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.     

Testosterone-Fatty Acid esterification: a unique target for the endocrine toxicity of tributyltin to gastropods

Over the past thirty years, a global occurrence of sexual aberrationhas occurred whereby females among populations of prosobranchsnails exhibit male sex characteristics. This condition, calledimposex, has been causally associated with exposure to the biocidetributyltin. Tributyltin-exposed, imposex snails typically haveelevated levels of testosterone which have led to the postulatethat this endocrine dysfunction is responsible for imposex.This overview describes recent evidence that supports this postulate.Gastropods maintain circulating testosterone levels and administrationof testosterone to females or castrates stimulates male sexdifferentiation in several snail species. Studies in the mudsnail (Ilyanassa obsoleta) have shown that gastropods utilizea unique strategy for regulating free testosterone levels. Excesstestosterone is converted to fatty acid esters by the actionof a testosterone-inducible, high capacity/low affinity enzyme,acyl-CoA:testosterone acyl transferase, and stored within theorganisms. Free testosterone levels are regulated during thereproductive cycle apparently due to changes in esterification/desterificationsuggesting that testosterone functions in the reproductive cycleof the organisms. Testosterone esterification provides a uniquetarget in the testosterone regulatory machinery of snails thatis altered by tributyltin. Indeed, imposex and free testosteronelevels were elevated in field collected snails containing hightin levels, while testosterone-fatty acid ester pools were reducedin these organisms. These observations indicate that tributyltinelevates free testosterone by reducing the retention of testosteroneas fatty acid-esters. This endocrine effect of tributyltin maybe responsible for imposex.     

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.     

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.     

THE ESTUARINE MUD SNAIL, NASSARIUS OBSOLETUS: ABNORMALITIES IN THE REPRODUCTIVE SYSTEM

Females of the dioecious snail Nassarius obsoletus may havethree male characteristics: a penis, a vas deferens, and theconvolution of a normally straight gonadial oviduct to resemblea stunted mimic of the vesicula seminalis in the male. The firstcharacteristic is the most common and the last is the leastcommon. Collectively these characteristics were called imposex.A system for evaluating its frequency of occurrence and intensityof expression was developed. The frequency and intensity ofimposex was similar among different age groupings of snails,though there may be a trend towards less imposex in juveniles.The differences in the frequency and intensity of imposex betweengroups of parasitized and unparasitized females were very small.In males the reduction of penis size due to parasites was muchlarger. This pattern ofimposition of male characteristics onfemales is a new syndrome which does not resemble reported formsof hermaphroditism or pseudohermaphroditism related to age orparasitism. ^*Current address: Community Health Program, Electric Power ResearchInstitute, P.O. Box 10412, Palo-Alto, California 94303, U.S.A. (Received 28 August 1979;     

Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds

Molluscs can conjugate a variety of steroids to form fatty acid esters. In this work, the freshwater ramshorn snail Marisa cornuarietis was used to investigate sex differences in endogenous levels of esterified steroids. Testosterone and estradiol were mainly found in the esterified form in the digestive gland/gonad complex of M. cornuarietis, and males had higher levels of esterified steroids than females (4-10-fold). Additionally, the ability of several xenobiotics, namely tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN) to interfere with the esterification of testosterone and estradiol was investigated. All three compounds induced imposex - appearance of male sexual characteristics in females. Exposure to TBT led to a decrease in both esterified testosterone (60-85%) and estradiol (16-53%) in females after 100 days exposure, but had no effect on males. Exposure to FEN and MT did not alter levels of esterified steroids in males or in females, although exposed females developed imposex after 150 days exposure. The decrease in esterified steroids by TBT could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, which catalyzes the esterification of steroids. In fact, ATAT activity was marginally induced in organisms exposed to TBT for 50 days (1.3-fold), and significantly induced in males and females exposed to MT for 50 days (1.8- and 1.5-fold, respectively), whereas no effect on ATAT activity was observed after 150 days exposure.     

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.     

