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.     

Sexual anomalies in the estuarine snail Ilyanassa obsoleta: imposex in females and associated phenomena in males

Summary The spatial distribution of the imposex phenomenon (the imposition of male characters on females) among populations of Ilyanassa obsoleta in southern Delaware, USA estuaries was studied. Imposex was most frequent among females in a segment of the Lewes and Rehoboth Canal (68%, n=376) and there was significant spatial heterogeneity of imposex frequencies (range=43–98%) even within this habitat. Two lines of evidence indicate that conditions which generated female imposex (organotin molecules in the water) also affected male sexuality: 1) healthy males normally lose the penis following reproduction but they tended to retain it where imposex-inducing conditions were strong; and 2) males parasitized by trematodes usually lose the penis but tended not to under strong imposex-inducing conditions. The altered biology associated with imposexinducing environments extends beyond female I. obsoleta to males and to other species. This must be recognized when studying organisms exposed to these conditions.     

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.     

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.     

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.     

三种前鳃亚纲海产腹足类性畸变现象的组织学研究

有机锡污染可以导致海产腹足类雌性个体产生性畸变现象。本文报道了阿文绶贝(Mauritia arabica)、褐棘螺(Chicoreus brunneus)和桶形芋螺(Conus betulinus)三种前鳃亚纲腹足类正常雄性个体和性畸变个休雄性生殖器官的组织学结构。结果表明,不同种间雄性个体的输精管和阴茎的结构存在开放和封闭两种类型,封闭型是由开放型进化而来。虽然性畸变个体的雄性生殖器官与正常雄性个体的在组织结构上无明显差异,但性畸变个体的雄性生殖器官并不完整,无法行使生殖功能。由于内分泌扰乱物质对人和动物影响的相似性,使得海产腹足类性畸变现象成受到人们的重视。     

软体动物维甲酸X受体研究进展

维甲酸X受体(retinoid X receptor,RXR)作为配体依赖的转录因子,是核受体超家族重要的一员.脊椎动物RXR与配体及其辅调节因子相互作用,调控基因的协调表达,在胚胎发育、细胞分化、新陈代谢等许多生理过程中起着重要作用.软体动物RXR的研究因其与腹足类性畸变的关系越来越受到关注.本文综述了目前获得的软体动物RXR基因的结构,比较了软体动物RXR基因各功能结构域与人类和其他动物RXR的相似性.以RXR编码区的氨基酸序列为基础,构建了系统进化树,发现软体动物RXR与脊索动物而不是其他无脊椎动物的RXR聚成一支.软体动物和甲壳动物不同RXR亚型的氨基酸序列比较发现,两类动物可能存在不同的剪切酶或剪切位点.此外论文还针对软体动物RXR的配体、二聚体伙伴以及生理功能等方面的研究进行了综述.     

Tributyltin pollution biomonitoring under the Water Framework Directive: Proposal of a multi-species tool to assess the ecological quality status of EU water bodies

The Water Framework Directive (WFD) is a key legislative action developed by the European Union in order to protect aquatic ecosystems. One of the concerning pollutants, listed in this directive as a priority hazardous substance, is tributyltin (TBT), a biocide largely used in antifouling paints and identified as a causative agent of imposex/intersex in gastropods. In order to integrate TBT pollution monitoring within this legislative framework, a practical exercise is here proposed to assess the evolution of surface water ecological status in Ria de Aveiro (NW Portugal). Three bioindicators – the caenogastropods Nucella lapillus, Nassarius reticulatus and Littorina littorea – were used under the general WFD benthic invertebrate quality element, and the vas deferens sequence index (VDSI) and the intersex index (ISI) were selected as biomarkers for the purpose of assessing the condition of this quality element regarding the impact of TBT pollution. Levels of VDSI in N. lapillus and N. reticulatus, and ISI in L. littorea, were surveyed in 2013 and compared with previous data available for the same species and study area in 1998 and 2005, providing a time lapse for a period of 15 years. VDSI and ISI values were converted into Ecological Quality Ratios (EQR) and EQR boundaries were set for each species in order to define the five ecological status classes (High, Good, Moderate, Poor and Bad). We propose N. lapillus as key bioindicator, however the combined use of further species is very useful to cover a wider study area. Based on the proposed method, it is concluded that the ecological status of the surface waters surveyed in Ria de Aveiro, concerning the impact of TBT pollution on the above benthic invertebrate taxa, improved considerably since 1998 and achieved a Good Ecological Status in 2013, thus meeting the WFD environmental objectives for this priority hazardous substance even before 2015.     

Ecdysone receptor homologs from mollusks, leeches and a polychaete worm

The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes.