José Miguel Ruiz

Decreased TBT pollution and changing bioaccumulation pattern in gastropods imply butyltin desorption from sediments.

Two monitoring surveys were repeated in Galicia (NW Spain): one in 2003 concerned the rocky shore gastropod Nucella lapillus (19 populations, the reference campaign in 1996), the other dealt in 2005 with the infaunal snail Nassarius reticulatus (25 sites, the previous one in 2000). Samples were subject to a standard protocol to determine the concentrations of butyltins (BTs) in tissues. Results show that pollution in most populations has considerably decreased over the last decade: for N. lapillus the mean descent ranged from 37% (for tributyltin -TBT-) to 66% (for monobutyltin -MBT-), and TBT concentrations were on average halved in N. reticulatus. However, derivatives generally increased in this latter species, to the extent that dibutyltin -DBT- in several 2005 samples exceeded the aggregate of all three BTs in 2000. As a consequence, a major change in the bioaccumulation patterns becomes evident, particularly when computing the butyltin degradation index [BDI: (DBT+MBT)/TBT]. This shift is most marked at sites where pollution has always been lowest, and it shows significant negative correlation between both gastropods. Since sources other than antifouling paints are not important in the area, it is proposed that observations are due to BT desorption from sediments through some interplay involving the different characteristics of the chemicals and the contrasting biology of the animals.

The use of Nucella lapillus (L.) transplanted in cages to monitor tributyltin (TBT) pollution

Specimens of the gastropod Nucella lapillus were transplanted from their natural habitat to sites affected by tributyltin (TBT) pollution to diverse degrees; individuals were held in cages that also included mussels as food. Subsamples were taken from the cages and the natural site at different time intervals (1, 2 and 5 months) to compare growth, imposex (a set of sexual anomalies caused by TBT) and butyltin concentrations accumulated in female tissues. Individuals in cages experienced negligible mortality and considerable growth. In addition, while the Relative Penis Size Index (RPSI, an index of imposex) remained consistent in the natural site through the experiment, it increased markedly at the implant sites; the same applies to the butyltin residues. These results are discussed to conclude that cage transplanting of N. lapillus is a suitable technique that can quickly (i.e. 1 month) detect differences in the level of TBT contamination among sites; however, extrapolation of experimental results to the field seems to require a longer exposure time (at least 5 months). Finally, the use of such an approach is deemed helpful to interpret the relationship between N. lapillus population dynamics and TBT pollution.

Imposex and gender-independent butyltin accumulation in the gastropod Nassarius reticulatus from the Cantabrian coast (N Atlantic Spain).

The gastropod Nassarius reticulatus has been used as a tributyltin (TBT) biomonitor over the last two decades, and it is now endorsed by Atlantic Europe environmental agencies. However, there is one important question debated by the earliest studies still unresolved, namely whether butyltin accumulation in tissues is sex-dependent or not. Thus, a field survey was conducted along the Cantabrian coast: samples were subject to both customary imposex and modern chemical analyses. No significant bioaccumulation differences between sexes were found for any of the three butyltin species examined (i.e. TBT plus derivatives di- and mono-butyltin). In addition, both lower-than-expected absolute butyltins and minor relative TBT proportions in tissues conform with decreasing pollution in nearby areas. Imposex, though, is generally still considerable due to a lesser responsiveness of population indexes. Finally, one sample showed no sign of imposex but quantifiable butyltins; these are good news indicating that TBT is getting back down to levels around the induction threshold of this specific biological effect.

Decade-long monitoring reveals a transient distortion of baseline butyltin bioaccumulation pattern in gastropods.

Worldwide measures to restrict tributyltin (TBT) in antifouling paints have been legislated for decades, and were upgraded to a total ban on September 2008. With a view to test the response of coastal biota to changing pollution, since 1996 we have determined the concentration of TBT and derivatives di- and mono-butyltin (DBT and MBT) in NW Spain populations of two gastropods of contrasting biology, the rock-snail Nucella lapillus (n=18) and the mud-snail Nassarius reticulatus (n=24). TBT pollution in the study area has decreased consistently and considerably over time. In addition, the baseline butyltin (BT) bioaccumulation patterns showed a marked but transient distortion. These field observations are consistent with BT desorption from sediments, a natural phenomenon that is now to be expected in developing countries recently subject to the global TBT ban.

MICROSATELLITE DEVELOPMENT IN RHODOPHYTA USING HIGH-THROUGHPUT SEQUENCE DATA 1

Shotgun genome sequencing is rapidly emerging as the method of choice for the identification of microsatellite loci in nonmodel organisms. However, to the best of our knowledge, this approach has not been applied to marine algae so far. Herein, we report the results of using the 454 next‐generation sequencing (NGS) platform to randomly sample 36.0 and 40.9 Mbp (139,786 and 139,795 reads, respectively) of the genome of two red algae from the northwest Iberian Peninsula [Grateloupia lanceola (J. Agardh) J. Agardh and a still undescribed new member of the family Cruoriaceae]. Using data mining tools, we identified 4,766 and 5,174 perfect microsatellite loci in 4,344 and 4,504 sequences/contigs from G. lanceola and the Cruoriaceae, respectively. After conservative removal of potentially problematic loci (redundant sequences, mobile elements), primer design was possible for 1,371 and 1,366 loci, respectively. A survey of the literature indicates that microsatellite density in our Rhodophyta is at the low end of the values reported for other organisms investigated with the same technology (land plants and animals). A limited number of loci were successfully tested for PCR amplification and polymorphism finding that they may be suitable for population genetic studies. This study demonstrates that random genome sequencing is a rapid, effective alternative to develop useful microsatellite loci in previously unstudied red algae.

