Relative growth and reproductive cycle in two ...

Biologia 67/4: 1—, 2012 Section Zoology DOI: 10.2478/s11756-012-0060-7

Relative growth and reproductive cycle in two populations of Bolinus brandaris (Gastropoda: Muricidae) from northern Tunisia (Bizerta Lagoon and small Gulf of Tunis) Sami Abidli, Youssef Lahbib & Najoua Trigui El Menif* University of Carthage, Faculty of Sciences of Bizerta (FSB), Laboratory of Environment Biomonitoring, Group of Fundamental and Applied Malacology (LEB/GFAM), University of Carthage, Faculty of Sciences of Bizerta (FSB), Laboratory of Environment Biomonitoring, Group of Fundamental and Applied Malacology (LEB/GFAM), 7021 Zarzouna, Bizerta, Tunisia; e-mail: [email protected]

Abstract: A study on the relative growth and reproductive cycle of the purple dye murex (Bolinus brandaris) was performed from March 2007 to February 2008 in two populations with contrasting imposex levels (Carthage Byrsa – CB in the small Gulf of Tunis and Menzel Abderrahmane – MA in the Bizerta Lagoon). Both populations presented balanced sex ratios. Comparison of allometric relationships established between linear and ponderal variables (sexes confounded) revealed higher growth in CB than in MA. Mature individuals were found throughout the year, except for September in MA. Spawning periods occurred from March-April to May and from June to August in both sites. Spawning was associated to an increase in seawater temperature. Besides increasing the current knowledge on the biology of B. brandaris from the southern Mediterranean, the information gathered in the present study constitutes a useful baseline for the sustainable management of local wild stocks, namely by prohibiting collection of snails during the spawning season. Key words: Bolinus brandaris; purple dye murex; Gastropoda; Muricidae; growth; reproductive cycle; imposex; Bizerta Lagoon; small Gulf of Tunis.

Introduction The purple dye murex, Bolinus brandaris (L., 1758), is a prosobranch neogastropod that inhabits on sandy and sandy-muddy bottoms down to 200 m depth (Poppe & Goto 1991; Martín et al. 1995; Houart 2001). This is a medium-sized species with total shell length up to 120 mm (Houart 2001). It is distributed throughout the Mediterranean Sea and in the NE Atlantic Ocean, from Morocco (as far south as Tangier) to Portugal (as far north as Cascais) (Martín et al. 1995). This muricid has been exploited since ancient times and used for producing a purple dye during the Roman Empire. Because of its high organoleptic quality, B. brandaris became a target species for artisanal fisheries along the coasts of France (Bartolome 1985), Spain, Italy and Turkey (Martín et al. 1995) and Portugal (Vasconcelos et al. 2008a). For example, in the Ria Formosa lagoon (southern Portugal), B. brandaris is greatly appreciated seafood with high commercial value (reaching values around 20 /kg) (Vasconcelos et al. 2008a). In Tunisia, especially after the impoverishment of the stocks of the clam Ruditapes decussatus (L., 1758) following overfishing, and also due to the prohibition of collection in several sites due to presence of biotoxins * Corresponding author

c 2012 Institute of Zoology, Slovak Academy of Sciences

in the flesh, the local consumption of B. brandaris increased and it started having higher commercial value. Like most neogastropods, B. brandaris is gonochoristic and has internal fertilization (Fretter & Graham 1994). During spawning, females lay eggs inside oothecas that attach to each other, forming large masses fixed to solid substrata on the sea bed (Ramón & Amor 2002). Like approximately 200 gonochoristic neogastropod species (Shi et al. 2005; Sternberg et al. 2010), B. brandaris is affected by imposex throughout its distributional range (Morcillo & Porte 1998; Solé et al. 1998; Ramón & Amor 2001; Chiavarini et al. 2003; Lemghich & Benajiba 2007; Vasconcelos et al. 2010, 2011; Abidli et al. 2009a, 2011, 2012). In B. brandaris sampled from two sites along the northern Tunisian coast (Bizerta Lagoon and small Gulf of Tunis), the stages of imposex development varied between VDS 1 and VDS 4.3, with all imposex indices being significantly higher in snails from the Bizerta lagoon (Abidli et al. 2011). Imposex frequency was also higher in gastropods collected from the Lagoon of Bizerta than from the small Gulf of Tunis (93.9% against 65.7%), corroborating TBT analysis in the whole tissues of female B. brandaris from the two collecting sites (Abidli et al. 2011).

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Fig. 1. Map of Tunisia showing the sampling sites of Bolinus brandaris: A – Bizerta Lagoon (Menzel Aberrahmane – MA); B – small Gulf of Tunis (Carthage Byrsa – CB).

Besides imposex induction, TBT can alter the reproduction and growth in some gastropod species. After an experimental exposure of Hexaplex trunculus (L., 1758) to TBT, Abidli et al. (2009b) detected that the number of spawning females decreased with increasing TBT concentration and that the egg-capsules laid in the control and low TBT concentration aquaria showed harder texture of the capsule wall compared to those exposed to high TBT concentration. These observations corroborated a previous study by Trigui El Menif et al. (2006) that showed that imposex development had an impact on the growth and fecundity of H. trunculus. While the biological, ecological and populational impacts of TBT on B. brandaris are well studied, little is known about its growth and reproductive biology, which are fundamental to the comprehension of the population dynamics. Among previous studies performed with this muricid species, we cite the fishery and biology (Bartolome 1985), the fishery and population structure (Martín et al. 1995; Vasconcelos et al. 2008a), growth of hatchlings and juveniles (Ramón & Flos 2001) and adults (Vasconcelos et al. in press), reproductive cycle (Ramón & Amor 2002; Vasconcelos et al. 2011) and gametogenesis (Amor & Durfort 1990; Amor et al. 2004). In the Mediterranean Sea, studies on the biology of B. brandaris are scarce and restricted to populations from the northern shore (Martín et al. 1995; Ramón & Flos 2001; Ramón & Amor 2002). On the southern shore, such type of investigations are not available, therefore, the main objective of this study

was to complete gaps of knowledge in the biology of B. brandaris from the southern Mediterranean. For this purpose, the relative growth and reproductive cycle of B. brandaris were described and compared between two populations from the northern Tunisian coast, collected at sites with different degrees of TBT pollution (Bizerta Lagoon and small Gulf of Tunis) and showing contrasting imposex frequencies and intensities. Material and methods Approximately 80–100 B. brandaris were collected monthly from March 2007 to February 2008 using fishing nets at two sites located in the small Gulf of Tunis and Bizerta Lagoon (Fig. 1). The collecting sites are situated in the western basin (Carthage Byrsa – CB in the small gulf of Tunis, and Menzel Abderrahmane – MA in the Bizerta Lagoon) and have depths around 20 m and 10 m, respectively. Surface seawater temperature was measured in both collecting sites at the time of sampling. In the laboratory, live individuals were frozen. After thawing, the shell length without siphonal canal (SLWS), shell breadth (SB) and shell aperture width (SAW) were measured to the nearest 0.1 mm using a vernier caliper. The shell was broken and the soft part of the organism was carefully removed and observed under a stereomicroscope. Sexual identification was based on the presence or absence of the vagina and capsule gland. After opening the mantle cavity, imposex analysis (including the measurement of penis length – PL) was performed as described by Abidli et al. (2009a, 2011). Macroscopic examination of the gonads in both sexes and of the capsule gland in females was

Growth and reproduction of Bolinus brandaris.

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Fig. 2. The purple dye murex Bolinus brandaris: A – Ventral view; B – Apex view; C – Soft parts of a female; D – Soft parts of a male. cg – capsule gland, dg – digestive gland, FP – female penis, g – gonad, MP – male penis, o – operculum, ot – ocular tentacle, vo – vaginal opening, Vc – visceral coil (complex digestive gland / gonad), vd – vas deferens, SLWS – shell length without siphonal canal, SAW – shell aperture width, SB – shell breadth, SC – siphonal canal, TSL – total shell length. Scale 1 cm.

