (June – July); they were caught by
means of “volante” nets. For each fish total and standard length were measured to the nearest
millimetre below. Gonads were removed and fixed in Bouin’s fluid and, after dehydration in
ethanol, embedded in paraffin. Sections (7 µm thick) were stained with Galgano’s trichrome and
prepared for histological examination (Beccari and Mazzi, 1972). All sections were observed with a
Leica Diaplan photomicroscope. On 10 oocytes for each development stage the cellular diameter
(P, to μm) was measured with an image analyzer (Lucia 4.5).
Ovarian follicles were classified into 5 development stages (I, chromatin nucleolar stage; II,
perinucleolar stage; III, yolk vesicle stage; IV, mature (vitellogenetic) stage, V, post-ovulatory
follicle stage according to West (1990). Because anchovies are asynchronous spawners (Wallace &
Selman, 1981) it was attributed for each specimen a maturity stage corresponding to the most
advanced vitellogenetic stage observed in the ovary sections. They were considered immature with
stages I-II oocytes, developing with stage III oocytes, mature with stage IV oocytes and spent with
post-ovulatory follicles (stage V).
Spermatogenic activity was assessed by evaluating the different types of gametocytes (from
spermatogonia to spermatozoa) in the seminiferous lobules of each testis. The testes maturity was
assessed by using a five development stages classifications according to Billard (1986): I immature
stage (spermatogonia and spermatogonial mitoses); II – III development stages (first and second
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meiotic divisions, respectively); IV mature stage (spermatozoal cysts); V post–reproductive stage
(collapsed lobules and residual spermatozoa).
Based on the histological maturity stage of the ovaries, the proportion of specimens within each half
centimetre size class that had gonads in stages 3 - 5 was calculated. The length at first maturity (L50)
representing the size at which 50 % of the specimens was mature, was determined by fitting the
logistic equation (Prager et al., 1994):
p = [1 + e – r (x – x50)] -1
(where p is the estimated proportion in size class, r is a fitted parameter, x is the total length, x 50 is
the length at which 50 % of the specimens was mature), to the proportion of fish in each size class.
An attempt was made considering the proportion of specimens with gonads in 4 – 5 stages in order
to establish the length at first spawning.
Results
Oogenesis
In anchovy, as commonly reported in other teleosts (Hoar, 1969), ovary is characterized by a
cystovarian structure as shown by an evident lumen (“ovocele”), in which, at ovulation, mature
oocytes were released and spread throught the genital pore. Ovaric laminar structures, enclosing and
spreading to internal parts of ovocele, hold ovaric follicles in different development stages.
Histological data
The gonads of 93 females ranging in size from 7 to 11.2 cm were histologically analysed. In
describing the ovaries histological maturity stages the specimens were divided into three sizeclasses: 7 - 8 cm, 8 - 9 cm, 9 - 11.2 cm. The 17 specimens, belonging to the first size-class, resulted
immature showing little previtellogenic chromatinic and perinucleolar oocytes (stage I – II,
respectively P = 37.4 and 83.2 µm Fig. 2 A) with a very basophilic cytoplasm and a central and
rounded nucleus occupying most of the cell and characterized by a great number of nucleoli located
at its periphery (Fig. 2 A). The 38 females belonging to the second size-class evidenced a more
variable histological condition: 14 of these were immature with stage II oocyte whereas 8 showed
maturing ovaries; at this stage (stage III, Fig. 2 B) oocytes start to increase considerably in size (P =
194.2 µm) and are characterized by the formation of chromophobic vesicles in the region adjacent
to the plasmatic membrane. These vesicles, the cortical alveoli, gradually increase in size and
number moving towards the centre of the cytoplasm. At last 15 appeared mature and close to
ovulation: in these ovaries batches of big advanced vitellogenesis (stage IV, P = 317.6 µm, Fig 2 C
- D) oocytes with yolk granules increasing in number and size and gradually filling the cytoplasm,
together with few post-ovulatory follicles were observed; in a single sampled in September
specimen the ovary quiescence (stage II) together with the presence of several post-ovulatory
follicles (stage V P = 173.3 µm, Fig. 2 F) suggested the end of the spawning process. The last sizeclass composed by 38 females displayed in almost all cases advanced maturity with stage IV
oocytes some post-ovulatory follicles and few atresias (Fig. 2 E); only 3 sampled in September
specimens were spent (stage V) with early development stage oocytes and post-ovulatory follicles.
