Gametogenic cycle and variations in oocyte size of Tapes philippinarum from the Lagoon of Venice

Aquaculture 240 (2004) 473 – 488 www.elsevier.com/locate/aqua-online

Gametogenic cycle and variations in oocyte size of Tapes philippinarum from the Lagoon of Venice F. Meneghetti, V. Moschino, L. Da Ros * Institute of Marine Science, ISMAR-CNR, Castello 1364/A, 30122, Venice, Italy Received 26 November 2003; received in revised form 15 April 2004; accepted 15 April 2004

Abstract The gametogenic cycle of the clam Tapes philippinarum was studied in two populations from different areas of the Lagoon of Venice: S. Angelo (central basin) and Chioggia (southern basin). Samples were collected at regular intervals from July 2000 to July 2001. The study was performed by microscopic observations of histological sections of gonadic tissue and by measuring follicular and oocyte sizes by image analysis. Gametogenesis began in January and was clearly evident until May. Ripe clams appeared in April, and most active spawning occurred in May, continuing during summer until September. Measurements performed using image analysis (i.e., follicular area, percentage of reproductive tissue, oocyte diameter, percentage of resorbing area) highlighted environmental differences between the two sampling sites, demonstrating the better condition of clams from Chioggia. D 2004 Elsevier B.V. All rights reserved. Keywords: Lagoon of Venice; Image analysis; Gametogenic cycle; Histology; Tapes philippinarum

1. Introduction Gonadal cycles in marine invertebrates are strongly influenced by exogenous and endogenous factors (Giese, 1959; Adiyodi and Adiyodi, 1983), temperature generally being considered the main environmental timing factor which regulates gametogenesis and spawning processes (Mann, 1979). However, other important environmental parameters must be taken into account, such as food availability, photoperiod and salinity (RodriguezMoscoso et al., 1992; Urrutia et al., 1999) as also confirmed in the Lagoon of Venice by

* Corresponding author. Tel.: +39-41-2-404-730; fax: +39-41-5-204-126. E-mail address: [email protected] (L. Da Ros). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.011

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the study of Breber (1980) performed on Tapes decussatus. The effect of these variables is complex, depending on the specific way in which they affect processes of energy acquisition and consumption. Moreover, some studies on marine bivalve molluscs have shown alterations in the structure and composition of reproductive tissue associated with adverse environmental conditions also due to anthropogenic activities. Lowe (1988) found an increase in gamete alterations and degenerations in the reproductive tissue of Mytilus edulis exposed to various contaminant concentrations (PAHs, PCBs, heavy metals). Ovarian lesions were observed in wild Asian clam Potamocorbula amurensis experimentally exposed to cadmium, and necrosis of testis and ovary was also revealed in clams from contaminated areas (Clark et al., 2000). Studies concerning the reproductive cycle of Manila clam Tapes philippinarum have assessed the importance of geographical locations in defining and controlling gametogenetic activity (Holland and Chew, 1974; Beninger and Lucas, 1984; Rodriguez-Moscoso et al., 1992; Laruelle et al., 1994; Robert et al., 1993), less attention being focused on the possible influence of various anthropogenic factors. T. philippinarum is indigenous to Japan and was introduced for aquaculture purposes in the Lagoon of Venice in 1983 (Cesari and Pellizato, 1990). Since then, this species has become widespread, not only for farming but also in the natural environment, displacing the native T. decussatus and becoming the principal bivalve species commercially harvested, as confirmed by landing data which peaked at about 36,000 tons per year after 1994 (Lovatelli, 2002). As a consequence, this lagoon has been suffering almost 10 years of intense and destructive fishing pressure (Pellizzato and Da Ros, in press). The aim of the present study was to characterise the gonadal cycle of T. philippinarum, comparing a natural and a farming population from two distinct lagoon areas identified by both different ecological conditions and resource management systems. Indeed, clams from free access sites and concession areas are differently influenced by fishing, mostly in terms of fishing pressure: the free access zone is extensively exploited both by legal and unauthorised dredging gears, the licensed site is subject to a lesser and more controlled impact, being fished only by the owner using controlled dredges (Chicharo et al., 2001).

