厦门海域岩相海岸带疣荔枝螺（Thais clavigera）与甲虫螺（Cantharus cecillei）性畸变

海产腹足类性畸变以其对有机锡的敏感性、特异性和不可逆性而成为海洋有机锡污染的理想指示种.调查研究了厦门海域岩相海岸带疣荔枝螺(Thais clavigera)和甲虫螺(Cantharus cecillei)的性畸变情况,通过生物学指标IOI(性畸变率)、RPSI(相对阴茎发展指数)、VDSI(输精管发展指数)及SRI(性比指数)综合评价其性畸变发展程度,间接指示厦门周边海域的有机锡污染程度.研究结果发现,在采集疣荔枝螺的17个站点中有5个站点种群性畸变率为100%,其中宝珠屿种群性畸变程度最为严重,VDSI达4,采集甲虫螺的4个站点的种群性畸变率均为100%.性畸变程度较严重的种群普遍集中于厦门西港,并且表现出由港内到港外逐渐减轻的趋势,而白城、会展中心和大嶝岛等位于开阔的厦门东海域的种群性畸变程度最轻,与往年厦门西海域有机锡污染监测数据相比一致,体现出性畸变程度与有机锡污染程度及海港码头远近之间的相互关系.     

Development of indices for nonsacrificial sexing of imposex-affected Hexaplex (Trunculariopsis) Trunculus (Gastropoda: Muricidae)

The muricid Hexaplex (Trunculariopsis) trunculus lacks externalsexual dimorphism and is highly affected by imposex, which furthercomplicates its sexual identification. In this context, theaim of this study was to develop sexual indices based on thedimensions of male and imposex-affected female penis, whichcould constitute a useful, simple and nonsacrificial tool forsexing live specimens of T. trunculus. The adoption of sexualindices consisting of penis dimensions of both sexes relativeto individual size revealed a high accuracy in the sexual identificationof sacrificed specimens (>95% correct sexing). Additionally,multivariate discriminant analysis allowed correct sexing of98.8% of the original 1053 sacrificed individuals, with accuratesexual identification being higher for males (99.5%) than forimposex-affected females (97.7%). An anaesthetization experimentwas performed to investigate the effects produced by the anaesthetic(MgCl₂) on penis measurements, to test this nonsacrificial approachand to validate the previously developed sexual indices. Theanaesthetic provoked an expected enlargement in penis dimensionsbut, despite this side effect, the sexual indices developedfor sacrificed specimens were still highly successful in sexinganaesthetized T. trunculus (generally more than 95% correctsexual identification). The practical application and some limitationsof developing and employing this kind of index for the sexualidentification of T. trunculus and other imposex-affected gastropodspecies are discussed. (Received 30 May 2005; accepted 17 January 2006)     

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.     

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.     

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.     

Survey of imposex in prosobranchs mollusks along the northern Mediterranean coast of Morocco

Imposex, i.e., the superimposition of male sex organs on female gonochoristic neogastropods has been observed in more than 140 species worldwide and suggested as a suitable biomarker of tributyltin pollution in the marine environment. In this study, an imposex survey for three neogastropod species: Murex trunculus, Murex brandaris and Thaïs haemastoma was conducted for the first time at five sites along the northern Mediterranean coasts of Morocco. The imposex phenomenon was observed in the all surveyed sites. Nevertheless, rates of occurrence and degree of imposex were more important in both harbours sites (Tangier and Mdiq) characterized with heaving shipping activity than in the three seaside stations (Martil, Azla and Amsa) where only sporadic and small boats are used in traditional fishing activities.     

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.     

Size matters: importance of penis length variation on reproduction studies and imposex monitoring in Bolinus brandaris (Gastropoda: Muricidae)

The present study aimed at analysing the monthly variation in penis length (PL) during the reproductive cycle of the purple dye murex (Bolinus brandaris). Two hypotheses were tested: PL variation reflects male maturation and reproductive activity; PL variation bias the calculation of imposex indices based on penis measurement (RPLI and RPSI). Sampling was performed during 1 year in a population from Ria Formosa lagoon (Algarve coast, southern Portugal) with high incidence of imposex. Penis-bearing individuals were measured for shell length (SL) and PL, and two bio-physiological indices were calculated for both sexes, the general condition index (K) and the gonadosomatic index (GSI). PL presented monthly variation in both sexes, but while female PL showed small and random oscillation, male PL exhibited significant variation throughout the reproductive cycle (as evidenced by the similar and synchronous trends between male PL and GSI). These findings have implications both for reproduction studies and imposex monitoring: calculation of standardised PL (PL/SL) allows for gender identification without killing the organism and constitutes a useful penial index for assessing male maturation and reproductive activity; RPLI and RPSI should be applied cautiously in spatial and temporal comparisons of imposex severity in B. brandaris.     