Spermatogenesis
The anchovy testes stucture shows a lobular pattern similar to the greatest part of teleosts
(Nagahama, 1983). Particularly the elongated and finger-like lobules, present a cystic structure in
which the different types of gametocytes development takes place. As typical in teleosts, testes
present the peculiar structure defined as “unrestricted spermatogonial testis type” (Grier et al.,
1980) with germinative cells distributed along the seminiferous lobules.
Histological data
A total of 96 males ranging from 7 and 11.2 cm of total length were histologically analysed. Testes
of 5 small specimens (until 7.3 cm) were immature (stage I) and characterised by spermatogonial
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cysts (fig 3 A); in two cases developing testes with meiotic spermatocytes II, with a deep-stained
cytoplasm, and spermatids (stage III) were found, whereas on the contrary 82 specimens were
caught at the peak of spermatogenic process displaying a massive confluence of spermatozoal cysts
towards lobule lumina (stage IV, Fig. 3 B - C). At last 7 fish appeared in post-spermiation condition
with collapsed lobules with some interstitial spermatogonia at their periphery, residual spermatozoa
in some lobules, apoptosis of gametocytes that had not completed the spermatogenic process (Fig 3
D).
% maturi
Length at first maturity (L50)
The length at first spawning was calculated only for females, by pooling together histological data
from the specimens sampled in 2003 and 2005 surveys. By fitting the logistic equation on the
available data the estimated length at first maturity was 8.14 cm (ASE = 0.0194, r2 = 0.97, Fig.1).
Moreover an estimated length of 8.37 was obtained considering only mature stages (length at first
spawning).
Unfortunately the very low number of available maturing (stages II – III) males did not allow us to
establish the L50 as well; therefore over the length of 7.5 cm all the sampled specimens resulted
mature.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
6
7
8
9
10
11
12
LT (cm)
Figure 1: Logistic curve fitted to the proportion of mature females (histological maturity stages III – V) in
relation to fish length. Dashed line indicates the length at first spawning estimate (L50).
Discussion
Anchovy (Engraulis encrasicolus, Linnaeus, 1758) is a little pelagic clupeoid widely distributed in
Mediterranen sea thanks to its great reproductive ductility that consists in modifying one or more
spawning tactics whenever the environmental condition require it, thus favouring better survival
chances to eggs and larvae (Alheit, 1989). Moreover as typical constituent of the pelagic fish fauna
living in temperate waters anchovy show some peculiar reproductive features already described in
other pelagic species: early sexual maturity, high fecundity, relatively small egg size at maturity,
multiple spawning events along the reproductive season (Williams & Clarke, 1982; Hunter &
Macewicz 1985; Schaefer, 1987; Alheit, 1989). These parameters indicate a clear ecological
orientation towards the “r – strategy” by maximizing the reproductive effort in the production of a
great number of eggs without any parental care. Although facing a big larvae mortality this strategy
should increase the probability of survival by favouring eggs dispersal over the most suitable
environmental conditions.
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Our histological data confirmed that anchovy is a batch spawner with asynchronous ovaries
containing several oocyte batches in different maturity stages, indicating many intermittent releases
of ripe eggs over a long spawning season (that may last from March – April to August – September
as reported by Piccinetti et al., 1979; Piccinetti et al., 1980; Regner et al., 1985; Regner & Dulcic,
1994). Further histological evidence of prolonged and intermittent spawnings was displayed by the
contemporary presence of transitional post-reproductive structures like atresias of non-deposed
oocytes and post-ovulatory follicles in mature (filled with vitellogenic oocytes) ovaries ready to
restart the spawning process. Regarding males, testes can be structurally defined as “lobular”
according to Billard (1986) or an “unrestricted spermatogonial” according to Grier (1980); in this
type of testis spermatogonia are confined to small, peripheral cysts within the lobule, which
subsequently enlarge and extend toward the tubule lumen as spermatogenesis takes place.
