THE MORPHOLOGICAL EXPRESSION OF IMPOSEX IN NUCELLA LAPILLUS (LINNAEUS) (GASTROPODA: MURICIDAE)

The prosobranch gastropod Nucella lapillus exhibits imposex(occurrence of male parts in addition to the female genitalduct) in response to tributyltin (TBT) pollution. Six stageswith three different types in the first three stages and twotypes in the last two stages can be distinguished and are documentedwith SEM-photographs for the first time. Furthermore, nine additionalalterations to the genital tract are shown. As a consequence of TBT exposure in early life stages a sexchange might occur. The oogenesis is suppressed and supplantedby spermatogenesis. The results are two observed specimens witha male genital tract (with a testis) and different female parts.Anatomical and histological details are given here. If TBT-expositionoccurs later, the vagina is replaced by a small prostate glandor the vaginal opening is occluded by proliferating tissue. The two established indices for PSH monitoring (VDS and RPS)in muricid gastropods are discussed. It is shown that the VDSis the more valid parameter. (Received 2 January 1991; accepted 1 March 1991)     

Comparison of imposex response in three Prosobranch species

A comparative study of the three gastropod species Nucella lapillus (L.), Ocenebra erinacea (L.) and Hinia (Nassarius) reticulata (L.) reveals that Nucella is the most TBT sensitive species while Hinia is the least sensitive. Of the two imposex indices VDSI and RPSI, good interspecies correlations were obtained only for VDSI. The three species can be considered as complementary not only in terms of their ecology but also for their levels of sensitivity. Indeed, Nucella and Ocenebra are useful test species at TBT concentrations below 2 ng Sn 1⁻¹ while Hinia is the more appropriate species at higher TBT levels. For the first time, two sterilised Hinia females are recorded. This sterilization does not seem to be due to proliferation of vas deferens tissue in the vaginal opening and further investigation is needed to find intermediary VDS stages between stage 4⁺ and sterilization. Studies using Nucella show that the use of narcotization in imposex analysis leads to an underestimation of RPSI compared with non-narcotization methods. Indeed, narcotization straightens the penis and increases its length when compared with non-narcotized animals, but this increase is proportionally higher in males than in females.     

TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level

Tributyltin (TBT) exposure at different concentrations (5, 60, and 100 ng TBT as Sn/l) induces a concentration- and time-dependent imposex (=pseudohermaphroditism) development in femaleNucella lapillus andHinia reticulata. In both species the average imposex stage, termed as vas deferens sequence (VDS) index, and the average female penis length increases with increasing TBT concentration and duration of TBT exposure. Testosterone added at a concentration of 500 ng/l induces a faster and more intensive imposex development compared to that induced by the TBT concentrations used in the present experiments. Radioimmunological determination of endogenous steroid content reveals increasing testosterone titres in female gastropods exposed to TBT which correlate with the TBT concentration used and the duration of the experiment. The most marked and highest increase of the endogenous testosterone level is exhibited by females, of both species exposed to testosterone. Simulataneous exposure to TBT and to the antiandrogen cyproterone acetate which suppresses imposex development completely inN. lapillus and reduces imposex development strongly inH. reticulata proves that the imposex-inducing effects of TBT are mediated by an increasing androgen level and are not caused directly by the organotin compound itself. Further-more, TBT-induced imposex development can be suppressed in both snails by adding estrogens to the aqueous medium. These observations suggest that TBT causes an inhibition of the cytochrome P-450 dependent aromatase system which catalyses the aromatization of androgens to estrogens. The increase of the androgen content or the shift of the androgen-estrogen balance in favour of androgens induces the development of pseudohermaphroditism in marine prosobranchs. Artificial inhibition of the cytochrome P-450 dependent aromatase system using SH 489 (1-methyl-1,4-androstadiene-3,17-dione) as a steroidal aromatase inhibitor and flavone as a nonsteroidal aromatase inhibitor induces imposex development inN. lapillus as well as inH. reticulata.     