Fig. 3. Macroscopic maturation stages based on gonads and capsule gland volume in Bolinus brandaris: A – immature gonad; B – male intermediate gonad; C – female intermediate gonad; D – male ripe gonad; E – female ripe gonad; F – small capsule gland of an immature female; G – intermediate capsule gland; H – large capsule gland of a mature female. cg – capsule gland, dg – digestive gland, g – gonad. Scale 1 cm.

Fig. 4. Visceral coil section: A – Female; B – Male. GA – Gonad area, DGA – Digestive gland area. Scale 1 cm.

performed to distinguish three maturity stages (immature, intermediate and mature) following Ramón & Amor (2002) (Figs 2–4). Briefly, in stage I (immature) the gonads of both sexes are indistinguishable from the digestive gland and females present an inconspicuous capsule gland; in stage II (intermediate) the gonads of both sexes are more developed and correspond approximately to one-third of the area of

the digestive gland; in stage III (mature / ripe) males show a well developed yellowish testicle corresponding to more than half of the area of the digestive gland, whereas females have a voluminous pinkish ovary and a large whitish capsule gland. The reproductive cycle was also analyzed using five bio-physiological indices on mature specimens (SLWS >

4 35 mm); it should be noted that maturation occurs around 30 mm SLWS, thus the choice of animals larger than 35 mm SLWS garantees that only sexually mature specimens are analyzed. For this purpose, after removing the operculum, the soft part of the organism was weighed (SPW). In females, the capsule gland was also separated and weighed (CGW). Then, the visceral coil (digestive gland/gonad complex) was dissected from the remaining soft parts directly under the stomach and photographed for measuring the gonad area (GA) and the area of the digestive gland/gonad complex (DGGA), using the software ImageJ 1.43u. Finally, meat dry weight and shell dry weight of each individual were determined after drying in the oven at 60 ◦C for 48 h. In both sexes, the bio-physiological indices were calculated using the following equations: general condition index, K = (meat dry weight / total dry weight) × 100; gonadosomatic index, GSI = (digestive gland-gonad complex dry weight / shell dry weight) × 100; gonad area index, GAI = GA/DGGA (Poore 1973; Vasconcelos et al. 2008b). In females, the capsule gland index was calculated through the equation: CGI = CGW/SPW (Giménez & Penchaszadeh 2003; Vasconcelos et al. 2008). In males, the penial index, PI = (PL/SLWS) × 100, was employed to ascertain the seasonal variation in penis length during the reproductive cycle following Vasconcelos et al. (2011). In order to describe relative growth, allometric relationships were established between morphometric variables [SLWS, SB, SAW, SPW and SW] by fitting a power function (Y = aX b ), where a and b are the parameters to be estimated, with b being the coefficient of allometry. In addition, to determine if b was different from 1 (linear parameters) or 3 (ponderal parameters), a Student t-test was performed following Mayrat (1959). Therefore, the type of growth between variables (negative allometry for b < 1 or b < 3; isometry for b = 1 or b = 3; positive allometry for b > 1 or b > 3) was determined. A chi-square test was employed to verify a balanced proportion (1M : 1F) in the sex ratio of both populations. Monthly variations in bio-physiological indices were analyzed by one-way ANOVA, after confirming data normality and homoscedasticity, using the software Statistica 8.0. Whenever ANOVA detected significant differences, post-hoc comparisons were made using the Newman–Keuls test. Statistical significance was considered for P < 0.05.

Results Seawater temperature The monthly variation in seawater temperature during the study period is shown in Fig. 5. In CB, the temperature ranged from 14.8 ◦C in February to 27.0 ◦C in July. In MA, extreme values were recorded in February (14.4 ◦C) and August (28.0 ◦C). Population sex ratio and size characteristics From a total of 1,028 and 1,103 individuals analyzed, in CB 510 were females and 518 were males, whereas in MA 520 were females and 583 were males. On the whole, both populations presented statistically balanced sex ratios (χ2 = 0.03, P > 0.05 in CB; χ2 = 1.71, P > 0.05 in MA). Most monthly samples presented balanced sex ratios, except May, June and November (female-biased) and July (male-biased) in CB, and January (in malebiased) in MA (Fig. 6). Both populations presented

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Fig. 5. Monthly variation in surface seawater temperature recorded in Carthage Byrsa (CB) and Menzel Abderrahmane (MA) during the study period (March 2007 – February 2008).

Fig. 6. Monthly variation in the sex ratio of Bolinus brandaris during the study period. Interrupted line represents balanced sex ratio (1M : 1F). Asterisks denote the monthly samples with sex ratios significantly different from parity (χ2 test, P < 0.05).

broad size ranges in SLWS, from 17.0 to 50 mm in CB and from 17.1 to 50 mm in MA. The frequency distribution of SLWS revealed that individuals from CB were more abundant at lengths between 30.0 and 42.0 mm (Fig. 7A), while in MA the highest frequency of individuals occurred between 28.0 and 38.0 mm (Fig. 7B). The comparison between sexes showed that males were more abundant than females especially in the size ranges 32.0–38.0 mm in CB and 30.0–34.0 mm in MA. Relative growth Both males and females from CB displayed positive allometry in the relationships SLWS versus SB, SAW and SPW, indicating that during B. brandaris ontogeny, SB, SAW and SPW grow at relatively faster rate than SLWS. In MA, the relationships SLWS vs. SB showed was isometric in both sexes, SLWS vs. SAW revealed a negative allometry in females and a positive allomety in males, while SLWS vs. SPW showed a positive allometry in both sexes. Concerning the relationship SLWS vs. SW, a negative allometry was registered in both sexes collected from CB and MA, indicating that B. brandaris grows faster in SLWS than in SW (Table 1). In CB, the comparison of the allometry coefficients of the relationships established between linear variables (SB vs. SLWS and SAW vs. SLWS) did not show significant differences (P > 0.05) between sexes, whereas in the population from MA, growth in SAW was higher in males than in females. The relationships between


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Fig. 7. Size frequency distribution (SLWS) of Bolinus brandaris during the study period. A – Carthage Byrsa; B – Menzel Abderrahmane.

Table 1. Allometric relationships established between morphometric parameters of Bolinus brandaris from Carthage Byrsa and Menzel Abderrahmane. Morphometric variables SB vs. SLWS SAW vs. SLWS SPW vs. SLWS SW vs. SLWS

y= r= SAW vs. SLWS y = r= SPW vs. SLWS y = r= SW vs. SLWS y = r=

SB vs. SLWS

Carthage Byrsa y= r= y= r= y= r= y= r=

0.713x1.023 0.967 0.201x1.186 0.917 3 × 10−6 x3.558 0.902 0.000x2.866 0.924

0.715x1.022 y = 0.971 F r= 0.198x1.190 y = 0.921 F r= 2×10−6 x3.615y = 0.914 F r= 0.000x2.850 y = 0.931 F r=

0.709x1.024 y = 0.963 M r= 0.205x1.180 y = 0.911 M r= 4×10−6 x3.480y = 0.884 M r= 0.000x2.891 y = 0.915 M r=

Menzel Abderrahmane y= r= y= r= y= r= y= r=

0.767x1.000 0.975 0.387x1.003 0.887 6 × 10−6 x3.341 0.901 0.000x2.890 0.945

0.796x0.989 y = 0.976 F r= 0.452x0.959 y = 0.875 F r= 5×10−6 x3.367y = 0.899 F r= 0.000x2.961 y = 0.945 F r=

t-test

P

2.18

P < 0.05

+/=

34.49

P < 0.001

+/–

120.27

P < 0.001

+/+

3.14

P < 0.005

–/–

P > 0.05

+/+/=/=

0.743x1.009 0.15CB 0.974 M 0.339x1.040 0.78CB 0.897 M 6×10−6 x3.31849.4CB 0.902 M 0.000x2.830 3.39CB 0.946 M

1.92MA

Type of growth

P > 0.05/ P < 0.001 20.36MA P < 0.001

+/+/+/+

15.73MA P < 0.001

–/–/–/–

6.59MA

+/+/–/+

Explanations: F – female; M – male; SLWS – shell length without siphonal canal; SB – shell breath; SAW – shell aperture width; SPW – soft parts weight; SW – shell weight; + positive allometry; – negative allometry; = isomet

linear and ponderal variables showed significant differences between males and females for SPW vs. SLWS and for SW vs. SLWS. In both sites, females grew more than males in these two morphometric variables (Table 1). The comparison between the two populations of B. brandaris (sexes confounded) showed that individuals from CB grew more in SB (t-test = 2.18, P < 0.05 and in SAW (t-test = 34.49, P < 0.001). Concerning the relationships between linear and ponderal variables, higher growth was recorded in CB for SLWS vs. SPW (t-test = 120.27; P < 0.001) and in MA for SLWS vs. SW (t-test = 3.14; P < 0.005). Accordingly, B. brandaris from CB grew more in both linear (SB and SAW) and ponderal parameters (SPW) than that from MA (Table 1). Reproductive cycle Both populations ofB. brandaris showed an annual re-

productive cycle. Mature individuals were found yearround, but at higher frequencies in CB. In MA, the highest percentages of ripe gonads were recorded in January (77.8% for females and 90.3% for males), whereas in CB, the frequency of mature individuals was higher in December, reaching 87.0% in females and 81.8% in males (Figs 8A–D). In both sites, females presented all three gonad maturation stages almost yearround, except for the absence of stage I (immature) in December, January and February in CB (Fig. 8A) and January in MA, and for the absence of stages II (intermediate) and III (mature) in September in MA (Fig. 8C). In CB, female maturation began in October and afterwards the frequency of more advanced maturation stages increased until April. Most ripe females were observed between December and April, preceding a spawning event between April and May (Fig. 8A). The main spawning period occurred from June to August and the resting phase between August and Oc-

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Fig. 8. Monthly variation in the frequency of gonad maturation stages in Bolinus brandaris during the study period. A, B – Carthage Byrsa; C, D – Menzel Abderrahmane. I – Immature, II – Active, III – Mature/Ripe.

tober (Fig. 8A). Male maturation started in September and then the percentage of mature individuals increased gradually until April, preceding two periods of gametic emission from April to May and from June to August (Fig. 8B). In males, the resting phase appeared to be very short and occurred between August and September (Fig. 8B). In MA, female maturation started in September and the highest frequencies of ripe females were registered from January to March and in June (Fig. 8C). Two spawning periods were observed in March-May and June-July and the resting phase occurred between July and September (Fig. 8C). In males, all maturation stages were recorded throughout the year (except for the absence of ripe gonads in September and immature gonads in January) (Fig. 8D). Male maturation began in October and continued until March. High frequencies of ripe gonads were observed mainly from January to March, preceding a short gamete release (between March and May). The main period of gametic emission was recorded from June to September and the resting phase occurred between August and October (Fig. 8D). The general condition index (K), presented similar trends in both sexes and significant monthly oscillations throughout the year, with the most pronounced variation occurring between June and July in both sexes. No significant differences were recorded between sexes (F = 0.0002; P = 0.98 in CB and F = 0.0005; P = 0.94 in MA) (Figs 9A, B). Spawning occurred twice in MA, from March to April and from June to August, as well as in CB, from April to May and from June to August. Significant differences in the K index between populations were recorded during April, June and October-February in females and during FebruaryJune and November in males (P < 0.05). The GSI and GAI indices showed similar temporal evolution and revealed synchrony in the gonadal devel-