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.     

Undernutrition Affects Cell Survival,Oxidative Stress,Ca2+ Handling and Signaling Pathways in Vas Deferens,Crippling Reproductive Capacity

Background

The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life.

Methodology/Principal Findings

Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca²⁺-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca²⁺ affinity and regulation of Ca²⁺ handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca²⁺ release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca²⁺ handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca²⁺-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation.

Conclusions/Significance

The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.     

Morphofunctional characterization of the reproductive system of the gastropods <Emphasis Type="Italic">Littorina brevicula,L. mandshurica</Emphasis>, and <Emphasis Type="Italic">Nucella heyseana</Emphasis> from uncontaminated and contaminated areas of Peter the Great Bay,Sea of Japan

The structure and state of the reproductive system were studied in the periwinkles Littorina brevicula and L. mandshurica from the coastal area of Vladivostok and in the dogwhelk Nucella heyseana from the protected area of Vostok Bay and from contaminated sites in Amursky Bay. These species were investigated for the presence of imposex and intersex (pathological changes caused by endocrine disruption under the influence of toxic substances). The pathological changes revealed in the gonads of Littorina were oocyte resorption and lipofuscin accumulation; an intersex condition was not found. Imposex specimens (with penis formation) were found among Nucella females from Amursky Bay. The relative penis length in females was 25% of the penis length in males. This kind of pathology was not found in Nucella from clean waters of Vostok Bay.     

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.     

Tyrosine-modified analogs of methionine-enkephalin and their effects on the mouse vas deferens

Eighteen analogs of Met-enkephalin were synthesized in order to examine those features of the N-terminal tyrosine (Tyr) residue responsible for activity on the mouse vas deferens. The most critical part of the tyrosine side-chain was its phenolic hydroxyl group which, in terms of biological activity, was highly sensitive to small changes and to the inclusion of fluorine or methyl groups in the aromatic ring. In contrast, the free amino group was not as sensitive to alterations. Single amino acid extensions had only modest effects on activity; however, beta and D-amino acid extensions virtually destroyed activity. Although the Tyr residue might be considered a promising part of the opiate peptides for the development of competitive antagonists, none of our inactive analogs were able to antagonize enkephalin and were, therefore, without binding affinity towards opiate receptors in the vas deferens.     

Interconversion of Agonist-Induced Responses of Prostatic and Epididymal Portions of the Rat Vas Deferens Induced by Castration

The influence of castration (30-90 days) on the effects of ATP, noradrenaline (Nor), and carbachol (CCh) were examined in prostatic and epididymal portions of the rat vas deferens. All agonist-induced contractions were greatly increased in the prostatic and suppressed in the epididymal portion of the vas deferens from castrated rats, as compared with those in preparations from normal animals. Responses to Nor showed a typical cumulative dose–effect curve, with an additional twitch at the minimal dose. Responses to CCh were paradoxically changed; the maximum of twitch responses was observed at threshold concentrations (10^-7 to 3 · 10^-7 M), and decreased at higher concentrations. In the threshold concentration (10^-7 M), ATP induced almost maximal responses in a non-cumulative manner. All agonist-induced responses were blocked by isradipine (10^-7 M) and increased by pretreatment with reserpine. In normal animals, injection of testosterone insignificantly decreased responses to all agonists. The ratio of the amplitude of high-potassium (80 mM)-induced contractions (mm) to the tissue wet mass (per mg) in preparations from castrated animals also showed oppositely directed modulation of the responses, as compared with that in normal animals. It is suggested that the lack of hormone modifies a negative feedback mechanism of elevation of the intracellular Ca²⁺ concentration, which exists in normal rats mostly in the prostatic portion and is the reason for regional variation along the vas deferens, to a positive feedback link amplified in castrates.     

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.     

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;     

Modulation of A-type potassium current in smooth-muscle cells of the rat Vas Deferens by Ca2+/calmodulin-dependent protein kinase II

Using a standard patch-clamp technique in the perforated patch configuration, we studied the effect of a highly specific membrane-permeable inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), KN-93, on fast outward A-type potassium current in isolated smooth-muscle cells (SMCs) of an epididymal region of the rat vas deferens. This inhibitor significantly changed the dynamics of the studied current; in particular, it increased the rate of inactivation and considerably slowed down the recovery after inactivation. In the presence of 5 μM KN-93, we observed a moderate (nearly by 20%) decrease in the peak amplitude of fast A-type current. Based on the data obtained, we conclude that voltage-sensitive fast A-type potassium current in SMCs of the epididymal part of the rat vas deferens can be significantly regulated by the activity of CaM-KII. Therefore, by influencing the kinetic characteristics of the above current, this enzyme can be indirectly involved in the control of electrical activity in SMCs. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 419–422, July–October, 2007.     

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.