Effects of tributyltin (TBT) and testosterone on the female genital system in the mesogastropodLittorina littorea (Prosobranchia)

Experiments were performed with the mesogastropodLittorina littorea on Helgoland, in Roscoff, and in the laboratory in order to evaluate the reaction of the female genital system to TBT, an environmental toxicant. The snails were either injected with 50 or 100 ng tributyltin (TBT) soluted in ethanol or exposed to artificial sea water treated with 5, 50, 100, and 200 ng TBT/l, and 33 ng testosterone/l. The duration of the experiments was either four or eight weeks. None of the analysed femaleL. littorea showed signs of imposex. Compared to results for the control groups, the size of the female glandular complex was significantly reduced if the pre-experimental toxication was already high, as is the case in snails collected around Helgoland. TBT-related gland complex reduction occurs also in femaleL. littorea from other sampling sites. In addition, injection of ethanol also causes a decrease in gland size. The experimental results demonstrate that the distal female genital system responds with significantly lower sensitivity to TBT than that of other prosobranchs. This behaviour is ascribed to the lack of an androgen receptor at the ovipositor. The results further strengthen the case of the extreme rarity of imposex described for femaleL. littorea in natural habitats. However, strong TBT-toxication may affectL. littorea populations significantly because of increasing masculinization of the females, which reduces reproduction ability.     

Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera

To clarify how tributyltin (TBT) and triphenyltin (TPT) interact with the retinoid X receptor (RXR) to induce growth of male sex organs in female gastropods, we treated female rock shells (Thais clavigera) with three different concentrations (0.1, 1, or 5 μg/g wet wt) of 9-cis-retinoic acid (9CRA) or with a single concentration (1 μg/g wet wt) of TBT, TPT, or fetal bovine serum (as a control). The effects of each treatment were measured as the incidence of imposex, the length of the penis-like structure, and the vas deferens sequence (VDS) index. 9CRA induced imposex in a dose-dependent manner; imposex incidence was significantly higher in the rock shells that received 1 (P < 0.05) or 5 μg (P < 0.001) 9CRA than in the controls. After 1 month, the rock shells treated with 5 μg 9CRA exhibited substantial growth of the penis-like structure that was not as evident in the other treated shells. The length of the structure differed between the 0.1- and 5-μg 9CRA treatment groups (P < 0.05) but not between the 1- and 5-μg 9CRA treatment groups (P > 0.05). Compared with the control, the VDS index increased significantly in the 1- (P < 0.05) and 5-μg (P < 0.001) 9CRA groups. The penis-like structures behind the right tentacle in female rock shells treated with 5 μg 9CRA were essentially the same as the penises and vasa deferentia of normal males and of TBT-treated or TPT-treated imposexed females. These results further support the hypothesis that imposex in gastropods could be mediated by RXR.     

Imposex in Hexaplex trunculus at Some Sites on the North Mediterranean Coast as a Base-Line for Future Evaluation of the Effectiveness of the Total Ban on Organotin based Antifouling Paints

Imposex – the superimposition of male sexual organs (penis and vas deferens) onto female Neogastropods such as Hexaplex trunculus (Linné, 1758) – is used world-wide as a biomarker of ecological impact of organotin based antifouling biocides (TBT and TPhT). To limit the impact of organotin pollution, since January 1, 2003, the International Maritime Organization (IMO) has enacted a global ban on the use of organotin compounds in antifouling systems. It is important to record imposex levels and organotin contamination before the implementation of the ban, in order to assess the current situation and be able, in the future, to verify the effects of the International Protocol. In this paper, recent imposex data measured in populations of Hexaplex trunculus from three different Mediterranean regions are compared: the Ligurian Sea (Italy), the Lagoon of Venice (Italy) and the western coast of Istria (Croatia). In the two former locations, a partial ban on TBT has been in force for vessels less than 25 m since 1982, while in the latter region no restrictions on organotin antifouling paints have been applied yet. Gastropod samples collected from the Venice lagoon were analysed with an acid extraction followed by Grignard derivatisation, clean up and GC-MS determination, in order to relate the levels of TBT, TPhT and their metabolites with the imposex degree detected. Biological data show that the levels of imposex were very high (VDS from 4.3 to 5) in all the sampling sites considered, particularly in the Croatian coast stations. The concentrations of organotin compounds – butyltins and phenyltins – measured in the samples from the lagoon of Venice were found to partition differently in the visceral coil and in the rest of the soft body of the analysed organisms.     

