Population Genomics Reveals the Underlying Structure of the Small Pelagic European Sardine and Suggests Low Connectivity within Macaronesia.

The European sardine (Sardina pilchardus, Walbaum 1792) is indisputably a commercially important species. Previous studies using uneven sampling or a limited number of makers have presented sometimes conflicting evidence of the genetic structure of S. pilchardus populations. Here, we show that whole genome data from 108 individuals from 16 sampling areas across 5000 km of the species' distribution range (from the Eastern Mediterranean to the archipelago of Azores) support at least three genetic clusters. One includes individuals from Azores and Madeira, with evidence of substructure separating these two archipelagos in the Atlantic. Another cluster broadly corresponds to the center of the distribution, including the sampling sites around Iberia, separated by the Almeria-Oran front from the third cluster that includes all of the Mediterranean samples, except those from the Alboran Sea. Individuals from the Canary Islands appear to belong to the Mediterranean cluster. This suggests at least two important geographical barriers to gene flow, even though these do not seem complete, with many individuals from around Iberia and the Mediterranean showing some patterns compatible with admixture with other genetic clusters. Genomic regions corresponding to the top outliers of genetic differentiation are located in areas of low recombination indicative that genetic architecture also has a role in shaping population structure. These regions include genes related to otolith formation, a calcium carbonate structure in the inner ear previously used to distinguish S. pilchardus populations. Our results provide a baseline for further characterization of physical and genetic barriers that divide European sardine populations, and information for transnational stock management of this highly exploited species towards sustainable fisheries.

Impact of polyethylene microplastics on the clam Ruditapes decussatus (Mollusca: Bivalvia): examination of filtration rate, growth, and immunomodulation.

The present study was conducted to assess, for the first time, the effects of a 14 days experimental exposure to polyethylene (PE) based MPs (40-48 µm) on the clam Ruditapes decussatus. Clams were exposed to three different concentrations of MPs in controlled laboratory conditions: 10 µg/L (low), 100 µg/L (medium), and 1000 µg/L (high). The effects of MPs were assessed using a multi-marker approach, including the filtration rate, growth, and the integrity of immune cells (such as haemocyte numbers, viability, and lysosomal membrane destabilization). The results revealed that as the concentration of PE-MPs increased, the filtration rate decreased, indicating that PE-MPs hindered the clams' ability to filter water. Furthermore, there was a noticeable decrease in the overall weight of the clams, particularly in the group exposed to 1000 µg/L. This decrease could be attributed to the impairment of their nutrient filtration function. In terms of immune system biomarkers, exposure to PE-MPs led to immune system disruption, characterized by a significant increase in the number of haemocytic cells, especially in the group exposed to the high concentration. Additionally, there was a notable reduction in the viability of haemocytes, resulting in the destabilization of their lysosomal membranes, particularly in the groups exposed to medium and high PE-MPs concentrations. The findings of this study indicate that the sensitivity of hemolymph parameter changes and filtration rate in R. decussatus exposed to PE-MPs (100 and 1000 µg/L), surpasses that of growth performance and can serve as reliable indicators to assess habitat conditions and contaminant levels.

Effects of environmentally relevant levels of polyethylene microplastic on Mytilus galloprovincialis (Mollusca: Bivalvia): filtration rate and oxidative stress.

The objective of the present study was to evaluate the potential toxic effects of polyethylene microplastics (PE-MPs) (40-48 µm) on the Mediterranean mussel Mytilus galloprovincialis in controlled laboratory conditions. The exposure was carried out for 14 days with three environmentally relevant PE-MPs concentrations of 1, 10, and 100 and a high concentration of 1000 µg/L. Effects of PE-MPs were assessed by evaluating the filtration rate (FR) after 7 and 14 days of exposure and by analyzing biochemical biomarkers of oxidative stress (catalase - CAT, glutathione S-transferase - GST, and the levels of lipid peroxidation - LPO) in the M. galloprovincialis digestive gland after 14 days of exposure. Results showed that M. galloprovincialis does not accumulate PE-MPs of 40-48 µm size in its whole tissues. The filtration rate was significantly reduced with the increase of PE-MPs concentrations. The biochemical biomarkers indicated that PE-MPs induced oxidative damage (LPO) at low concentrations (1 and 10 µg/L) with a significant reduction in females of 1000 µg/L treated group and inactivate antioxidative system (CAT and GST) in the digestive gland of both sexes at high concentrations (100 and 1000 µg/L). This study demonstrates that PE-MPs have biological effects on M. galloprovincialis at environmentally relevant concentrations thus brings new insights on the potential impacts of PE-MPs in marine bivalves.