Almost all our specimens presented, after the histological overview, the typical reproductive pattern
with spermatozoal cysts within the seminiferous lobules. Moreover in both females and males,
specimens sampled in September resulted spent confirming, as reported in literature (Gamulin,
1940, 1964; Regner, 1972), that in this species the spawning activity attains a peak in the summer
months rapidly declining with the start of autumn.
Regarding the length at first maturity in the Adriatic sea Sinovcic (1999) reported a size of about 9
cm slight differing from our data obtained by fitting a logistic curve on histological results, that
indicated as females attained maturity at about 8.1 - 8.2 cm; for males although it wasn’t possible to
fit the logistic curve histological data permitted to establish a first spawning length at about 7.5 cm;
over this size testes appeared fully mature.
These results put themselves among the reported lengths in west Mediterranean (11 – 13 cm in both
males and females) and in the Black sea (6.5 – 7 cm for both sexes), thus delineating a sort of
decreasing spawning length gradient from west to east. As widely reported anchovy reproductive
parameters are deeply influenced by abiotic and biotic factors of the habitat; particularly many
authors underlined the positive correlation between food abundance, high temperature moderate
hydrodinamism and the increasing of spawning rhythms in this species (Vucetic, 1975; Palomera,
1992; Giraldez & Abad, 1995; Motos, 1996; Regner 1996, Sinovcic, 1999). In this context both
Adriatic and Black sea represent favourable habitats thanks their environmental characteristics (e.g.
high superficial temperature, moderate idrodinamism and big seasonal productivity). Hence these
factors could influence anchovy first spawning length it’s likely that the populations of Adriatic and
Black sea be able to accumulate the necessary trophic resources for reproduction in rather short
period than the west Mediterranean ones thus anticipating the sexual maturity.
Acknowledgements
We wish to thank Dr. M. La Mesa of the CNR-ISMAR - Ancona (Italy) for collaboration in
statistical analysis.
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Females
Maturity stages
N.of specimens
TL (cm)
1
2
3
4
5
6
25
8
50
4
7 - 7.3
7.3 - 8.5
7.8 - 8.2
8.3 - 11.2
8.9 - 9.6
Table 1: Reproductive and morphometric data of females. Maturity stages correspond to the most
advanced vitellogenic stage observed in the ovary sections.
Males
Maturity stages
N.of specimens
TL (cm)
1
3
4
5
5
2
82
7
7 - 7.3
7.5 - 7.7
7.5 - 11.2
9.5 - 11
Table 2: Reproductive and morphometric data of males. Maturity stages correspond to the most advanced
spermatogenetic stage observed in the testis sections.
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Figure 2: A: Cross-section of an immature ovary showing stage II oocytes; double arrow indicates
peripheral nucleoli. Scale bar = 40 µm. B: Maturing ovary: stage III oocytes with yolk vesicles (double
arrow); scale bar = 100 µm. C: Cross-section of a mature ovary showing both stage III (double arrow
indicates yolk vesicles) and stage IV (double asterisk ) oocytes together with reabsorbed post-ovulatory
follicles (single asterisk). Scale bar = 100 µm. D. Particular of a large stage IV oocyte with evident
vitellogenetic granules; scale bar = 150 µm. E: A large atresia of non-deposed oocyte (double asterisk) can
be observed; scale bar = 100 µm. – F: Post-reproductive ovary with large post-ovulatory follicles (double
arrow) together with some previtellogenetic oocytes; scale bar = 75 µm.
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Figure 3: A: Cross-section of a small immature specimen testis showing cysts filled with spermatogonia;
scale bar = 15 µm. B:.Mature testis with free spermatozoa within the seminiferous lobules scale bar = 35 µm.
C: Particular of a seminiferous lobule completely filled with spermatozoa; scale bar = 50 µm. D: Crosssection of a post reproductive testis: note the collapsed lobules with some some residual spermatozoa
(asterisks); scale bar = 50 µm.
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