On the hoplonemertean,Kameginemertes parmiornatus (Iwata, 1957) gen. n., comb. n. from Sagami Bay,Japan

Two different tributyltin (TBT)-induced virilisation phenomena in prosobranch snails – intersex in Littorina littorea and imposex in Nucella lapillus – are compared in order to facilitate their simultaneous use in geographical large scale effect monitoring surveys. Imposex in dogwhelks is a more sensitive biomarker and should be used in areas that are only slightly or moderately contaminated with TBT (ambient TBT concentrations < 2.0 ng as Sn l-1). The assessment of intersex intensities in periwinkle populations has considerable advantages in areas with higher TBT concentrations and should be used also wherever dogwhelks are absent irrespective of the TBT exposure level. The intersex index (ISI) and vas deferens sequence (VDS) index are proposed as the most suited parameters for effect monitoring purposes. The geographical uniformity of intersex and imposex is analysed and proven for the coasts of Ireland, France, and Germany. A relative loss of TBT sensitivity in females can be found, but to a varying extent in both species. The implications of this result for biological TBT effect monitoring programmes are discussed in light of the fact that intersex and imposex have both been found to be irreversible. Because it is the objective of these programmes to assess current TBT contaminations and resulting biological effects, only relatively young specimens should be considered in the sampling strategy.     

Ultrastructure of oogenesis in imposex females of <Emphasis Type="Italic">Babylonia areolata</Emphasis> (Caenogastropoda: Buccinidae)

During a tributyltin (TBT)-exposure experiment, the ultrastructural features of oogenesis have been examined in TBT-induced imposex females of Babylonia areolata and compared with those of the normal female. The results obtained from such experiment demonstrates that B. areolata exhibits a low to moderate intensity of imposex because all VDSI values are never higher than 3. Ultrastructures of germ cell development including oogonia, pre-vitellogenic, early vitellogenic, late vitellogenic and mature oocytes show that oogenesis in imposex female is similar to that of normal females except for the presence of numerous lipid droplets in the cytoplasm of the oocytes and the follicle cells in imposex females, indicating the degeneration of their oocytes. Vitellogenesis in B. areolata involves both auto- and heterosynthetic processes that resemble those of the basal gastropods and the pulmonates. In addition, the presence of cortical granules and microvilli are unique structures of this species.     

New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in <Emphasis Type="Italic">Hexaplex trunculus</Emphasis> (Gastropoda,Muricidae) with different stressors

Imposex, i.e. the development of additional male sex organs (penis and/or vas deferens), in females of gonochorist marine and freshwater gastropods, is known to be caused by tributyltin (TBT), and it has been widely used as a biomonitoring tool in environmental surveys for TBT pollution assessment. In this study, we experimentally tested the potential to induce imposex by another endocrine disruptor (polychlorinated biphenyls [PCBs] mixture—Aroclor 1260). Adults of Hexaplex trunculus with low imposex level, coming from an Italian Marine Protected Area, were injected separately with different doses of tributyltin chloride (TBTCl) and Aroclor 1260. The compounds were dissolved in ethanol and the organisms were narcotised by immersion in MgCl₂ solution before injection. Before and after the experiment, butyltin compounds (BuTs) and PCB tissue concentrations were determined. A significant increase in imposex with respect to non-treated organisms was observed in all treatments, including artefact controls. No clear correlation was observed between BuTs and PCB tissue concentrations and indices of imposex incidence. Based on these results, no assumption can be formulated about PCB effect on imposex development. Nevertheless, they suggest that the imposex level increase, at least in H. trunculus, in laboratory conditions might not be caused by TBT only, but it would rather be a non-specific response to different stress stimuli.     

Acute toxicity of tributyltin to encapsulated embryos of a marine gastropod

The marine gastropod Buccinanops globulosus is known to have high imposex incidence in areas moderately polluted by tributyltin (TBT). Acute toxicity was previously studied in adults but no information is known about embryonic intracapsular development. To estimate the potential effects of organotin pollution on the progeny of B. globulosus, acute toxicity tests were conducted on encapsulated and excapsulated pre-hatching embryos. The lethal median concentration estimated for 96?h (LC₅₀ 96?h) in B. globulosus excapsulated embryos was 196.70?µg?TBTCl?L^?1, while in encapsulated embryos it was 2951.28?µg?TBTCl?L^?1. The LC₅₀ 96?h was 15-fold higher for encapsulated embryos compared to excapsulated embryos, denoting egg capsule protection against pollutants from the external environment. Our results show that TBT pollution can have significant effects in molluscs other than the chronic effect of imposex.     