Microplastics in commercial molluscs from the lagoon of Bizerte (Northern Tunisia).

Microplastic (MP) pollution was investigated, for the first time, in six commercial molluscs collected from the lagoon of Bizerte during March 2018. The objective of this study was to determine the bioavailability of MPs to marine organisms and their risk for consumers of seafood. MP concentrations varied from 703.95 ±â€¯109.80 to 1482.82 ±â€¯19.20 items kg-1 wet weight. Three types of coloured MPs, including fibres, fragments and films were recovered. Fibres were the most common MP type isolated in each species. The most common size class was 0.1-1 mm. The FTIR-ATR analysis confirmed the presence of two polymer types polyethylene and polypropylene. Our results suggest that MP pollution was widespread and exhibited a relatively high level in commercial molluscs collected from Bizerte lagoon, suggesting trophic transfer in the food web and human exposure risks by diet. More investigations on MPs should be conducted in seafood and other marine organisms.

Microplastics in freshwater environment: the first evaluation in sediments from seven water streams surrounding the lagoon of Bizerte (Northern Tunisia).

Microplastic (MP) concentrations were determined, for the first time, in surface sediment of seven streams around the lagoon of Bizerte (Northern Tunisia), using a saturated NaCl flotation technique. Microplastics were categorised according to type, colour and size using a stereoscopic microscope. Results showed that all sediment samples contained MPs. The greatest MP abundance was observed at Jedara stream (6920 ± 395.98 items kg-1 dry weight), while the lowest mean value was 2340 ± 227.15 items kg-1 dry weight at Khima stream. The highest numbers of MPs were from streams near populated areas and municipal and industrial effluent discharges. Samples were made up entirely of secondary MPs mainly fibres, followed by fragments and films. The predominant colours were as follows: black > clear > white > red > blue > green > yellow for fibres, white > blue > black > red for fragments and red > white > clear > green > blue = black for films. Microplastic particles in the samples ranged from 0.2 to 5 mm in length. FTIR analysis revealed that the abundant polymers were polypropylene and polyethylene. This work contributes to the growing evidence that MP contamination is widespread even in freshwater ecosystems and provides a baseline for future studies and risk assessments.

First assessment of the effectiveness of the international convention on the control of harmful anti-fouling systems on ships in Tunisia using imposex in Hexaplex trunculus as biomarker.

The global ban on the use of tributyltin (TBT) forced in September 2008 was ratified in Tunisia as late as June 2011. In this context, the present study aims to highlight the consequences of TBT contamination by monitoring 22 Tunisian sites before (2007) and after the ban (2012 and 2016) using as biomarker the occurrence of imposex in Hexaplex trunculus. From 2004 to 2016, complete recovery from imposex was reported in 11 sites. All average imposex indices calculated for each sampling year also decreased: imposex incidence from 61 to 27%, VDSI from 2 to 0.7, and RPLI from 15.7 to 1.6%. Ecological Quality Ratios (EQR) revealed that the sampling sites are in moderate to good ecological status. Overall, the present study confirms the effectiveness of the enacted legislation in reducing the impact of TBT pollution along the Tunisian coast.

Imposex development in Hexaplex trunculus (Gastropoda: Caenogastropoda) involves changes in the transcription levels of the retinoid X receptor (RXR).

In order to further demonstrate that TBT-induced imposex through RXR signaling is not species-specific, Hexaplex trunculus was selected as an experimental model species. We first isolated RXR in H. trunculus, and determined gene transcription through quantitative real-time PCR in key tissues (e.g., penis/penis-forming area and central nervous system:- CNS), upon exposure to tributyltin (TBT) (5 and 50 ng TBTL(-1)). Two months of exposure to TBT induced imposex and led to a significant increase in the severity of the phenomenon in females and an increase in male penis lengths. Exposure to TBT altered RXR gene transcription in a tissue and sex-specific manner. In the CNS, there were no significant changes in RXR gene transcription between control and TBT-exposed females. A similar trend was observed in male CNS. On the contrary, in the penis-forming area/penis of females exposed to TBT, a significant increase in RXR gene transcription was observed in the 50 ng TBTL(-1) group. Furthermore, a positive correlation was observed between overall female penis lengths and RXR gene transcription. In males, although a trend towards an increase in RXR gene transcription in penis was observed, differences did not reach significance. Overall, the results of the present study give further support to a local role of RXR in the penis-forming area during the development of imposex by TBT, thus suggesting a conserved function of RXR in penis formation at least within prosobranch gastropods.