Dumpton Syndrome reduces the tributyltin (TBT) sterilising effect on Nucella lapillus (L.) by limiting the development of the imposed vas deferens

TBT induces imposex and sterilisation in female Nucella lapillus. Interestingly, there are populations where individuals (both males and imposexed females) may suffer from a genetic aberration [Dumpton Syndrome (DS)] which results in the development of no penis (i.e. aphally). This anomaly confers a so far unexplained protection against TBT. Six populations with contrasting DS frequency were sampled; the imposex stage in females and the mean calibre of every vas deferens (VDC) were assessed. While the sex ratio in the 600 specimens examined was close to 1, aphally was more frequent in females than in males (31 and 7%, respectively). VDC was significantly higher in normal than in aphallic (DS) specimens, independently of sex and/or site. It is concluded that the vas deferens imposed by TBT onto DS females is less prone to develop thick enough so as to interfere with normal egg laying.

Bi-species imposex monitoring in Galicia (NW Spain) shows contrasting achievement of the OSPAR Ecological Quality Objective for TBT

Imposex is decreasing worldwide after the total ban on tributyltin (TBT) from antifouling paints. In order to assess improvement in the NE Atlantic, the OSPAR Convention designed an Ecological Quality Objective (EcoQO) based on the VDSI (vas deferens sequence index, an agreed measure of imposex) in the rock snail Nucella lapillus; wherever this is not available, the mud snail Nassarius reticulatus was proposed as a proxy. We determined VDSI in Galician populations of rock (n≥34) and mud (n≥18) snails at regular intervals from pre-ban times until 2009 and 2011, respectively. While imposex in the former started decreasing in 2006 and by 2009 the EcoQO had been met in the area, VDSI in the latter was not significantly reduced until 2011 and values contradict such an achievement. This suggests that the OSPAR imposex bi-species scheme may not be of direct application in the current post-ban scenario.

A combined whelk watch suggests repeated TBT desorption pulses

Environmental quality in coastal Europe has improved since the complete 2003 ban on the use of tributyltin (TBT) in antifouling paints. However, there is evidence that TBT is entering the water column, presumably from illegal practices. We determined the concentration of butyltins (BTs: TBT and derivatives) in populations of two gastropods, the rock snail Nucella lapillus (n=17) and the mud snail Nassarius reticulatus (n=18) at regular intervals from pre-ban times until 2009 and 2011, respectively, in NW Spain. Although a substantial decline in TBT occurred shortly after the ban, no significant changes were observed in either species over the last 3-year period of study. In addition, the proportion of TBT relative to the sum of BTs (a marker of recent pollution) in the most recent rock snail samples unexpectedly increased; this proportion therefore showed a generally decreasing but oscillatory trend over time. The results are consistent with the theoretical expectation of BT desorption from sediments; however, this natural phenomenon is now interpreted as a recurrent episode rather than a unique, transient event. Evidence of this subtle input improves our understanding of TBT persistence in the environment in Europe and worldwide.

MULTISCALE GENETIC STRUCTURE OF AN ENDANGERED SEAWEED AHNFELTIOPSIS PUSILLA (RHODOPHYTA): IMPLICATIONS FOR ITS CONSERVATION(1).

Although marine macroalgae have recently entered the lists of endangered species, conservation efforts are still limited by a lack of data, particularly for naturally rare species. One example is the turf‐forming Ahnfeltiopsis pusilla (Mont.) P. C. Silva et DeCew. Albeit cataloged as vulnerable in the Northwest Iberian Peninsula (NWIP), where it occurs only at five enclaves separated by 1,200 km from the closest recorded presence of the species, nothing is known about its genetic diversity and population connectivity. We used amplified fragment length polymorphism (AFLP) and sequences of the intergenic region between the mitochondrial cytochrome oxidase subunit 2 and subunit 3 genes (cox2‐3) to investigate its genetic structure at large (1,200 km), regional (<125 km), fine (<250 m), and patch (<1 m) scales. While cox2‐3 variability was too low for the intraspecific study, AFLP revealed that most of the genetic diversity was due to differences between populations. Locally, genetic diversity was always low, and clones were frequent, suggesting that asexual reproduction may be common; patches of turf, however, often were composites of various genetic individuals. Genetic structure at local, regional, and large scales indicated that A. pusilla is a poor disperser, and an assignment test found no evidence of real‐time dispersal between NWIP sites. Therefore, it is proposed that the five NWIP enclaves are designated independent management units (MUs). Bayesian‐clustering approaches suggested that the three southernmost sites are particularly valuable for conservation since they concentrate most of the genetic heritage of A. pusilla in NWIP. Our study shows that the approaches of conservation genetics may provide useful insights for endangered seaweeds.

Discovery of imposex in the gastropod Cyclope neritea now invading Galicia (north-west Spain)

A field survey to re-assess imposex and tributyltin (TBT) bioaccumulation in the gastropod Nassarius reticulatus over Galicia uncovered three populations of the invasive species Cyclope neritea , all showing some advanced imposex. One female was found to carry a mass of aborted egg capsules. The TBT pollution, now decreasing in the area, is the most likely explanation for these reproductive anomalies, and it may bear some relationship to the described population dynamics of C. neritea .