Tributyltin-induced imposex in stenoglossan gastropods: Pathological effects on the female reproductive system

• 1.1. The imposition of male sex organs on female stenoglossan gastropods—“imposex”—is a worldwide phenomenon now known to be associated with the usage of marine antifouling paints containing tributyltin (TBT) compounds.
• 2.2. The effect of the imposex response on the female reproductive system varies according to species: in some, breeding appears unaffected, whilst in others the anatomy of the oviduct may be so modified that the female is effectively sterilised.
• 3.3. New evidence suggests that TBT interrupts steroid metabolism, notably the conversion of testosterone to oestradiol 17β.

     

Beyond imposex: other sexual alterations in a nassariid snail from Patagonia

The occurrence of a penis in female gastropods has commonly been accepted as an indicator of tributyltin (TBT) pollution. During the surveys performed in Patagonian waters between 2002 and 2010, individuals of Buccinanops deformis with a short penis and an external vas deferens were initially considered as showing imposex. Doubt arose when different manifestations of the phenomenon were identified at the two sites with the densest populations of the species (San Antonio Bay, SAB, and Villarino). This led us to search at both locations for: (a) histological confirmation of sex, (b) butyltin measurements in sediments and snail tissues, and (c) incidence of trematode infestation. We found that both populations have individuals with a short penis. While all the inspected individuals from SAB were histologically identified as females (imposex), those from Villarino were identified as males. Trematodes were absent in individuals from SAB and showed very low prevalence in those from Villarino, with metacercariae found in the digestive gland. In snail tissues, butyltins were found mainly in the form of dibutyltin (DBT) in SAB and at negligible levels in Villarino. In sediments, butyltins were found only in the form of TBT, at higher levels in SAB than in Villarino. Thus, it became clear that our finding of a short penis is a manifestation of imposex on females from SAB related to the high TBT levels, whereas in Villarino it is a male trait, which is not related to TBT or a trematode infestation but may be a Dumpton syndrome-like abnormality.     

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel,Nucella lapillus

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this ‘nonmodel’ species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator–activated receptor (PPAR) pathways. Application of rosiglitazone, a well‐known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT‐induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR‐responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.     

Imposex expression in Hexaplex trunculus from the North Tunis Lake transplanted to Bizerta channel (Tunisia)

Adult specimens of the muricid gastropod Hexaplex trunculus were collected from their natural habitat: the Lake Club station located in the North Tunis Lake, a sampling site characterized by the absence of shipping activity and an imposex incidence of 12.9%. These specimens were transplanted to a sampling site with intense shipping traffic: the Bizerta channel, where a preliminary survey detected an imposex incidence of 100%. The transplanted specimens were tagged prior to release and periodical recaptures were made every 60 days during a study period of 8 months (from July 2004 to March 2005) in order to compare the imposex development between both sampling sites. After 5 months of exposure in the Bizerta channel, all transplanted females were affected by imposex (with the development of an incomplete male genital tract). Microscopic examinations showed that imposex begun by the development of a small portion of vas deferens located halfway between the expected position of the future penis and the vagina. The imposex indices used in this study were the imposex frequency (I (%)), the vas deferens sequence index (VDSI), the relative penis length index (RPL), the average female penis length index (FPL) and the average female vas deferens length (VDL). All these indices increased in the transplanted population as compared to the population from the original sampling site, but VDSI and VDL were considered the best indices to assess the environmental pollution by tributyltin (TBT) and its deleterious effects in H. trunculus.     