The rough tingleOcenebra erinacea (Neogastropoda: muricidae): An exhibitor of imposex in comparison toNucella lapillus

The muricid gastropodOcenebra erinacea exhibits imposex (occurrence of male parts in addition to the female genital duct), a phenomenon which is caused by tributyltin (TBT)-compounds leached from ships’ antifouling paints. Five stages of imposex development (1–5) with two different types at stage 1 can be distinguished and are documented with SEM-photographs for the first time. Four additional alterations of the genital tract are shown. Close to harbours and marinas,O. erinacea females exhibit malformations of the pallial oviduct, which seem to inhibit copulation and capsule formation resulting in sterilization. The TBT accumulation in the whole body and the accumulation pattern in single tissues are described; contrary to other prosobranchs no sexrelated differences were found. The VDS, uncubed RPS and average female penis length of a population were analysed regarding their quality as parameters for TBT biomonitoring. On the background of the ecology of this species the VDS is chosen as the best index. Only in highly polluted areas should the uncubed RPS be used as a secondary parameter. A statistical study, based on the analysis of natural populations ofNucella lapillus andO. erinacea, allows a comparison of the specific TBT sensitivity of the two bioindicators. Dogwhelks exhibit a greater TBT sensitivity, namely at slightly polluted sites, nevertheless even here rough tingles develop obvious imposex characteristics.     

Imposex and butyltin burden in Bolinus brandaris (Mollusca, Gastropoda) and sediment from the Tunisian coast

The present study aimed at analyzing the imposex incidence and the presence of butyltins namely tributyltin (TBT) with its di- and mono-substituted metabolites in Bolinus brandaris whole tissues and in surface sediments at seven sites from the Tunisian coast during one campaign in May 2010. Butyltin levels were evaluated using isotope dilution GC–MS. Except the population collected from Zarat site, imposex was found in snails from the remained six sites with a maximal incidence and sterility (closure of the vaginal opening) registered in Carrier bay. Both imposex indices VDSI and RPLI showed a positive correlation with tissue concentrations of TBT. Total butyltin concentrations in sediments were higher in sites located in the vicinity of shipping areas with levels of TBT high enough to cause environmental concern if there is no legislative restriction and enforcement for the sale and use of these chemicals in Tunisia. These results further confirmed that B. brandaris is a good bioindicator of butyltin pollution in the studied areas. In addition, this study provided recent and new data on sediment butyltin concentrations that could serve for long-term monitoring of TBT pollution in Tunisia and the Mediterranean Sea.     

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.     

桶形芋螺和菖蒲芋螺的性畸变

2 0 0 1年 9月和 2 0 0 3年 8月在广东湛江的硇洲岛和 2 0 0 3年 6月在广东阳江的闸坡渔港采集桶形芋螺 (Conus betulinus)和菖蒲芋螺 (Conus vexillum) ,发现两个海区的芋螺雌性个体均发生性畸变 ,性畸变率均为 10 0 % ,但雌 /雄性比仍大于 1.0。两种芋螺的畸变阶段和类型多 ,桶形芋螺有 S3b、S3c、 S4 、S*4 、S5b、S5c,而菖蒲芋螺有 S1 c、S3b、S4 、S*4 、S6 b。 2 0 0 3年 6月在阳江采集的桶形芋螺畸变程度最高 ,种群 RPSI为 5 3.8% ,VDSI高达 4 .9,雌性不育率达 4 4 .0 %。 2 0 0 1年 9月在硇洲岛外海深水区采集的菖蒲芋螺的种群 RPSI虽然只有 14 .7% ,但性畸率为 10 0 % ,VDSI也达 4 .1%。由此可见 ,两种芋螺对有机锡污染均比较敏感 ,而且有个体大、易采集、性畸变率高、畸变阶段跨度大、畸变类型多、畸变特征易于鉴别等特点 ,是中国东南沿海低潮线和潮下带有机锡污染生物监测的理想指示种。如与潮间带有机锡污染指示种疣荔枝螺 (Thaisclavigera)结合起来 ,便可相互补充 ,能更加全面和准确地反映近岸海域有机锡污染的现状     