opment of both sexes at the two collecting sites (Figs 9A–D). Gonad maturation occurred earlier in CB. Regarding GSI, no significant differences were recorded between sexes (F = 0.009; P = 0.92 in CB and F = 0.13; P = 0.71 in MA) (Figs 9C, D), while significant differences in GSI between populations were recorded during April–May and October–February in females and during April and October–February in males (P < 0.05). Concerning GAI, no significant differences were recorded between sexes (F = 0.49; P = 0.48 in CB and F = 0.046; P = 0.83 in MA), while significant differences in GAI between populations were recorded during April and November–December in females and during February–April and November in males (P < 0.05). Both sexes showed significant monthly oscillation in these indices, especially during the periods of maturation and gamete release (Figs 9C–F). In MA, spawning occurred from March to April and from June to August, against April–May and June–August in CB. In males, decreases in these indices are indicative of gamete release, but also of transfer of sperm from the testis to the seminal vesicle. The monthly variation in the capsule gland index (CGI) at the two sampling sites generally displayed similar trends as K, GSI and GAI. Significant decreases in CGI, which are indicative of spawning periods, occurred twice in both sites, from April to May and from June to August in MA, against March-May and June–August in CB (Fig. 9G). No significant differences in GAI were recorded between populations. The penial index (PI) presented monthly variation throughout the year in both populations, with a progressive decrease, indicative of gametic emission, recorded from March to August in MA and from April to August in CB (Fig. 9H). No significant differences in PI were recorded between populations.


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Fig. 9. Monthly variation in the general condition index (K), gonadosomatic index (GSI), gonad area index (GAI), capsule gland index (CGI) and penial index (PI) of Bolinus brandaris during the study period. Error bars represent standard deviation. Asterisks denote statistically significant differences (P < 0.05) between consecutive monthly samples, detected by one-way ANOVA followed by Newman–Keuls test.

Discussion The overall sex ratio is balanced in the two populations of B. brandaris. This observation agrees with those reported for B. brandaris populations collected from Vilanova i la Geltrú and Barcelona (Spain) (Ramón & Amor 2001). In contrast, the same authors (Ramón & Amor 2002), as well as others (Vasconcelos et al. 2011), reported that females were more abundant than males in populations of B. brandaris collected off Sant Carles

de la Rapit` a (Tarragona, Spain) and from the Ria Formosa lagoon (Portugal), respectively. Differences in the sex-ratios can be explained by distinct depth distribution between sexes that might affect their availability (Ramón & Amor 2002). Concerning the SLWS frequency distribution, results showed that B. brandaris larger size classes are more frequent in CB, where individuals were abundant at lengths between 30.0 and 42.0 mm, whereas in MA the most represented size classes were between 28.0 and

8 38.0 mm. These slight differences in size distribution between collecting sites can be also explained by differences in depth, because for the same species caught in Saint Carles de la R` apita (Catalan coast), Martín et al. (1995) showed that the size distribution was significantly correlated with depth. The present results corroborate Ramón & Amor (2002), who found that both sexes were distributed from 26 mm to 52 mm SLWS. Females were more abundant at lengths between 33 and 40 mm SLWS, but the size structure was similar in both sexes. This difference in shell length distribution was associated with depth, which varied between 15 m and 30 m (Ramón & Amor 2002). Concerning the differences in growth between these two populations of B. brandaris, it seems that growth features are potentially influenced by factors that differ considerably between sampling sites, namely exposure to currents, predation and degree of pollution. In fact, water circulation in the small Gulf of Tunis is controlled by currents and tidal waves that cause an intense coastal erosion (Ben Charrada, 1997). This site is exposed to heavy pollution due to urban, industrial and agricultural waste coming from the region of Tunis (Souissi et al. 2000). Regarding the Bizerta Lagoon, the intensity of water masses movement is normal given the presence of the tip of Echara` a that forms a barrier protecting the MA site (Frisoni et al. 1986). This site receives freshwater inflows through eight channels and is affected by various anthropogenic activities, including domestic sewage, industrial waste, atmospheric pollution, farmland and bivalve aquaculture. Except for shell weight, all the other morphometric parameters displayed higher growth in the population collected from CB. This difference in growth can be explained as an adaptative behavior against predation by crabs and sea stars, as suggested by Bourdeau (2009). In fact, this author showed that snails responded to crabs predation by increasing refuge use and producing a thicker and rotund shell, while in response to sea stars predation, snails increased refuge use but produced elongated shells with higher spires that allowed for greater retraction of the body. Similarly, the examination of the shell morphology of B. brandaris from the two sites revealed that those sampled from CB had bigger and larger spines, which increased shell breadth (authors, personal observation). Some authors associated differences in growth between gastropod populations with the imposex phenomenon. Indeed, in Italian populations of H. trunculus, Terlizzi et al. (1999) reported that those with advanced imposex stages have greater shell length and width, compared with normal and partially affected populations. In the same species, Trigui El Menif et al. (2006) and Lahbib et al. (2009) showed that individuals collected from Menzel Jemil (low incidence of imposex) grew more in terms of soft body weight and shell aperture, whereas gastropods from Bizerta Channel (high incidence of imposex) grew more in terms of shell weight. The present data corroborates these later findings, but contradict those of Terlizzi et al. (1999)

