Global warming scenarios for the Eastern Adriatic Sea indicate a higher risk of invasiveness of non-native marine organisms relative to current climate conditions.

Globally, marine bioinvasions threaten marine ecosystem structure and function, with the Mediterranean Sea being one of the most affected regions. Such invasions are expected to increase due to climate change. We conducted a risk screening of marine organisms (37 fishes, 38 invertebrates, and 9 plants), both extant and 'horizon' (i.e., not present in the area but likely to enter it). Based on expert knowledge for the Eastern Adriatic Sea coasts of Slovenia, Croatia, and Montenegro, screenings were conducted under both current and predicted climate conditions indicating with an increase in sea surface temperature and salinity of the Adriatic Sea together with changes in precipitation regime. Our aims were to: (1) identify non-native extant and horizon marine species that may pose threats to native biodiversity and (2) evaluate the risk of invasiveness of the selected species under current and predicted climate conditions. Of the 84 species screened, there was an increase in those ranked as 'high risk' from 33 (39.3%) under current climate conditions and to 47 (56.0%) under global warming scenarios. For those ranked as 'very high' risk, the increase was from 6 (7.1%) to 21 (25.0%). Amongst the screened species, the already established high-risk species Pacific oyster Magallana gigas and Atlantic blue crab Callinectes sapidus represent a threat to ecosystem services. Given the under-representation of marine species in the current European Union List, the species we have ranked as high to very high risk should be included. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00196-9.

Development and application of a second-generation multilingual tool for invasion risk screening of non-native terrestrial plants.

Under the increasing threat to native ecosystems posed by non-native species invasions, there is an urgent need for decision support tools that can more effectively identify non-native species likely to become invasive. As part of the screening (first step) component in non-native species risk analysis, decision support tools have been developed for aquatic and terrestrial organisms. Amongst these tools is the Weed Risk Assessment (WRA) for screening non-native plants. The WRA has provided the foundations for developing the first-generation WRA-type Invasiveness Screening Kit (ISK) tools applicable to a range of aquatic species, and more recently for the second-generation ISK tools applicable to all aquatic organisms (including plants) and terrestrial animals. Given the most extensive usage of the latter toolkits, this study describes the development and application of the Terrestrial Plant Species Invasiveness Screening Kit (TPS-ISK). As a second-generation ISK tool, the TPS-ISK is a multilingual turnkey application that provides several advantages relative to the WRA: (i) compliance with the minimum standards against which a protocol should be evaluated for invasion process and management approaches; (ii) enhanced questionnaire comprehensiveness including a climate change component; (iii) provision of a level of confidence; (iv) error-free computation of risk scores; (v) multilingual support; (vi) possibility for across-study comparisons of screening outcomes; (vii) a powerful graphical user interface; (viii) seamless software deployment and accessibility with improved data exchange. The TPS-ISK successfully risk-ranked five representative sample species for the main taxonomic groups supported by the tool and ten angiosperms previously screened with the WRA for Turkey. The almost 20-year continuous development and evolution of the ISK tools, as opposed to the WRA, closely meet the increasing demand by scientists and decision-makers for a reliable, comprehensive, updatable and easily deployable decision support tool. For terrestrial plant screening, these requirements are therefore met by the newly developed TPS-ISK.

Limited population genetic variation but pronounced seascape genetic structuring in populations of the Mediterranean mussel (Mytilus galloprovincialis) from the eastern Adriatic Sea.

Population genetic analysis of variation at five neutral microsatellite loci for Mediterranean mussels (Mytilus galloprovincialis) from 18 sites along the eastern Adriatic Sea revealed little or no spatial variation. In contrast, seascape genetics analysis revealed a pronounced locus-specific gradient in allelic and genotypic frequencies across the study region. At a sixth locus, MGE7, the frequencies of two alleles, MGE7243 and MGE7249, were strongly associated, negatively and positively, respectively, with a single environmental variable - minimum salinity (minSAL). The frequency of the MGE7243/243 homozygous genotype was strongly negatively associated with minSAL, whereas the frequencies of the MGE7246/249 and the MGE7249/249 genotypes were strongly positively correlated with minSAL. Interpretation of these pronounced gradients is confounded by the fact that minSAL and another environmental variable, maximum sea surface temperature (maxSST), are highly correlated (R = -.911) and are therefore not necessarily acting independently. BLAST searches of the MGE7 locus against M. galloprovincialis whole genome shotgun sequence returned an alignment with contig mg10_S01094 (accession UYJE01010330.1) and 7 predicted M. galloprovincialis proteins VDI82194.1 - VDI82200.1. Conserved domain searches revealed a similar structure to the transcriptional regulator Msx2-interacting protein. The BLASTp search also returned significant alignments to Msx2-interacting proteins in Mytilus coruscus, Crassostrea virginica, and Haliotis rubra. The existence of the MGE7 gradient highlights the role that environmental variation may play in retarding gene flow among wild M. galloprovincialis populations, and also how the success of collection of young mussels (spat) from one site and their transfer to another site (the farm) may be influenced by a single factor such as minSAL or maxSST on a localized scale.