2. Material and methods 2.1. Sampling and sites Samplings were carried out monthly from July 2000 to July 2001 in two different sites of the Lagoon of Venice: a free fishing area in S. Angelo and a clam farm in Chioggia (Fig. 1). S. Angelo is located in the central basin of the Lagoon of Venice, the most deeply influenced by human activities, considering its nearness to both the industrial and port area of Marghera and the city of Venice. Although fishing is prohibited in the S. Angelo area due to high pollution loads of nutrients (Bianchi et al., 1990), hydrocarbons and heavy metals (Zatta et al., 1992), this environment is highly productive and thus extensively exploited by clam fishermen. Indeed, physical habitat assessment of both sediment (mostly sandy-silty) and waters (higher temperatures due to the cooling wastes of a nearby power

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Fig. 1. Lagoon of Venice.

plant) indicated favourable conditions for T. philippinarum growth (Barillari et al., 1990; Socal et al., 1999). The licensed farm of Chioggia, located in the southern basin, is less affected by industrial activities and mainly used for aquaculture purposes. Chioggia, located near the southern mouth of the Lagoon, is particularly influenced by tidal currents which increase the sand component of the bottom and the salinity of water. During the sampling period, temperature and salinity were also recorded. 2.2. Histology The gonads of 10 individuals per sample were freshly dissected and immediately fixed in Baker’s formol for 24 h at 4 jC. The tissues were subsequently dehydrated in a series of increasing concentrations of ethyl alcohol solutions, then clarified in CleareneR and embedded in paraffin wax (ParaplastR at 56 jC). Sections (5– 6 Am thick) were cut in the mid-region of the gonad, and the resulting slides were stained using Papanicolaou’s method. Microscopic examination under the light microscope aimed at determining the stages of the gametogenic cycle. Gonadal stages were graded following Valli et al. (1996) with slight modifications, and are summarised here: stage 0, sexual inactivity; stage I,

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early developing; stage II, late developing; stage III, ripe; stage IV, spawning; stage V, spent. The gonadal index (Seed, 1980) was then calculated for each sample in the following manner: the number of clams at each stage was multiplied by the numerical score attributed to that stage, the products were added, and the result divided by the total number of individuals sampled. The gonadal index ranged from 0 (all individuals in the sample are in resting or spent stages) to 3 (all individuals are ripe). 2.3. Image analysis Histological slides were examined under a microscope (magnification 400  ) and reproductive tissue images, acquired on a Leica DMLB microscopy video camera, were subsequently analysed by Image-Pro Plus software (version 4.0.0.9). The purpose was to measure variations in area fractions, other than in sizes of developing and ripe gametes during gonadal development. The image analysis method suggested by Xie and Burnell (1994), slightly modified, was applied in this study. The boundaries of gonadal structures were first defined semiautomatically and areas were then calculated automatically and converted into theoretical diameters expressed in micrometers. For each specimen, at least five randomly chosen fields were considered to measure the following parameters: follicular areas (at least 10 follicles); ripe oocyte areas (at least 50 free oocytes in the lumen, with a well-defined germinal vesicle); unripe oocytes (at least 40 maturing oocytes either exhibiting a clear nucleolus, when in the follicle wall, or showing a pedunculus, when in the lumen); percentages of areas filled with resorbing oogonia/oocytes, and of areas filled with male gametes. The percentage of reproductive tissue was subsequently calculated as the ratio of total follicular area to total gonadic area. The measurements of follicular and oocyte area were carried out only during the reproductive period, when gonads were active, and when the number of individuals was adequate for statistical analysis. 2.4. Statistical analysis For each sample, the normality of data was tested using the Shapiro –Wilk test. Since the assumptions of the parametric test were violated, Kruskal – Wallis one-way, Mann – Whitney and Kolmogorov –Smirnov tests for two independent samples were used for comparisons (Sokal and Rohlf, 1981).

