Variability of imposex development and reproductive alterations in the Patagonian gastropod Buccinanops globulosus inhabiting a polluted harbourarea

The aim of this work was to study the reproductive output and imposex parameters of the caenogastropod Buccinanops globulosus inhabiting a harbour area, with high maritime traffic and pollution. The results showed higher imposex incidence for non-gravid females than for gravid females (carrying egg capsules). Among imposex-affected females, mean penis size was significantly larger in non-gravid (3.4 mm) than gravid (1.5 mm) females. In B. globulosus usually a single juvenile hatches per egg capsule, but in this study capsules with multiple embryos were found. Overall, these results suggest a relationship between imposex development and alterations on the reproductive output of B. globulosus females living in adverse environmental conditions (polluted area).


INTRODUCTION
Harbour activities, in particular maritime traffic, imply the release of toxic substances such as tributyltin (TBT) (Fent, 2003). This toxic compound can be accumulated, persisting in the sediments for up to 2.5 years (de Mora et al., 1995). In female caenogastropods, the neoformation of a penis or vas deferens in individuals exposed to TBT is known as imposex (Gibbs & Bryan, 1996). Because imposex is irreversible (Stroben et al., 1992) researchers worldwide have used this phenomenon as a marine pollution biomarker associated to maritime traffic (Axiak et al., 2003, Ketata et al., 2008. In areas highly polluted with TBT, sterile females and popula-tion declines have been observed (Oehlmann et al., 2007). In Argentinean harbour areas, several gastropod species have been reported to be affected by imposex, including Buccinanops globulosus, and its congeners B. monilifer and B. cochlidium (Penchaszadeh et al., 2001, Bigatti et al., 2009, Averbuj & Penchaszadeh, 2010a. Pollutants such as TBT, heavy metals and polyaromatic hydrocarbons (PAHS) were detected in Nuevo Gulf at north Patagonia, Argentina (Gil et al., 2006, Massara Paletto et al., 2008, Bigatti et al., 2009. The highest concentrations of TBT were reported in the harbour area of Nuevo gulf, with up to 174.81ng (Sn).g -1 in sediments and 345.27ng (Sn).g -1 in body tissues of the gastropod Odontocymbiola magellanica (del Brío, 2011 Buccinanops globulosus presented high values of imposex parameters in moderately polluted areas with TBT concentrations of 0.4 ng (Sn).g -1 dry sediment; thus, it was classified as a good indicator due to its high sensitivity to low concentrations of TBT (Bigatti et al., 2009). Buccinanops globulosus lives mostly buried in sandy or muddy bottoms (Scarabino, 1977). This species is edible and is part of an expanding artisanal fishery (Narvarte, 2006, Averbuj et al., 2014. Adult females spawn once a year a variable number of egg capsules that are attached to their own shells (Penchaszadeh, 1971). Generally one B. globulosus crawling juvenile, measuring between 3-7 mm, hatches from each egg capsule (Penchaszadeh, 1971, Narvarte, 2006, Averbuj et al., 2014.
The aim of this work was to study the reproductive output and imposex parameters of B. globulosus inhabiting a polluted area.

MATERIALS AND METHODS
Females of B. globulosus were collected during February 2012 when egg capsules with embryos in late stages of development are most abundant (Averbuj et al., 2014).The "gravid females" occur mainly from October to March, when the water temperature ranges between 11°C and 18°C (Averbuj et al., 2014). This work was performed at Luis Piedra Buena Harbour (LPBH, 42º45ˈ45" S and 64º1ˈ51" W), a high maritime traffic area with ~730 vessels per year (APPM, 2013). In this area sporadic catches of B. globulosus are carried out for human consumption.
Buccinanops globulosus individuals were captured by SCUBA diving, then placed in glass aquaria and sexed by presence (female) or absence (male) of the accessory glands. The gastropods were counted and classified into three categories: female, male and gravid female (carrying egg capsules). This species was chosen considering its sensitivity to TBT and the fact that the egg capsules are attached to the female's own shell (Penchaszadeh, 1971, Averbuj & Penchaszadeh, 2010b, allowing for a straightforward estimation of reproductive output of each gravid female. Total shell length (TSL), female penis length (FPL) and male penis length (MPL) were measured. The imposex percentage (I%), was estimated and the relative penis length index (RPLI) was calculated according to (Gibbs & Bryan, 1994). Reproductive output was estimated for gravid females (Fig. 1) when embryos were at hatching stage (Averbuj et al., 2014). The fol-lowing parameters were considered for reproductive output: a) number of egg capsules per female (# EC), b) number of embryos per egg capsule, c) total number of embryos per female and d) mean embryo size per female. Egg capsules with lysed eggs/embryos or undeveloped embryos were not considered but special attention was taken for cases of embryo malformation. To estimate the mean embryo size (d), 10 hatchling embryos were randomly removed from each of 15 gravid females and measured to determine embryo shell length (ESL) under a stereomicroscope. The Mann-Whitney test was used to test for differences in the mean female shell length and mean female penis length (FPL), between gravid and non-gravid imposex females. In all cases the STATISTICA 7 ® software was used.