A protocol for screening potentially invasive non-native species using Weed Risk Assessment-type decision-support tools.

There is increasing use worldwide of electronic decision-support tools to identify potentially invasive non-native species so as to inform policy and management decisions aimed at preventing or mitigating the environmental and socio-economic impacts of biological invasions. This study reviews the analytical approaches used to calibrate scores generated by the Weed Risk Assessment and subsequent adaptations thereof and provides a protocol for: (i) the identification of the assessor(s) who will carry out the screenings; (ii) the definition of the risk assessment area; (iii) the criteria for selection of the species for screening; and (iv) the a priori categorisation of the species into invasive or non-invasive necessary to compute the thresholds by which to distinguish between high-risk and medium-risk non-native species. This analytical approach represents an evidence-based and statistically robust means with which to inform decision-makers and stakeholders about policy and management of potentially invasive species and is expected to serve as a general reference of forthcoming screening applications of Weed Risk Assessment-type toolkits.

High stocking density alters growth performance, blood biochemical profiles, and hepatic antioxidative capacity in gibel carp (Carassius gibelio).

This study investigated the effect of stocking density on growth performance, blood biochemical profiles, antioxidative capacity, and muscle quality of gibel carp (Carassius gibelio). Gibel carps (initial body weight 57.04 ± 1.89 g) were reared at high stocking density (HSD, 10.85 kg m-3), medium stocking density (MSD, 5.06 kg m-3), and low stocking density (LSD, 1.47 kg m-3) for 60 days. The LSD group exhibited the highest growth rate, while HSD significantly inhibited fish growth. The muscular compositions of crude fat, crude ash, and moisture were significantly changed by stocking density, but crude protein content did not differ significantly. The stocking density altered the muscular texture profiles of gibel carp. Compared to either the HSD group or the MSD group, the highest levels of resilience and springiness occurred in the LSD group. Significant differences were observed in the levels of plasma glucose, alanine aminotransferase, aspartate aminotransferase, cholesterol, and creatinine among three groups. The fish exhibited the highest level of plasma cortisol as well as the lowest levels of triiodothyronine and thyroxine in the HSD group. The fish stocked in the LSD group showed the highest activities of superoxide dismutase, glutathione peroxidase, and catalase as well as the highest content of glutathione in liver. The significant highest total antioxidant capacity occurred in the fish stocked in the LSD group. The results showed that HSD resulted in chronic crowding stress, and exerted negative impact on growth performance, muscle quality, and antioxidative capacity of gibel carp.

Sugar transporter genes in grass carp (Ctenopharyngodon idellus): molecular cloning, characterization, and expression in response to different stocking densities.

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m-2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.

Potentially toxic elements in muscle tissue of different fish species from the Sava River and risk assessment for consumers.

Fish from the Sava River are consumed daily by the local people: therefore, concern has been raised about the health implications of eating contaminated fish. In the present study, potentially toxic elements (PTE), such as Zn, Cu, Cr, Ni, Cd, Pb, As, Hg, and methylmercury (MeHg), were determined in ichthyofauna that are commonly consumed. PTE were determined in the fish muscle tissue. Fish were sampled at 12 locations from the source of the Sava River to its confluence with the Danube River during two sampling campaigns, namely; in 2014 under high water conditions and in 2015 under normal water conditions. Due to the different water regimes, different fish species were collected for chemical analysis. We observed that the concentrations of elements analysed in the fish muscle tissue were generally very low, except for those of Hg. Moreover, more than 90% of Hg present in the fish was in its most toxic form, namely MeHg. Especially in fish from the 2015 sampling campaign, Hg and MeHg concentrations increased with fish size, trophic level, and in the downstream direction. In addition, for Pb and As, and to some extent for Cd and Cr, spatial differences were detected in both years. The highest concentrations of PTE were detected in fish from sites with intensive industrial and agricultural activities. The consumption of fish in general does not pose a health risk for the PTE studied, except for Hg/MeHg at selected contaminated sites.

Long-term analysis of fish assemblage structure in the middle section of the Sava River - The impact of pollution, flood protection and dam construction.