Monitoring of organotin pollution in Bizerta channel (northern Tunisia): temporal trend from 2002 to 2010.

Imposex and tributyltin (TBT) body burden were quantified in the gastropod Hexaplex trunculus collected from the Bizerta channel between 2002 and 2010. Except for the imposex frequency that remained maximal (100%), all the other imposex indices decreased throughout the study period. Similarly, TBT levels also decreased over time, being the less frequent compound among butyltins, with a proportion of 22.2%, against 42.9% for dibutyltin (DBT) and 34.9% for monobutyltin (MBT). These findings reflect the effectiveness of new generation of TBT-free antifouling paint introduced in the Tunisian market and global ban of TBT on reducing the environmental impact of this biocide.

Imposex and butyltin concentrations in Bolinus brandaris (Gastropoda: Muricidae) from the northern Tunisian coast.

The imposex incidence and butyltin concentration i.e. tributyltin with its di- and mono-substituted metabolites were investigated in the muricid Bolinus brandaris sampled from two sites on the northern Tunisian coast (the Lagoon of Bizerta and the small Gulf of Tunis). Both populations had imposex, with stages of imposex development varying between VDS 1 and VDS 4.3. All imposex indices (imposex frequency (I %), female penis length, female vas deferens length, vas deferens sequence index, relative penis length index, and vas deferens length index) were significantly higher in snails from the Bizerta lagoon. Butyltins were detected in the whole tissues of both sexes from the two sites. TBT levels were higher in gastropods collected from the lagoon of Bizerta (12.65 ±1.48 ng Sn g( - 1) dw in female and 15.21 ±1.13 ng Sn g( - 1) dw in male) than in individuals from the Gulf of Tunis (10.71 ±1.26 ng Sn g( - 1) dw in female and 11.65 ±1.63 ng Sn g( - 1) dw in male), corroborating the data of imposex analysis. These results confirmed that B. brandaris could be used as a bioindicator species of butyltin pollution in the studied areas. In addition, this study provided baseline data that could serve for long-term monitoring of TBT pollution in Tunisia, since legislation to reduce the use of TBT-based antifouling paints has not been introduced yet.

Potential of Nassarius nitidus for monitoring organotin pollution in the lagoon of Bizerta (northern Thunisia).

Imposex and butyltin burden were assessed in Nassarius nitidus, Bolinus brandaris and Hexaplex trunculus collected at five stations in the Bizerta lagoon. Biological analysis showed that imposex followed type (a) in N. nitidus (distal evolution), against type (d) in the two muricids (proximal evolution). Imposex indices were higher in sites located nearby sources of tributyltin and N. nitidus was the least affected species of the five sites, followed by B. brandaris and H. trunculus. Butyltin analysis showed lower accumulation in N. nitidus followed by H. trunculus and B. brandaris. This study has allowed the gathering of data on imposex in a snail studied for the first time in Tunisia (N. nitidus). It suggests the possibility of using such snail as a complementary species for organotin monitoring programs in the Mediterranean and further confirmed that H. trunculus is the most suitable species for such investigations.

First record of butyltin body burden and imposex status in Hexaplex trunculus (L.) along the Tunisian coast.

We have assessed for the first time the current status of tributyltin (TBT) contamination in Hexaplex trunculus along Tunisian coastal waters. Two sampling campaigns have been performed in July 2004 and July 2007 at seven different sites. The snails were analyzed for imposex status, i.e. Imposex incidence, Relative Penis Length Index (RPLI) and Vas Deferens Sequence Index (VDSI), followed by the quantification of butyltins in their tissues. Imposex was detected in six populations from the 2004 samples and in five populations from the 2007 samples. The imposex incidence ranged from 0 (no imposex) to 100%, the RPLI from 0 to 56.7 and the VDSI from 0 to 4.4. TBT was also detected in six populations in 2004 and five populations in 2007. The concentrations ranged from 1.5 to 73.7 ng Sn g(-1) dw in 2004 samples and from 1.2 to 23.9 ng Sn g(-1) dw in 2007 samples. Temporal comparison of the data indicates some evidence of imposex recovery in most affected populations. Improvements in reduced body concentrations of TBT in both sexes were also seen in most stations. These TBT concentrations were only significantly correlated to imposex indices in samples from 2007 (r > 0.8, n = 7, p < 0.05). The present results will serve as a reference for long-term monitoring of butyltin contamination in the Tunisian coast, where TBT restrictions were not yet implemented.