S. Abidli et al. concerning shell length and width. In fact, the population from the site with higher TBT pollution (MA) grew less in all morphometric parameters (except shell weight). After a laboratory exposure of live H. trunculus and empty shells to 5 and 50 ng TBT L−1 during five months, all females with high imposex level and the shells in the contaminated groups presented thinner and more fragile shells compared to the control group (authors, unpublished data). Thus, it is possible that TBT might influence the thickness of gastropod shells. In addition, Márquez et al. (2011) observed clear differences on shell shape and structure in the gastropod Odontocymbiola magellanica (Gmelin, 1791) from TBT polluted and non-polluted areas. In fact, these authors detected a loss in shell weight of 20% in animals from the TBT polluted area. Regarding the reproductive cycle of B. brandaris, results showed a reasonable agreement between the macroscopic classification of gonad maturation stages and the calculation of different bio-physiological indices. These results confirm previous findings by Vasconcelos et al. (2008b), who reported that biophysiological indices (especially GAI and CGI) constitute simple, practical and efficient indices for the routine assessment of reproductive activity. B. brandaris showed the same timing of ovary and testis development in both sexes, which were similar to B. brandaris caught in the Ria Formosa lagoon (southern Portugal) (Vasconcelos et al. 2011) and to H. trunculus collected from the Bizerta Lagoon (Lahbib et al. 2009), but distinct to those observed in the population from the Spanish coast (Ramón & Amor 2002). The annual cycle started with early maturation stages in September–October, afterwards the frequency of more developed maturation stages increased until March and April preceding small emission periods in MA and CB, respectively. The main emission periods occured between June and August in both sexes in CB, and from June to July in females and from June to August in males from MA. The resting phases appeared to be very short and occurred between August and September–October in both sexes in CB and between August and September in both sexes in MA. The slight asynchrony in gamete release between populations is probably related to local differences in seawater temperature, which in MA increased by 3.2 ◦C between March and April, while in CB it increased later, by 4.1 ◦C between April and May. However, this is just a hypothesis, because these values measured in situ at the time of sampling might not accurately reflect the real monthly variation in seawater temperature in both collecting sites. Lahbib et al. (2009) associated the asynchrony in the reproductive cycle between two populations of H. trunculus to differences in seawater temperature, which showed deviations above 2 ◦C between collecting sites. In Tegula eiseni (Jordan, 1936), the maturation and release or spawning times seem to be closely related to seasonal temperature rises and abrupt changes in surface sea water temperature (Vélez-Arellano et al. 2009).