At the beginning of the 20th century, the middle section of the Sava River in Croatia was unaffected by major human activities and rich in ichthyofauna. The Sava River was important for commercial and recreational fishing for the local population, which still remains today. However, the 1920s mining industry was established in Slovenia, which emitted carbon dust into the Sava River. At the same time, the construction of embankments to mitigate flooding started in the middle section. Furthermore, in the 1980s, the Krsko nuclear power plant (NPP), and in the 2010s, the Krsko hydropower plant (HPP) were built in Slovenia. These activities could have an impact on the composition of fish communities downstream from the major sources of disturbances. Therefore, the main aim of this paper were to analyze the changes in fish assemblages of the Sava River from 1978 to 2017, prior to and after the construction of Krsko NPP and HPP at the Medsave site on the Sava River, 20 km downstream from the major construction operations. Collected data were divided into four sampling periods (SP): SP1, from 1978 to 1980; SP2, from 1991 to 1994; SP3, from 2001 to 2006, and SP4 from 2011 to 2017. Besides alien fish species, water quality and hydromorphological modifications were identified as significant stressors. In SP1 and SP2 limnophilic and eurytopic fish groups were predominant, and 26 different fish species were identified, but in SP3 and SP4 rheophilic fish groups become dominant, and the diversity has declined to 21 species. Threatened species blageon, Telestes souffia seems to be missing from the main course of the Sava River in last 20 years. It can be concluded that disturbances in the fish assemblage pattern have coincided with the presence of multiple stressors of human origin.

First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea.

The Mediterranean Sea is increasingly under threat from invasive species that may negatively affect biodiversity and/or modify ecosystem structure and function. The bivalve mollusc Pinctada imbricata radiata is listed among the 100 most invasive species in the Mediterranean. A first finding of an established population of P. imbricata radiata in the coastal waters of the eastern Adriatic Sea, is presented in this paper. Six and then 30 live specimens were collected in 2015 and in 2017, respectively, at depths of 5 to 15m from the island of Mljet, Croatia. DNA sequencing of the mitochondrial cytochrome c oxidase I gene (COI) revealed three different haplotypes. All samples showed greatest similarity (98 to >99%) to P. radiata COI sequence records in GenBank (=P. imbricata radiata as used in this paper). A Neighbour Joining tree placed all Croatian samples within the 100% bootstrap supported clade for P. imbricata radiata.

Characterization of sections of the Sava River based on fish community structure.

Sampling was undertaken, with the same fishing gear and along the Sava River, from its source to its confluence, in September 2014 and September 2015. In total, 44 fish species were identified, of which 37 were native species and 7 were alien. Fish samples revealed independence in terms of both species composition and their abundance under different hydrological conditions. During flooding and high water levels in 2014, pelagic fish species were sampled in greater proportion than at lower water levels in 2015 when benthic fish species were more abundant. The flood wave in 2014 was accompanied by catch of common carp, Cyprinus carpio, a typical lower rhithron fish species in the upper course, and of tench, Tinca tinca, a typical potamon fish species of backwaters, in the main channel of the lower Sava River. One specimen of bighead goby, Ponticola kessleri, which is common in the potamon fish community, was caught during the 2015 sampling close to the boundary between the upper and middle sections of the Sava. This is the first record of Ponto-Caspian gobies in the inland waters of Slovenia. Its finding far upstream indicates a strong effect of an as yet unidentified stress along the Sava River up to the spot where the bighead goby was sampled. Finally, these results indicate that pelagic fish species are more resistant to the stressful effect of flooding than benthic species, and that the structure of fish communities is influenced/affected by flooding as a short-term stressor. The progressively increasing number of alien fish species downstream in the Sava River point to the effects of long-term human-induced stressors in the area.

Evaluation of Genotoxic Pressure along the Sava River.

In this study we have performed a comprehensive genotoxicological survey along the 900 rkm of the Sava River. In total, 12 sites were chosen in compliance with the goals of GLOBAQUA project dealing with the effects of multiple stressors on biodiversity and functioning of aquatic ecosystems. The genotoxic potential was assessed using a complex battery of bioassays performed in prokaryotes and aquatic eukaryotes (freshwater fish). Battery comprised evaluation of mutagenicity by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg-modified comet assay) was studied in blood cells of bleak and spirlin (Alburnus alburnus/Alburnoides bipunctatus respectively). Result indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. The standard and Fpg-modified comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with micronucleus assay and SOS/umuC test. Our data represent snapshot of the current status of the river which indicates the presence of genotoxic potential along the river which can be traced to the deterioration of quality of the Sava River by communal and industrial wastewaters. The major highlight of the study is that we have provided complex set of data obtained from a single source (homogeneity of analyses for all samples).