3. Results 3.1. Environmental variables Monthly temperature and salinity in surface water at the time of sample collection are reported in Table 1: salinity ranged from 27.6xto 33xat S. Angelo and from 30xto 37x at Chioggia, with a minimum in May; temperature values fluctuated between 5 jC in January– February and 27 jC in June –July.

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Table 1 Temperature and salinity in the study areas at the time of sample collection July August September October December January February March April May June July 2000 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2001 Temperature (C) S. Angelo 24.2 24.1 Chioggia 23.0 22.0

20.8 20.0

16.2 16.0

20.5 14.0

5.0 5.0

6.0 5.0

18.2 13.5

15.3 25.8 24.5 27.5 16.2 19.9 26.0 24.5

Salinity (x) S. Angelo 31.6 31.2 Chioggia 34.9 34.0

31.5 33.5

28.6 33.0

30.6 33.0

33.0 30.0

30.1 32.0

31.9 23.2

27.7 28.9 30.2 31.0 32.0 32.1 32.8 33.0

3.2. Gametogenic cycle Data regarding microscopic observations in gonadal tissues of T. philippinarum collected in the two sampling sites were first recorded separately. Following an exploratory analysis (Spearman correlation factor) showing that the correlation coefficient was not significantly different between the gonadic indices of clams from S. Angelo and from Chioggia (R = 0.87; p < 0.001), results were pooled. Gonadal stages are reported in Fig. 2. Sexual activity was quiescent from October to January (prevalence of stage 0), exhibiting the highest percentages in December. The onset of gametogenesis, revealed by the first appearance of individuals exhibiting gonads at stage I (early developing), was recorded in January. In the following months, the decreasing number of individuals at this stage was related with the increasing number of organisms at stage II (late developing), which started from March. Moreover, this stage

Fig. 2. Stages of reproductive cycles of T. philippinarum. Percentages of clams corresponding to each gonad stage are identified by different bar patterns. Legend: 0, resting; I, early developing stage; II, late developing stage; III, ripe stage; IV, spawning stage; V, spent stage.

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was always recorded between March and July. Stage III (sexual maturity) was first detectable in April and always recorded until August. The onset of spawning (stage IV) started in May and continued during summer until September, when the first organisms at stages V and 0 (partially spent and sexually inactive) were observed. The monthly gonadal index (Fig. 3) was calculated according to the method of Seed (1980). This index, which briefly describes the maturation level reached by the population, peaked in July and August, fitting the period of sexual maturity and spawning; in autumn, it decreased sharply to reach its minimum values in winter, when most of the animals were sexually inactive. From February onwards, the index rose slowly, indicating the end of sexual rest and the gradual onset of gametogenesis. The female/male ratio calculated by pooling all samples collected over the whole sampling period was 1.67; no cases of hermaphroditism were detected among examined clams. 3.3. Image analysis The values of several parameters measured by computer-aided image processing (mean follicular area, percentage of reproductive tissue, mean oocyte diameter, percentages of areas filled with resorbing oogonia/oocytes, and of areas filled with male gametes) are shown in Table 2 and their statistical comparisons between S. Angelo and Chioggia samples are reported in Table 3. The trends of the mean follicular area were similar at the two sampling sites, the highest values being reached in summer, when the gonads were at their maximum development, and lowest in September. When gametogenesis started, the follicles progressively enlarged, reaching a maximum area of about 40,000 Am2 at S. Angelo in August 2000 and 50,000 Am2 at Chioggia in May 2001. Higher values were generally observed at Chioggia, showing significant differences in May ( p < 0.01). This measurement was no longer feasible from October to January, due to follicular fragmentation caused by the progressive evolution towards the resting phase. A similar trend was evidenced for the percentage of reproductive tissue (as the ratio of follicular to total gonadic area), showing values increasing throughout spring– summer

Fig. 3. Gonadal index of T. philippinarum.