RESULTS
A total of 67 individuals of Buccinanops globulosus were studied. The minimum TSL of gravid females was 37.78 mm and the total shell length (TSL) was not significantly different (U=243.5; p=0.713; n=43) between gravid (41.88±2.40 mm) and non-gravid (41.58±2.56 mm) females. Imposex parameters (I%, FPL and RPLI) were higher in females without egg capsules than in gravid females (Table 1). Furthermore, all females without imposex incidence were gravid. Penis length was significantly larger in non-gravid than gravid females (U=44.0, p<0.0001, n=41) ( Table  1). None of the studied females were sterilized or exhibited an occluded gonopore.
On average 47.89±2.7 egg capsules occurred per female, with 56.73±4.3 total embryos per females and 1.19±0.07 embryos per egg capsule. A high proportion (74%) of gravid females carrying egg capsules with multiple embryos (ME: >1 embryo/capsule) was recorded. On average, 17.3% of the egg capsules contained multiple embryos. The mean embryo shell length (ESL) was

DISCUSSION
The reproductive output, defined as the mean number of embryos (and their sizes) spawned per female, has been shown to be susceptible to variability in different environmental conditions (Ramirez Llodra, 2002). The effects of pollutants on gastropod reproductive output has been associated with histological changes in gonads and aborted egg capsules in females (Gibbs et al., 1991, Axiak et al., 2003, Oetken et al., 2004. Although generally in B. globulosus only one embryo is developed per egg capsule (Penchaszadeh, 1971), a population recently studied in Golfo Nuevo showed 0.22% of egg capsules with multiples embryos (Averbuj et al., 2014). Our results showed that gravid females presented on average a large proportion of egg capsules with multiple embryos (17.3%). Differences in imposex parameters between gravid and non-gravid females are remarkable. Imposex-affected gravid females showed significantly smaller penis than nongravid ones, suggesting that the later may be more severely affected by imposex, since exposition to the endocrine disrupter tributyltin (TBT) induces a concentration -and time-dependent imposex development in gastropod females (Bettin et al., 1996). Similarly, in B. monilifer from Mar del Plata (northern Argentina), females that succeed in spawning showed significantly lower imposex incidence (imposex % and penis length) than the rest of the female population (Averbuj & Penchaszadeh, 2010a). It is important to note that the larger penis recorded in non-gravid females were not related to female sterilization by gonopore occlusion, although it clearly evidence major imposex affection. This suggests either major exposure or individual sensibility to TBT (and probably other pollutants that might affect reproductive output) in B. globulosus.
Higher penis development infemales with reduced reproductive capacity may be suggesting an alteration of TBT on the reproductive physi- ology, in particular affecting gonadic proceses (Ramirez Llodra, 2002, Oehlmann & Schulte-Oehlmann, 2003. The studied population of B. globulosus could be negatively affected, limiting the number of females capable of reproduction and, complementarily altering its reproductive output under unfavorable environmental conditions as reported for other gastropods species (Marshall et al., 2008, Chatzinikolaou & Richardson, 2010, Collin & Spangler, 2012. This pattern, evident in the genus Buccinanops due to its particular breeding behavior, may be occurring in other gastropod species inhabiting similar polluted environments.