WITHDRAWN: First record and distribution of the black pygmy mussel, Limnoperna securis (Lamarck, 1819) in the eastern Adriatic and Ionian seas.

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Dietary items as possible sources of (137)Cs in large carnivores in the Gorski Kotar forest ecosystem, Western Croatia.

The mountain forest ecosystem of Gorski Kotar is distant from any significant sources of environmental pollution, though recent findings have revealed that this region is among the most intense (137)Cs contaminated area in Croatia. Therefore, the aim of this study was to investigate (137)Cs and (40)K load in three large predator species in the mountain forest ecosystem. Radionuclides mass activities were determined by the gamma-spectrometric method in the muscle tissue of brown bear (47), wolf (7), lynx (1) and golden jackal (2). The highest (137)Cs mass activity was found in lynx (153 Bq kg(-1)), followed by brown bear (132 Bq kg(-1)), wolf (22.2 Bq kg(-1)), and golden jackal (2.48 Bq kg(-1)). Analysis of 63 samples of dietary items suggests that they are not all potentially dominant sources of (137)Cs for wildlife. The most important source of radionuclides for the higher parts of the food-chain from the study area were found to be the mushroom species wood hedgehog (Hydnum repandum), with a transfer factor TF of 5.166, and blueberry (Vaccinium myrtillus) as a plant species (TF=2.096). Food items of animal origin indicated higher mass activity of radionuclides and therefore are possible moderate bioindicators of environmental pollution. The results also revealed that possible unknown wild animal food sources are a caesium source in the study region, and further study is required to illuminate this issue.

Biochemical indicators and biomarkers in chub (Squalius cephalus L.) from the Sava River.

Biochemical indicators and biomarkers were analyzed in the liver and gills of chub caught in three localities along the Sava River exposed to different environmental impacts. Sampling sites were: downstream from Zagreb (Zgd), downstream Sremska Mitrovica (SM) and downstream from Belgrade (Bgd). We observed that the relative amounts and levels of activity of Cu, Zn containing superoxide dismutase and glutathione in both the liver and gills, and the relative amounts of heat shock protein (HSP90) and metallothioneins in the gills were highest in the Zgd locality, suggesting a higher impact of metal pollution. The Zgd locality had higher concentrations of trace metals in the water, especially iron. In the SM and Bgd localities, higher relative levels of glutathione peroxidase and catalase were recorded (especially in SM) as compared to the Zgd locality, pointing to the presence of hydrogen peroxide and different classes of organic peroxides. Low water oxygen and high temperature levels in the Bgd locality suggesting different metabolic activity between examined locations. Our results suggest that different presence and concentrations of individual environmental factors (total environment) influence the way how fish establish homeostasis.

Dietary habits of invasive Ponto-Caspian gobies in the Croatian part of the Danube River basin and their potential impact on benthic fish communities.

Invasive Ponto-Caspian (P-C(1)) gobies have recently caused dramatic changes in fish assemblage structures throughout the Danube basin. While their presence in the Croatian part of the basin has been noted and distribution studied, their dietary habits and impacts on native fish communities have, until now, been unknown. In 2011, 17 locations in the Sava River Basin were sampled for fish and 15 for benthic invertebrates. Fish population monitoring data, available for nine seasons (2003-2006 and 2010-2014) and 12 locations, were used to analyse the impacts of P-C gobies on benthic fish abundance. Gut content analysis indicates that the monkey goby Neogobius fluviatilis diet is very diverse, but dominated by Trichoptera, Chironomidae, Bivalvia and Odonata. The diet overlaps considerably with the round goby Neogobius melanostomus diet, although Gastropoda are dominant in the latter's diet. Small fish and Gammarus sp. dominate the bighead goby Ponticola kessleri diet. Comparison of gut content with the prey available in the environment indicates that monkey and round gobies exhibit preference for Trichoptera, Megaloptera and Coleoptera, and bighead goby for Trichoptera, Gammarus sp. and Pisces. P-C gobies in the Sava River are spreading upstream, towards the reaches with lower fish diversity. Analyses indicate potentially positive impacts of P-C gobies' presence on some fish populations: round and bighead goby on Balkan golden loach Sabanejewia balcanica and monkey goby on common carp Cyprinus carpio, crucian carp Carassius carassius, burbot Lota lota and Balkan loach Cobitis elongata. However, there are also indications that bighead and round goby could adversely impact the native chub Squalius cephalus and zingel Zingel zingel populations, respectively. As P-C gobies are still in the expansionary period of invasion and the ecosystem still adapting to new circumstances, continued monitoring of fish population dynamics in the Sava basin is needed to determine the outcome and impacts of this invasion.