Table 2 Monthly variations in several gonadic parameters (follicular areas, percentage of reproductive tissue, unripe and ripe oocyte diameters percentages of degenerate and male oocyte areas, values are median F absolute mean score) July 2000

August 2000

September 2000 February 2001 March 2001

April 2001

May 2001

June 2001

July 2001

Follicular area (lm ) S. Angelo 28,869 F 6421 39,406 F 13,741 9692 F 7163 Chioggia 41,207 F 11,681 42,176 F 12,388 10,012 F 6180

9586 F 2334

Reproductive tissue (%) S. Angelo 73.7 F 7.8 Chioggia 65.8 F 14.2

33.4 F 17.8

57.9 F 10.6 35.4 F 7.5

57.7 F 13.6 27.7 F 8.8

66.6 F 10.6 65.0 F 10.4

73.6 F 6.0 60.9 F 10.3

84.8 F 12.6 57.3 F 6.9

13.1 F 0.86

18.0 F 3.0 15.6 F 8.3

22.8 F 2.4 14.4 F 0.6

19.1 F 1.7 19.7 F 4.1

21.2 F 1.8 25.6 F 4.5

24.6 F 5.0 19.8 F 1.0

27.3 F 4.7

40.6 F 3.8

32.0 F 2.7 39.5 F 5.8

33.4 F 2.7 39.3 F 1.6

37.3 F 2.3 41.1 F 1.4

5.5 F 2.3

11.7 F 4.7

11.8 F 5.9 10.6 F 3.3

15.8 F 5.1 13.1 F 2.8

11.6 F 3.1 14.0 F 3.1

40.4 F 21.7 22.4 F 7.0

93.8 F 14.0 66.4 F 25.5

90.3 F 3.7 92.5 F 3.0

93.9 F 17.5

99.8 F 0.9 99.5 F 0.5

86.3 F 6.1 74.0 F 3.9

40.2 F 15.9 51.6 F 27.4

Unripe oocytes mean diameter (lm) S. Angelo 20.7 F 2.0 35.2 F 4.6 Chioggia 25.8 F 7.6 Ripe oocytes mean diameter (lm) S. Angelo 37.9 F 3.3 42.2 F 2.0 Chioggia 45.7 F 2.8 Degenerate area (%) S. Angelo 13.0 F 4.0 Chioggia 7.8 F 0.3 Male oocyte areas (%) S. Angelo 94.4 F 1.8 Chioggia 99.7 F 0.4

9.8 F 3.8

2.5 F 1.7

98.3 F 1.1 99.8 F 0.3

92.8 F 7.0

44.6 F 9.0

18,785 F 9585 28,777 F 8224 26,354 F 6639 23,788 F 6564 30,85 F 18,088 14,660 F 5524 12,185 F 6538 49,295.1 F 29,454 29,731 F 8141 43,218 F 20,111

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Table 3 Statistical comparison of follicular areas, percentage of reproductive tissue, unripe and ripe oocyte diameters, percentages of degenerate and male oocyte areas between clams collected from S. Angelo and from Chioggia

Follicular area Reproductive tissue Unripe oocytes Ripe oocytes Degenerate oocyte area Male oocyte area

July 2000

August 2000

September 2000

March 2001

April 2001

May 2001

June 2001

July 2001

n.s. n.s. n.s. n.s. * n.s.

n.s. n.s.

n.s. n.s.

n.s. * n.s.

* ** *

n.s. n.s. n.s. ** n.s.

n.s.

n.s.

** n.s. n.s. n.s. n.s. n.s.

n.s. * n.s. n.s. n.s. n.s.

n.s.

Kruskal – Wallis test: *: p < 0.05; **: p < 0.01; ***: p < 0.001.