COPULATORY ACTIVITY AND SPERM PRODUCTION IN BULINUS (PHYSOPSIS) GLOBOSUS (GASTROPODA: PLANORBIDAE)

Young Bulinus (Physopsis) globosus performed male copulatoryactivity and cross-fertilized other snails before their femalereproductive tracts were mature. The two most immature snailsshowed preputial eversion when secretion was present only inthe muciparous gland and at the carrefour region of the oviduct.Sixteen snails showed preputial eversion and four snails cross-fertilizedother snails when their oothecal glands and/or major portionof the oviducts contained either no secretion or only scantyamounts. When paired with a partner snail for 12 or 20 consecutive days,adult snails copulated as males on approximately 60% of thedays paired and up to 8 consecutive days. Virgin snails raisedin isolation copulated as male at the same rate as non-virgin,community-raised snails. Ability to copulate as male was notdependent upon previous experience as male or female. Aftera single copulation as male after 7 days isolation, the hermaphroditicducts of maleacting snails contained 87 000 sperm. Sperm productionoccurred at approximately 50 000 sperm.d-¹, until at 10 dayspost-copulation, snails contained 639 000 sperm. (Received 25 May 1982;     

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.     

The effect of organotin compounds on gender specific androstenedione metabolism in the freshwater ramshorn snail Marisa cornuarietis

In a recent study, we demonstrated that androstenedione was mainly converted to testosterone (T) and 5alpha-dihydrotestosterone (DHT) by digestive gland/gonad complex microsomal fractions isolated from male Marisa cornuarietis, whereas it was primarily metabolized to 5alpha-dihydroandrostenedione (DHA) by females. In the present work, the sexual dimorphic metabolism of androstenedione was further investigated, and attributed to a higher 17beta-hydroxysteroid dehydrogenase activity in males than in females. Thereafter, the hypothesis was tested that the metabolism of androstenedione might be affected by exposure to tributyltin (TBT) and triphenyltin (TPT), which are known to induce the development of imposex in several gastropod species. The in vitro metabolism of androstenedione, particularly the formation of DHA and DHT, was inhibited by both compounds. However, in vivo experiments showed no significant alteration in the metabolism of androstenedione in males, but a marginal (TBT) and a significant (TPT) inhibition of the formation of DHA in females exposed for 150 days to concentrations that had significantly induced the development of imposex. The ratio DHT+T/DHA, a possible indicator of metabolic androgenization, tended to increase (0.43 versus 0.35, p=0.06) in TPT exposed females. However, this ratio never reached values comparable to those found in males (11+/-1).     

Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus

Recent studies performed under laboratory conditions have shown that single exposure to high levels of several xenoestrogens is able to induce imposex in at least two neogastropod species. In an attempt to evaluate if xenoestrogens, at environmentally relevant conditions, do contribute to imposex induction, we have tested the effects of a mixture containing xenoestrogens (municipal sewage effluents) on imposex development in the dogwhelk Nucella lapillus. Exposure for 3 months to the raw (0.25% and 1%) and the final sewage effluent (12.5% and 50%) rendered no increase in the severity of imposex. Conversely, as exposure to high concentrations of natural steroids, estradiol and estrone, had previously been shown to partially rescue imposex development under laboratory conditions, we have also tested if exposure to the final sewage effluent could ameliorated the severity of imposex induction by tributyltin (TBT). The results demonstrated that co-exposure to the final sewage effluent leads to a decrease trend in the severity of imposex in the presence of TBT. Within the studied imposex parameters, the Relative Penis Size index (RPSI) was the most affected with a 50% decrease in the effluent 12.5% plus TBT exposed group and 25% decrease in the effluent 50% plus TBT, if compared with the TBT alone. Overall, our results give further support to the use of imposex in N. lapillus as a specific biomarker of TBT contamination. However, in areas of high inputs of sewage effluents, the assessment of TBT contamination by the use of the imposex phenomenon should ideally also include data on the tissue levels of butyltins.     

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)     

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.