Growth and reproduction of Bolinus brandaris. The comparison of the present results with data based on macroscopic maturation stages of B. brandaris from the Mediterranean Spanish coast (Ramón & Amor 2002) reveals a similar temporal evolution in the reproductive cycle. In that study, the reproductive cycle had two spawning peaks (April and June–July), but the first seems to be less important because in April immature females outnumbered mature females, and egg masses were only collected from the sea bed during June–July. Concerning the population of B. brandaris from the southern Portuguese coast (Atlantic Ocean), the results reported by Vasconcelos et al. (2011) based on K and GAI indices showed one main spawning season in June–July. This difference might be explained by differences in seawater temperature between the Mediterranean Sea and the Atlantic Ocean, with a longer warm season and higher seawater temperature in Tunisia than in southern Portugal. The comparison of the reproductive cycle of B. brandaris with that of H. trunculus from the Bizerta Lagoon (Menzel Jemil) reveals differences both in the number and period of gamete release. In fact, Lahbib et al. (2009) registered only one period in both sexes between January and May–June. Gharsallah et al. (2010) reported that in both sexes ofH. trunculus, gamete release occurred mainly from March to May (with a spawning peak between March and April), followed by a period of empty gonads between June and August. Moreover, another study on the reproductive cycle of H. trunculus from the Gulf of Gabès detected just one period of gametic emission in both sexes (January–May for males and April–May for females) (Elhasni et al. 2010). Overall, these studies confirm differences in the reproductive cycle of two species belonging to family Muricidae that coexist in the same environment. Besides seawater temperature, another factor that might influence the reproductive cycle is imposex. In H. trunculus from the Bizerta Channel, Lahbib et al. (2009) showed that advanced imposex stages and sterile females could have influenced the GAI because the gonad did not show normal development. The capsule gland index (CGI) of B. brandaris from the two sampling sites displayed the same general trend of the remaining bio-physiological indices. The same index was used by Lahbib et al. (2009) in two populations of H. trunculus and results also showed the same general trend of the GAI. The CGI based on capsule gland weight must be used with caution because in extreme cases of imposex this organ can be fissured (Trigui El Menif et al. 2006), therefore altering its weight. Trigui El Menif et al. (2006) reported that the frequency of individuals emitting egg-capsules (30%) was higher in the Menzel Jemil site (imposex incidence of 65.8%) than in the Channel of Bizerta (imposex incidence of 100%), where the proportion was only 12%. This difference was explained by partial or total obstruction of the female spawning orifice and by fissuration of the capsule gland. Lahbib et al. (2009) showed that variation in CGI was more irregular and presented more oscillations in the Bizerta Channel than in Menzel Jemil, because some

9 females of H. trunculus from the first site were sterile by closure of the vaginal opening and by fissuration of the capsule gland. Regarding the penial index (PI) used for assessing maturation and reproductive activity in male B. brandaris, results showed a significant decrease in JuneJuly coinciding with the main spawning period, but the small gametic emission was not detected by this index. Nevertheless, the present data corroborates previous findings that variation in B. brandaris male penis length is clearly related to sexual maturation, increasing continuously during the extended period of gonadal maturation and decreasing sharply after copulation and gamete release (Ramón & Amor 2002; Vasconcelos et al. 2011). In B. brandaris from the Ria Formosa lagoon, Vasconcelos et al. (2011) showed that monthly variation in PI was similar to that of GAI, i.e. intimately related to the dynamics of male reproductive cycle. Nevertheless, this index must be used cautiously in B. brandaris from highly TBT-polluted sites, since in Nucella lapillus (Linnaeus, 1758) it has been shown that TBT also increases male penis length (Castro et al. 2007), thus can alter seasonal variation in male PL during the reproductive cycle. The information gathered in this study suggests that environmental conditions in CB (including the lower level of TBT-pollution) appear to be more suitable for the growth and reproduction of B. brandaris than in MA. Nevertheless, due to the reasons mentioned above (differences in shell ornamentation between collecting sites), the results on relative growth should be carefully interpreted, especially those involving shell breadth and weight. Indeed, both abiotic and biotic factors (e.g. environmental conditions and predation) play a key-role in shell morphometrics and relative growth, probably overlapping and masking some effects that might result from TBT pollution and imposex. Although the use of bio-physiological indices provided satisfactory results, ideally they should be confirmed through histological analyses of the gonads. The improved knowledge on the species reproductive cycle can be used for proposing management measures of local wild stocks, namely by prohibiting harvesting during the main spawning season (June-August). In addition, these data provide helpful insights on the most adequate timing to induce spawning in captivity, which is valuable information to assess the potential of B. brandaris for aquaculture. Acknowledgements Authors are grateful to Dr. John Bulger (Canada) for the correction of the English text. We acknowledge the Editor and two anonymous referees for valuable comments and suggestions that greatly improved the revised version of the manuscript. References Abidli S., Lahbib Y. & Trigui El Menif N. 2009a. Imposex and genital tract malformations in Hexaplex trunculus and Boli-

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