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.     

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.     

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.     

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.     

Sensitivity of Asellus aquaticus (L.) and Proasellus coxalis Dollf. (Crustacea,Isopoda) to Copper

The effects of Copper on the life-cycle of two crustacean isopods, Asellus aquaticus (L.) and Proasellus coxalis Dollf., are studied. ST₅₀ indicates that females and males are differently sensitive in comparison with juveniles. The two species do not show differences in sensitivity in the range of 0.01–15 mg·1^-1 between adults and juveniles. The effect of 0.005 mg·1^-1 Copper sulphate was strongest on embryonic and larval stages. Ecological implications are discussed.     

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.     

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.     

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.     

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.     

TBT effects on the female genital system of Littorina littorea: a possible indicator of tributyltin pollution

Specimens of the prosobranch Littorina littorea (L., 1758) collected along the East Frisian North Sea coast in summer 1993 exhibited alterations of the pallial oviduct termed as intersex in response to tributyltin (TBT) pollution. The range of TBT body burden was between 150.9 and 1289.5 μg as Sn kg⁻¹ (dry wt.). Five stages of intersex development (0–4) could be distinguished and are documented with scanning electron micrographs. In stages 2–4, which can be found in the direct vicinity of harbours and marinas, the morphological malformations of the oviduct inhibit successful copulation and capsule formation, resulting in sterilization. The intersex index (ISI, calculated as the average intersex stage of a population) and the average prostate length of females were used as parameters for the determination of intersex intensities in the populations. Both indices show significant and positive correlations to the TBT body burden of L. littorea and are promising parameters for TBT biomonitoring. A comparison of TBT bioconcentration factors with populations from England and France indicates that the threshold concentration for intersex development is in the range of 15 ng TBT as Sn/l. Morphometric analyses of the midgut gland revealed no significant differences between sampling stations. In the ovary a retardation and blockage of maturation (atresia) was observed in populations close to harbours. Lytic processes in ovary follicles were observed not only at TBT exposed sites but also at reference stations.     

Seasonal changes in plasma membrane H<Superscript>+</Superscript>-ATPase activity of <Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> (L.) and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.)

Seasonal changes in vanadate sensitive plasma membrane H⁺-ATPase activity of bilberry (Vaccinium myrtillus L.) and Scots pine (Pinus sylvestris L.) were studied in a period from February to August in northern Finland. The plasma membrane isolation was performed by sucrose gradient centrifugation, and the H⁺-ATPase activity was assayed by spectrophotometrical determination of released inorganic phosphate. The studied species showed seasonal changes from high winter to low spring activity, indicating probable physiological changes between hardened and dehardened tissue. ATPase activity of bilberry peaked up at the beginning of the growth period, obviously due to active phloem loading of photosynthates.     

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.     

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.     

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.     

Determination of root biomasses of three species grown in a mixture using stable isotopes of carbon and nitrogen

A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Celtis laevigata Willd. (C₃), Prosopis glandulosa Torr. (C₃, legume) and Schizachyrium scoparium (Michx.) Nash (C₄), or Gossypium hirsutum L. (C₃), Glycine max (L.) Merr. (C₃ legume), and Sorghum bicolor (L.) Moench (C₄) were grown together in separate, three-species combinations. Surface roots (0–10 cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of ¹⁵N and ¹⁴N and ¹³C and ¹²C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the δ ¹³C, % ¹⁵N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C₄ monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of ¹⁵N to ¹⁴N and ¹³C to ¹²C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

Olfactory response of three parasitoid species (Hymenoptera: Braconidae) to volatiles of guavas infested or not with fruit fly larvae (Diptera: Tephritidae)

The olfactory responses of the native parasitoids Doryctobracon areolatus (Szépligeti) and Asobara anastrephae (Muesebeck) and of the exotic parasitoid Diachasmimorpha longicaudata (Ashmead) to guava (Psidium guajava L.) infested or not with fruit fly larvae were evaluated. D. areolatus and D. longicaudata females responded to the odors of uninfested rotting guavas, although D. areolatus was also attracted to fruits at the initial maturation (turning) stage. The females of these species recognized the volatiles of guavas containing Ceratitis capitata (Wied.) larvae. However, in bioassays involving fruits with larvae of different instars, D. longicaudata females were not able to separate between fruits containing C. capitata larvae at the initial instars and larvae at the third instar. In the evaluations of volatiles released by guavas containing C. capitata and Anastrepha fraterculus (Wied.) larvae, the D. longicaudata females were oriented toward the volatiles of fruits containing both host species, but differed significantly from volatiles of guavas containing C. capitata larvae. The D. areolatus females also showed responses to both species, although with a preference for volatiles of fruits containing A. fraterculus larvae. The A. anastrephae females were oriented toward the odors of fruits infested with both fruit fly species. In the shade house, D. longicaudata females were oriented to volatiles of rotting fruits containing larvae or not, but could not significantly differentiate between hosts. D. areolatus females were not attracted toward fruits on the ground in the shade house, regardless of host, suggesting that this parasitoid does not forage on fallen fruits.     

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.