and decreasing in winter. In both stations the highest values (up to 86% and 74% at S. Angelo and Chioggia, respectively) were observed in August when the gonads were at their maximum expansion and the lowest in February at S. Angelo and in April at Chioggia due to the highest degree of cellular desegregation and reabsorbed follicles. Comparing the two sampling sites, S. Angelo showed significantly higher values in March, April and July 2001 ( p < 0.05). Monthly variations of both unripe and ripe oocyte diameters showed the higher average values over the period June – August. Significantly higher values for unripe oocyte diameters were observed at S. Angelo in April 2000 ( p < 0.05) and at Chioggia in July 2000 ( p < 0.05). Ripe oocytes had higher diameters in Chioggia clams, but appeared 2 months earlier in S. Angelo (March vs. May, respectively) (Table 2). With regard to the ‘‘degenerate areas’’, calculated on the basis of the mean follicular area occupied by resorbing oogonia and/or oocytes, they were always found whenever female gametes were present in the follicles, without revealing a clear monthly trend. The observed values were in the range of 2.5 –15.8% (Table 2), and were significantly greater at S. Angelo in July 2000. As for the percentages of male gametes occupying the follicles (Table 2), it peaked late spring – summer whereas minima were observed during the onset of gametogenesis (February and March) in both sampling sites. Table 4 lists values related to the size of ripe and unripe oocytes, obtained considering the averages of total annual measurements. Unripe oocytes showed greater diameters at S. Table 4 Descriptive parameters of measurements performed on total number of oocytes sampled ( F s.e.) in two sampling areas S. Angelo

No. of oocytes Minimum diameter (Am) Maximum diameter (Am) Mean diameter (Am) Median (Am) Asymmetric index

Chioggia

Unripe

Ripe

Unripe

Ripe

2974 7.9 62.2 23.0 ( F 0.2) 19.43 1.15

1698 9.4 61.8 35.8 ( F 0.2) 36.3 0.03

1286 7.4 69.0 22.7 ( F 0.3) 17.4 1.44

778 16.3 68.1 41.6 ( F 0.3) 42.2 0.33

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Angelo (median value 19.6 Am) than at Chioggia (median value 17.4 Am), whereas ripe oocytes were significantly larger in Chioggia (median value 42.2 Am) than in S. Angelo (median value 36.3 Am). With regard to the size-frequency distributions of oocyte diameters, data were obtained by pooling measurements from all samples (Fig. 4). The distribution of unripe oocyte diameters was considerably asymmetric in both sites, as shown by the asymmetry index and was significantly different ( p < 0.001) between the two populations. The most representative size-class fell in the range 15– 20 Am for both areas, although a number of greater size-classes but low in percentage did also occur. The size-frequency distribution of ripe oocytes (Fig. 4) was less asymmetric and significantly different ( p < 0.001) in the two stations: at Chioggia, diameter values fell more frequently in the higher size-classes than at S. Angelo, as evidenced by mode values (respectively in the ranges 45 – 50 and 35 –40 Am). Fig. 5 groups frequency distributions of diameters according to reproductive stage. Oocyte sizes rose according to degree of maturation: during gametogenesis (stages I and II), distribution was right-skewed, indicating the increased frequency of larger oocytes. At

Fig. 4. Relative frequency distribution percentages of unripe and ripe oocyte diameters of T. philippinarum collected from S. Angelo and Chioggia. Statistical comparison p < 0.001 (Kolmogorov – Smirnov test).

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Fig. 5. Percentages of relative frequency distribution of oocyte diameters in each stage of maturation of T. philippinarum collected from S. Angelo and Chioggia.

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stages III and IV, the highest frequencies were related to the largest diameters, although small diameters were always recorded as well.

4. Discussion Several studies on the reproductive cycle of T. philippinarum have demonstrated that the start and duration of the gametogenetic phases of maturity, spawning and resting mostly depend on latitude and temperature (Yamamoto and Iawata, 1956; Ohba, 1959; Holland and Chew, 1974). In northern Europe, the onset of gametogenesis and maturity begins later throughout the year (Xie and Burnell, 1994), whereas in the Mediterranean area gamete production always starts in January –February, and sexual maturity is reached in late spring (Sarasquete et al., 1990; Rodriguez-Moscoso et al., 1992). In the present investigation, the gametogenetic cycle was studied separately in the two clam populations of S. Angelo and Chioggia, but results from histological observations were combined, no differences being preliminarily detected when comparing data obtained independently from the two areas. On this basis, we inferred that gametogenesis (stage I) for T. philippinarum from the Lagoon of Venice started in January, confirming the results obtained by Valli et al. (1996). As observed by Devauchelle (1990) for populations from various temperate areas, the onset of gametogenesis seems to be related more to the rise in photoperiod than to variations in water temperatures (quite stable in lagoon waters, at values around 5 – 6 jC throughout January and February 2001). However, neither rapid nor repeated changes in temperature at S. Angelo (Table 1), probably due to industrial cooling waters discharged nearby, seemed to influence the reproductive cycle, underlining the great tolerance of this clam to fluctuations in hydrological parameters (Mann, 1979). The length of the gametes ripening period (stages I and II) was extended throughout spring and summer, sexual maturity (stage III) being reached in April, one month earlier than that reported by Valli et al. (1996). This is probably due to the progressive rise in water temperature required for complete ripening of gonads (24 – 27 jC) effectively recorded in this study. The emission phase (stage IV), extended from May to October according to Valli et al. (1996), was shorter in our study (May –September), with no evidence of emission peaks. These findings are consistent with the hypothesis of a number of incomplete emissions, brought about by subsequent gamete meiosis, following one another throughout summer. This feature is also confirmed by the number of individuals at stage II (late developing) and observations of oogonia on the follicular wall during the whole emission period. Moreover, as shown by other authors (Rodriguez-Moscoso and Arnaiz, 1998; Robert et al., 1993), a clear-cut relation was observed between reproductive and biochemical cycles: the onset of spawning coincided with the decrease in glycogen content studied in the same organisms by Marin et al. (2003). Furthermore, the results of the present study fit those obtained by Valli et al. (1996): stage 0 (sexual rest or quiescent) starts in late summer – early autumn, when water temperature decreases, and continues throughout winter until February. The few individuals in the resting stage observed in April and June 2001 would thus indicate possible inhibition of normal gonadic development due to environmental and/or endogenous stress. Moreover, the number of specimens showing gonads filled with connective tissue, haemocytes, and sometimes granulocytoma-

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like lesions which may reveal pathological conditions (Lowe and Moore, 1979; Grizel, 1990) was similar over the whole sampling period (about 10%) at both sites. Similar anomalies, described in previous papers for mussels M. edulis (Lowe, 1988), and clams (Navas et al., 1992; Park and Choi, 2001), have been related both to parasitic infections and environmental pollution. Hypnospores quite similar to those of Perkinsus atlanticus (Azevedo, 1989) were observed in most of the histological slides, and confirmed by Perkinsus body burdens quantified in the same populations according to the Ray’s technique (Meneghetti et al., 2003). The results of this investigation highlighted a significantly higher Perkinsus infection at Chioggia than at S. Angelo, emphasising that the greater population density in the clam farming area represents favourable conditions for the spread of the parasites. Thus, we hypothesize that Perkinsus infection may be at least partially responsible for the observed pathological alterations in the Chioggia clams. The same histological anomalies recorded in clams from S. Angelo could be more effectively interpreted as a consequence to xenobiotics exposure. The relatively higher level of both organic and inorganic micropollutants nearby S. Angelo is documented by large amounts of data acquired by the local environmental agencies in the frame of routine monitoring investigations, and mostly confirmed by scientific papers dealing with fate and effect in water (Dalla Valle et al., 2003), sediments (Bellucci et al., 2002; Frignani et al., 2001; Sfriso et al., 1995) and biota (Livingstone et al., 1995; Nasci et al., 1998) of pollutants originated from the adjacent industrial area. Image analysis, as a quantitative method of measuring gametogenetic activity in bivalves largely applied to T. decussatus and M. edulis (Lowe et al., 1982; RodriguezMoscoso and Arnaiz, 1998), is a useful tool which yields very precise information about the histology of reproductive tissues by analysing various cell types. Measuring oocyte size by image analysis is also considered to be more accurate and less tedious than the traditional method, which uses an eyepiece graticule to measure proportional areas. Previous studies showed that monthly variations in oocyte size are good descriptors of the annual trend of the reproductive cycle (Laruelle et al., 1994; Xie and Burnell, 1994; Lango-Reynoso et al., 2000). However, one drawback of this method is the difficulty of obtaining realistic measurements in tissues modified by pathological situations. As a consequence, our results do not consider that part of the population (about 10%) severely affected by gonadal anomalies. Measurements regarding follicular area and areas occupied by reproductive tissues showed similar trends to those described for the same species by Rodriguez-Moscoso et al. (1992), obtained following a different stereological method. Follicular area and percentage of reproductive tissue increased in the period of greatest gonadal development and then decreased towards the resting phase. The same pattern was observed in the male gamete areas. Comparing the two sampling sites, Chioggia clams showed generally larger follicular diameters than S. Angelo ones. The same trend was also observed for the diameter of ripe oocytes, although they matured later (first appearance in May, whereas at S. Angelo in March). Mean diameter values of ripe oocytes (35.8 Am at S. Angelo, 41.6 Am at Chioggia; Table 4) were similar to those observed by Xie and Burnell (1994) in T. philippinarum from Southern Ireland. In littoral species, the intra-specific range in oocyte and follicular sizes may correspond to a flexible response to phytoplankton availability (Le