Distribution and bioaccumulation of butyltins in the edible gastropod Odontocymbiola magellanica

Butyltins (BTs) were found in sediments and body tissues of the edible gastropod Odontocymbiola magellanica, in which imposex has been recorded since 2000. BTs in sediments ranged from < MDL to 174.8?ng (Sn) g^?1 for TBT, < MDL to 19.2?ng (Sn) g^?1 for DBT, and < MDL to 71.8?ng (Sn) g^?1 for MBT. In body tissues BTs varied from??1 for TBT, DBT and MBT, respectively. BT concentrations were higher in gonads and digestive glands than in the albumen gland and foot (edible). The highest concentrations of BTs in both sediments and gastropods were found in the harbour area, decreasing with distance to the harbour and areas with less maritime traffic. The Biota-Sediment Accumulation Factor (BSAF) in the different organs was between 0.02–0.42, 0.09–0.35 and 0.08–5.25 for TBT, DBT and MBT, respectively. There were positive correlations between concentrations of BTs in sediments and gastropod body tissues, suggesting that xenobiotic accumulation in O. magellanica occurs mainly through contaminated sediments, rather than water or the food chain. Considering current sediment quality guidelines, our results indicate that acute toxic effects would be expected from TBT exposure, which represents a serious environmental threat for the benthic community. Although the levels of BTs found in the foot of this edible gastropod did not exceed the recommended Tolerable Daily Intake in polluted areas, they should be monitored to ensure the safety of seafood consumers. The alternative antifouling biocides Irgarol and Diuron were not detected in sediments.     

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.     

Description of imposex and butyltin burden in Nassarius mutabilis from the lagoon of Bizerta (northern Tunisia)

Imposex occurrence in the changeable nassa Nassarius (Sphaeronassa) mutabilis (Linnaeus, 1758) was confirmed in the present study. Advanced imposex stages were discovered for the first time in this nassarid starting from the initial appearance of the vas deference sequence or the bud penis (VDS1), to the point where this duct reaches the vaginal opening (VDS4). Three types of imposex pathways were found for stage 1 (VDS1a, VDS1b and VDS1d), two for stage 2 (VDS2a and VDS2d) and stage 3 (VDS3a and VDS3b). Affected females were found in the three studied stations with imposex frequency ranging from 10.5 to 47.2%, VDSI from 0.1 to 0.8, FPL from 0 to 0.1 mm, RPLI from 0 to 0.9, and VDL from 0.2 to 1.3 mm. Organotins body burden ranged from 0.8 to 16 ng/g d.w. for MBT, 2.5–14.9 for DBT, and 3.7–14.4 for TBT. To our knowledge, this is the first detailed report on imposex in N. mutabilis suggests its inclusion to list of imposex-affected gastropods.     

The Neuropeptide APGWamide as a Penis Morphogenic Factor (PMF) in Gastropod Mollusks

The goal of this study was to further investigate the role ofendogenous APGWamide levels in imposex induction in snails.APGWamide is a common neurotransmittor/neuromodulator peptidefound in many species of molluscs, and is often related to sexorgan growth or reproductive behavior. Mud snails (Ilyanassaobsoleta) were collected from the Rachel Carson Estuarine Reservenear Beaufort, NC, and were dosed with the environmental contaminanttributyltin (TBT), testosterone (T), or solvent vehicle (EtOH/saline) controls. Both TBT and T have been shown previouslyto induce female snails to grow penises (a condition termedimposex), and to increase male penis size. Male normalized penislength was correlated to endogenous APGWamide levels (as measuredby Western blotting of whole animal homogenates, r² = 0.475),and control males had significantly higher APGWamide levelsthan control females. All TBT-treated animals, (male, female,and imposex) had levels of APGWamide similar to control malesand significantly higher than control females. In testosteronetreated animals, APGWamide levels were the same as controlsand it is likely that testosterone interferes with a downstreamsignaling event to induce imposex. In addition, immunohistochemistryfor APGWamide expression in abdominal areas was done on female,male and imposex snails collected from the wild. The patternof APGWamide in imposex snails was similar to male snails, showinglarge patches of immuno-reactive areas in the top portion ofthe visceral mass. In female snails, no areas of cross-reactivitywere found.     

The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta

We investigated whether neuropeptides which control sexual differentiation in mollusks can induce imposex-a condition where female snails grow male accessory sex organs after exposure to tributyltin (TBT). Mud snails, Ilyanassa obsoleta, were dosed with one of four neuropeptides: APGWamide, conopressin, LSSFVRIamide, or FMRFamide for seven or fourteen days. TBT and testosterone (T) were used as positive controls and induced imposex as expected. APGWamide significantly induced imposex, with a threshold dose near 10(-)(16) moles. The other neuropeptides had no effect on imposex induction. We propose that TBT could act as a neurotoxin to induce imposex via abnormal release of APGWamide.