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Pennec and Benninger, 2000). However, due to the observed similar trophic conditions in both studied sites (Marin et al., 2003) other causes must be considered for the Chioggia farming area, e.g., the lower fishing impact and/or different human manipulation of resources. Furthermore, difficult to interpret is the lack of a clear monthly trend in ripe oocyte diameters observed in the same licensed area (Table 2), which led us to hypothesise about occasional and unfortunately unverifiable introduction there of clams collected nearby. This observation is also confirmed by biometric measurements evaluated in the same samples: continuous seeding, harvesting and re-seeding caused uneven trends of the mean length of the populations which were particularly clear from January to July 2001 (IMPACTO, 2002). As regards the size-frequency distribution of oocyte diameters, in both sampling areas a large range of values was observed (from 7 to 70 Am), reflecting high inter- and intraindividual variability, as also found by Xie and Burnell (1994). As expected, in unripe oocytes, the very strong right asymmetry of the size distribution (Fig. 4) indicated a growing population. Moreover, considering monthly observations, the presence of small unripe oocytes in late summer confirmed gametogenesis as a continuous process throughout the warm season. The high variability also observed in the size distributions of ripe oocytes (Fig. 4) was explained by the presence of some small oocytes (diameter < 35 Am) classified as ripe, since direct evidence of their being unripe oocytes detached from the follicular wall was lacking (Xie and Burnell, 1994). A high fraction of resorbing areas inside follicles may indicate stress conditions, as demonstrated by Lowe (1988) in the mussel Mytilus edulis after exposure to Cd and Zn. In our two populations, the percentage of resorbing areas was always high (15%). Moreover, in some months it was significantly higher in S. Angelo with respect to Chioggia, perhaps indicating exposure to a more polluted environment, therefore confirming the findings of the histopathological observations. Our results evidenced that histological measurements obtained by image analysis (i.e., follicular area, percentage of reproductive tissue, oocyte diameter, percentage of resorbing areas) can provide accurate information about the general reproductive state of clam populations and at the same time supply knowledge of differing ecological conditions and/ or anthropic impact at the sampling sites. Specifically, quantitative variations of follicular areas, oocyte diameters and resorbing areas proved to be suitable indicators of physiological impairment due to adverse environment conditions. Instead, the gonadic index, as expected, is not sensitive to the small range of variations in temperature, photoperiod and trophism, as observed in the two sampling sites throughout the study period. In conclusion, as suggested in recent papers (Barber and Blake, 1991; Lango-Reynoso et al., 2000), the most complete approach in verifying reproductive events concerning gamete development in marine invertebrates must take into account both qualitative and quantitative aspects.

Acknowledgements This study was carried out with funding from the European Commission DGXIV (Studies 99/062): ‘‘Assessing the impact of bivalve fisheries on the benthic ecosystems of

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the Ria Formosa lagoon (Portugal), Venice lagoon (Italy), Aegean Sea (Kavala-Greece) and on the juvenile flatfish in the South coast of Portugal (IMPACTO)’’. Special thanks are also due to an anonymous reviewer for providing very fruitful comments.

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